1.
Eukaryotic transcription
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Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of RNA replica. Gene transcription occurs in eukaryotic and prokaryotic cells. Unlike prokaryotic RNA polymerase that initiates the transcription of all different types of RNA, RNA polymerase in eukaryotes comes in three variations, each encoding a different type of gene, a eukaryotic cell has a nucleus that separates the processes of transcription and translation. Eukaryotic transcription occurs within the nucleus where DNA is packaged into nucleosomes, the complexity of the eukaryotic genome necessitates a great variety and complexity of gene expression control. Transcription is the process of copying genetic information stored in a DNA strand into a complementary strand of RNA. Eukaryotic transcription takes place in the nucleus of the cell and proceeds in three stages, initiation, elongation, and termination. The transcriptional machinery that catalyzes this reaction has at its core three multi-subunit RNA polymerases. RNA polymerase I is responsible for transcribing RNA that codes for genes that become structural components of the ribosome, protein coding genes are transcribed into messenger RNAs that carry the information from DNA to the site of protein synthesis. Although mRNAs possess great diversity, they are not the most abundant RNA species made in the cell, the so-called non-coding RNAs account for the large majority of the transcriptional output of a cell. These non-coding RNAs perform a variety of important cellular functions, eukaryotes have three nuclear RNA polymerases, each with distinct roles and properties RNA polymerase I catalyses the transcription of all rRNA genes except 5S. These rRNA genes are organised into a single unit and are transcribed into a continuous transcript. This precursor is then processed into three rRNAs, 18S,5. 8S, and 28S, the transcription of rRNA genes takes place in a specialised structure of the nucleus called the nucleolus, where the transcribed rRNAs are combined with proteins to form ribosomes. RNA polymerase II is responsible for the transcription of all mRNAs, some snRNAs, siRNAs, many Pol II transcripts exist transiently as single strand precursor RNAs that are further processed to generate mature RNAs. For example, precursor mRNAs are extensively processed before exiting into the cytoplasm through the pore for protein translation. RNA polymerase III transcribes small non-coding RNAs, including tRNAs, 5S rRNA, U6 snRNA, SRP RNA, RNA Polymerases I, II, and III contain 14,12, and 17 subunits, respectively. All three eukaryotic polymerases have five core subunits that exhibit homology with the β, β’, αI, αII, an identical ω-like subunit is used by all three eukaryotic polymerases, while the same α-like subunits are used by Pol I and III. The three eukaryotic polymerases share four other common subunits among themselves, the remaining subunits are unique to each RNA polymerase. The additional subunits found in Pol I and Pol III relative to Pol II, are homologous to Pol II transcription factors, crystal structures of RNA polymerases I and II provide an opportunity to understand the interactions among the subunits and the molecular mechanism of eukaryotic transcription in atomic detail
2.
Gene expression
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Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are proteins, but in non-protein coding genes such as transfer RNA or small nuclear RNA genes. The process of gene expression is used by all known life—eukaryotes, prokaryotes, several steps in the gene expression process may be modulated, including the transcription, RNA splicing, translation, and post-translational modification of a protein. Gene regulation gives the cell control over structure and function, and is the basis for differentiation, morphogenesis. In genetics, gene expression is the most fundamental level at which the genotype gives rise to the phenotype, the genetic code stored in DNA is interpreted by gene expression, and the properties of the expression give rise to the organisms phenotype. Such phenotypes are expressed by the synthesis of proteins that control the organisms shape. Regulation of gene expression is critical to an organisms development. A gene is a stretch of DNA that encodes information, genomic DNA consists of two antiparallel and reverse complementary strands, each having 5 and 3 ends. This RNA is complementary to the template 3 →5 DNA strand, therefore, the resulting 5 →3 RNA strand is identical to the coding DNA strand with the exception that thymines are replaced with uracils in the RNA. A coding DNA strand reading ATG is indirectly transcribed through the non-coding strand as AUG in RNA, in prokaryotes, transcription is carried out by a single type of RNA polymerase, which needs a DNA sequence called a Pribnow box as well as a sigma factor to start transcription. RNA polymerase I is responsible for transcription of ribosomal RNA genes, RNA polymerase II transcribes all protein-coding genes but also some non-coding RNAs. Pol II includes a C-terminal domain that is rich in serine residues, when these residues are phosphorylated, the CTD binds to various protein factors that promote transcript maturation and modification. RNA polymerase III transcribes 5S rRNA, transfer RNA genes, transcription ends when the polymerase encounters a sequence called the terminator. These include 5 capping, which is set of reactions that add 7-methylguanosine to the 5 end of pre-mRNA. The m7G cap is then bound by cap binding complex heterodimer, another modification is 3 cleavage and polyadenylation. They occur if polyadenylation signal sequence is present in pre-mRNA, which is usually between protein-coding sequence and terminator, the pre-mRNA is first cleaved and then a series of ~200 adenines are added to form poly tail, which protects the RNA from degradation. Poly tail is bound by multiple poly-binding proteins necessary for mRNA export, a very important modification of eukaryotic pre-mRNA is RNA splicing. The majority of eukaryotic pre-mRNAs consist of alternating segments called exons and introns, in certain cases, some introns or exons can be either removed or retained in mature mRNA
3.
DNA
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Deoxyribonucleic acid is a molecule that carries the genetic instructions used in the growth, development, functioning and reproduction of all known living organisms and many viruses. DNA and RNA are nucleic acids, alongside proteins, lipids and complex carbohydrates, most DNA molecules consist of two biopolymer strands coiled around each other to form a double helix. The two DNA strands are termed polynucleotides since they are composed of simpler units called nucleotides. Each nucleotide is composed of one of four nitrogen-containing nucleobases—cytosine, guanine, adenine, or thymine —a sugar called deoxyribose, and a phosphate group. The nucleotides are joined to one another in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. The nitrogenous bases of the two polynucleotide strands are bound together, according to base pairing rules, with hydrogen bonds to make double-stranded DNA. The total amount of related DNA base pairs on Earth is estimated at 5.0 x 1037, in comparison the total mass of the biosphere has been estimated to be as much as 4 trillion tons of carbon. The DNA backbone is resistant to cleavage, and both strands of the double-stranded structure store the same biological information and this information is replicated as and when the two strands separate. A large part of DNA is non-coding, meaning that these sections do not serve as patterns for protein sequences, the two strands of DNA run in opposite directions to each other and are thus antiparallel. Attached to each sugar is one of four types of nucleobases and it is the sequence of these four nucleobases along the backbone that encodes biological information. RNA strands are created using DNA strands as a template in a process called transcription, under the genetic code, these RNA strands are translated to specify the sequence of amino acids within proteins in a process called translation. Within eukaryotic cells DNA is organized into structures called chromosomes. During cell division these chromosomes are duplicated in the process of DNA replication, eukaryotic organisms store most of their DNA inside the cell nucleus and some of their DNA in organelles, such as mitochondria or chloroplasts. In contrast prokaryotes store their DNA only in the cytoplasm, within the eukaryotic chromosomes, chromatin proteins such as histones compact and organize DNA. These compact structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed, DNA was first isolated by Friedrich Miescher in 1869. DNA is used by researchers as a tool to explore physical laws and theories, such as the ergodic theorem. The unique material properties of DNA have made it an attractive molecule for material scientists and engineers interested in micro-, among notable advances in this field are DNA origami and DNA-based hybrid materials. DNA is a polymer made from repeating units called nucleotides
4.
RNA
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Ribonucleic acid is a polymeric molecule essential in various biological roles in coding, decoding, regulation, and expression of genes. RNA and DNA are nucleic acids, and, along with proteins and carbohydrates, like DNA, RNA is assembled as a chain of nucleotides, but unlike DNA it is more often found in nature as a single-strand folded onto itself, rather than a paired double-strand. Cellular organisms use messenger RNA to convey information that directs synthesis of specific proteins. Many viruses encode their genetic information using an RNA genome, some RNA molecules play an active role within cells by catalyzing biological reactions, controlling gene expression, or sensing and communicating responses to cellular signals. One of these processes is protein synthesis, a universal function where RNA molecules direct the assembly of proteins on ribosomes. This process uses transfer RNA molecules to deliver amino acids to the ribosome, analysis of these RNAs has revealed that they are highly structured. Unlike DNA, their structures do not consist of long double helices, in this fashion, RNAs can achieve chemical catalysis. For instance, determination of the structure of the enzyme that catalyzes peptide bond formation—revealed that its active site is composed entirely of RNA. Each nucleotide in RNA contains a sugar, with carbons numbered 1 through 5. A base is attached to the 1 position, in general, adenine, cytosine, guanine, adenine and guanine are purines, cytosine and uracil are pyrimidines. A phosphate group is attached to the 3 position of one ribose, the phosphate groups have a negative charge each, making RNA a charged molecule. The bases form hydrogen bonds between cytosine and guanine, between adenine and uracil and between guanine and uracil, however, other interactions are possible, such as a group of adenine bases binding to each other in a bulge, or the GNRA tetraloop that has a guanine–adenine base-pair. An important structural feature of RNA that distinguishes it from DNA is the presence of a group at the 2 position of the ribose sugar. The A-form geometry results in a deep and narrow major groove. RNA is transcribed with only four bases, but these bases, pseudouridine, in which the linkage between uracil and ribose is changed from a C–N bond to a C–C bond, and ribothymidine are found in various places. Another notable modified base is hypoxanthine, a deaminated adenine base whose nucleoside is called inosine, inosine plays a key role in the wobble hypothesis of the genetic code. The specific roles of many of these modifications in RNA are not fully understood, the functional form of single-stranded RNA molecules, just like proteins, frequently requires a specific tertiary structure. The scaffold for this structure is provided by secondary structural elements that are hydrogen bonds within the molecule and this leads to several recognizable domains of secondary structure like hairpin loops, bulges, and internal loops
5.
Messenger RNA
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Messenger RNA is a large family of RNA molecules that convey genetic information from DNA to the ribosome, where they specify the amino acid sequence of the protein products of gene expression. As in DNA, mRNA genetic information is in the sequence of nucleotides, each codon encodes for a specific amino acid, except the stop codons, which terminate protein synthesis. It should not be confused with mitochondrial DNA, the brief existence of an mRNA molecule begins with transcription, and ultimately ends in degradation. During its life, a molecule may also be processed, edited. Eukaryotic mRNA molecules often require extensive processing and transport, while prokaryotic mRNA molecules do not, a molecule of eukaryotic mRNA and the proteins surrounding it are together called a messenger RNP. Transcription is when RNA is made from DNA, during transcription, RNA polymerase makes a copy of a gene from the DNA to mRNA as needed. This process is similar in eukaryotes and prokaryotes, the short-lived, unprocessed or partially processed product is termed precursor mRNA, or pre-mRNA, once completely processed, it is termed mature mRNA. Processing of mRNA differs greatly among eukaryotes, bacteria, and archea, non-eukaryotic mRNA is, in essence, mature upon transcription and requires no processing, except in rare cases. Eukaryotic pre-mRNA, however, requires extensive processing, a 5 cap is a modified guanine nucleotide that has been added to the front or 5 end of a eukaryotic messenger RNA shortly after the start of transcription. The 5 cap consists of a terminal 7-methylguanosine residue that is linked through a 5-5-triphosphate bond to the first transcribed nucleotide and its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other, shortly after the start of transcription, the 5 end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the reactions that are required for mRNA capping. Synthesis proceeds as a biochemical reaction. In some instances, an mRNA will be edited, changing the composition of that mRNA. An example in humans is the apolipoprotein B mRNA, which is edited in some tissues, the editing creates an early stop codon, which, upon translation, produces a shorter protein. Polyadenylation is the covalent linkage of a moiety to a messenger RNA molecule. In eukaryotic organisms most messenger RNA molecules are polyadenylated at the 3 end, the poly tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, MRNA can also be polyadenylated in prokaryotic organisms, where poly tails act to facilitate, rather than impede, exonucleolytic degradation
6.
Enzyme
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Enzymes /ˈɛnzaɪmz/ are macromolecular biological catalysts. Enzymes accelerate, or catalyze, chemical reactions, the molecules at the beginning of the process upon which enzymes may act are called substrates and the enzyme converts these into different molecules, called products. Almost all metabolic processes in the cell need enzymes in order to occur at rates fast enough to sustain life, the set of enzymes made in a cell determines which metabolic pathways occur in that cell. The study of enzymes is called enzymology, enzymes are known to catalyze more than 5,000 biochemical reaction types. Most enzymes are proteins, although a few are catalytic RNA molecules, enzymes specificity comes from their unique three-dimensional structures. Like all catalysts, enzymes increase the rate of a reaction by lowering its activation energy, some enzymes can make their conversion of substrate to product occur many millions of times faster. An extreme example is orotidine 5-phosphate decarboxylase, which allows a reaction that would take millions of years to occur in milliseconds. Chemically, enzymes are like any catalyst and are not consumed in chemical reactions, enzymes differ from most other catalysts by being much more specific. Enzyme activity can be affected by other molecules, inhibitors are molecules that decrease enzyme activity, many drugs and poisons are enzyme inhibitors. An enzymes activity decreases markedly outside its optimal temperature and pH, some enzymes are used commercially, for example, in the synthesis of antibiotics. French chemist Anselme Payen was the first to discover an enzyme, diastase and he wrote that alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells. In 1877, German physiologist Wilhelm Kühne first used the term enzyme, the word enzyme was used later to refer to nonliving substances such as pepsin, and the word ferment was used to refer to chemical activity produced by living organisms. Eduard Buchner submitted his first paper on the study of yeast extracts in 1897, in a series of experiments at the University of Berlin, he found that sugar was fermented by yeast extracts even when there were no living yeast cells in the mixture. He named the enzyme that brought about the fermentation of sucrose zymase, in 1907, he received the Nobel Prize in Chemistry for his discovery of cell-free fermentation. Following Buchners example, enzymes are usually named according to the reaction they carry out, the biochemical identity of enzymes was still unknown in the early 1900s. Sumner showed that the enzyme urease was a protein and crystallized it. These three scientists were awarded the 1946 Nobel Prize in Chemistry, the discovery that enzymes could be crystallized eventually allowed their structures to be solved by x-ray crystallography. This high-resolution structure of lysozyme marked the beginning of the field of structural biology, an enzymes name is often derived from its substrate or the chemical reaction it catalyzes, with the word ending in -ase
7.
RNA polymerase
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RNA polymerase, also known as DNA-dependent RNA polymerase, is an enzyme that produces primary transcript RNA. In cells, RNAP is necessary for constructing RNA chains using DNA genes as templates, RNA polymerase enzymes are essential to life and are found in all organisms and many viruses. In chemical terms, RNAP is a transferase that polymerizes ribonucleotides at the 3 end of an RNA transcript. RNAP was discovered independently by Charles Loe, Audrey Stevens, the 2006 Nobel Prize in Chemistry was awarded to Roger D. Kornberg for creating detailed molecular images of RNA polymerase during various stages of the transcription process. Therefore, it is surprising that the activity of RNAP is long, complex. In Escherichia coli bacteria, more than 100 transcription factors have been identified, RNAP can initiate transcription at specific DNA sequences known as promoters. It then produces an RNA chain, which is complementary to the template DNA strand, the process of adding nucleotides to the RNA strand is known as elongation, in eukaryotes, RNAP can build chains as long as 2.4 million nucleotides. RNAP will preferentially release its RNA transcript at specific DNA sequences encoded at the end of genes, products of RNAP include, Messenger RNA —template for the synthesis of proteins by ribosomes. Non-coding RNA or RNA genes—a broad class of genes that encode RNA that is not translated into protein, the most prominent examples of RNA genes are transfer RNA and ribosomal RNA, both of which are involved in the process of translation. However, since the late 1990s, many new RNA genes have been found and it is able to do this because specific interactions with the initiating nucleotide hold RNAP rigidly in place, facilitating chemical attack on the incoming nucleotide. Such specific interactions explain why RNAP prefers to start transcripts with ATP, in contrast to DNA polymerase, RNAP includes helicase activity, therefore no separate enzyme is needed to unwind DNA. There are multiple interchangeable sigma factors, each of which recognizes a set of promoters. After binding to the DNA, the RNA polymerase switches from a complex to an open complex. This change involves the separation of the DNA strands to form a section of DNA of approximately 13 bp. Ribonucleotides are base-paired to the template DNA strand, according to Watson-Crick base-pairing interactions, supercoiling plays an important part in polymerase activity because of the unwinding and rewinding of DNA. Because regions of DNA in front of RNAP are unwound, there are compensatory positive supercoils, regions behind RNAP are rewound and negative supercoils are present. As noted above, RNA polymerase makes contacts with the promoter region, however these stabilizing contacts inhibit the enzymes ability to access DNA further downstream and thus the synthesis of the full-length product. Once the open complex is stabilized, RNA polymerase synthesizes an RNA strand to establish a DNA-RNA heteroduplex at the active center, which stabilizes the elongation complex
8.
Nucleic acid
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Nucleic acids are biopolymers, or large biomolecules, essential to all known forms of life. They are composed of monomers, which are made of three components, a 5-carbon sugar, a phosphate group, and a nitrogenous base. If the sugar is a simple ribose, the polymer is RNA, if the sugar is derived from ribose as deoxyribose, Nucleic acids are arguably the most important of all biomolecules. They are found in abundance in all living things, where they function to create and encode, the encoded information is contained and conveyed via the nucleic acid sequence, which provides the ladder-step ordering of nucleotides within the molecules of RNA and DNA. Using amino acids and the known as protein synthesis, the specific sequencing in DNA of these nucleobase-pairs enables storing and transmitting coded instructions as genes. In RNA, base-pair sequencing provides for manufacturing new proteins that determine the frames and parts, Nucleic acids were discovered by Friedrich Miescher in 1869. In 1889 Richard Altmann discovered that nuclein has acidic properties, and it became called nucleic acid In 1938 Astbury, in 1953 Watson and Crick determined the structure of DNA. The term nucleic acid is the name for DNA and RNA, members of a family of biopolymers. Nucleic acids were named for their initial discovery within the nucleus, all living cells contain both DNA and RNA, while viruses contain either DNA or RNA, but usually not both. The basic component of nucleic acids is the nucleotide, each of which contains a pentose sugar, a phosphate group. Nucleic acids are generated within the laboratory, through the use of enzymes. The chemical methods also enable the generation of altered nucleic acids that are not found in nature, Nucleic acids are generally very large molecules. Indeed, DNA molecules are probably the largest individual molecules known, well-studied biological nucleic acid molecules range in size from 21 nucleotides to large chromosomes. In most cases, naturally occurring DNA molecules are double-stranded and RNA molecules are single-stranded, Nucleic acids are linear polymers of nucleotides. Each nucleotide consists of three components, a purine or pyrimidine nucleobase, a sugar, and a phosphate group. The substructure consisting of a nucleobase plus sugar is termed a nucleoside, Nucleic acid types differ in the structure of the sugar in their nucleotides–DNA contains 2-deoxyribose while RNA contains ribose. Also, the found in the two nucleic acid types are different, adenine, cytosine, and guanine are found in both RNA and DNA, while thymine occurs in DNA and uracil occurs in RNA. The sugars and phosphates in nucleic acids are connected to other in an alternating chain through phosphodiester linkages
9.
Base pair
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A base pair is a unit consisting of two nucleobases bound to each other by hydrogen bonds. They form the blocks of the DNA double helix. Dictated by specific hydrogen bonding patterns, Watson-Crick base pairs allow the DNA helix to maintain a regular helical structure that is dependent on its nucleotide sequence. The complementary nature of this structure provides a backup copy of all genetic information encoded within double-stranded DNA. Many DNA-binding proteins can recognize specific base pairing patterns that identify particular regulatory regions of genes, intramolecular base pairs can occur within single-stranded nucleic acids. The size of a gene or an organisms entire genome is often measured in base pairs because DNA is usually double-stranded. Hence, the number of base pairs is equal to the number of nucleotides in one of the strands. The haploid human genome is estimated to be about 3.2 billion bases long and to contain 20, a kilobase is a unit of measurement in molecular biology equal to 1000 base pairs of DNA or RNA. The total amount of related DNA base pairs on Earth is estimated at 5.0 x 1037, in comparison, the total mass of the biosphere has been estimated to be as much as 4 TtC. Hydrogen bonding is the interaction that underlies the base-pairing rules described above. Appropriate geometrical correspondence of hydrogen donors and acceptors allows only the right pairs to form stably. Purine-pyrimidine base pairing of AT or GC or UA results in proper duplex structure, the only other purine-pyrimidine pairings would be AC and GT and UG, these pairings are mismatches because the patterns of hydrogen donors and acceptors do not correspond. The GU pairing, with two bonds, does occur fairly often in RNA. Higher GC content results in higher melting temperatures, it is, therefore, on the converse, regions of a genome that need to separate frequently — for example, the promoter regions for often-transcribed genes — are comparatively GC-poor. GC content and melting temperature must also be taken into account when designing primers for PCR reactions, the following DNA sequences illustrate pair double-stranded patterns. By convention, the top strand is written from the 5 end to the 3 end, thus and this is due to their isosteric chemistry. One common mutagenic base analog is 5-bromouracil, which resembles thymine, most intercalators are large polyaromatic compounds and are known or suspected carcinogens. Examples include ethidium bromide and acridine, an unnatural base pair is a designed subunit of DNA which is created in a laboratory and does not occur in nature
10.
Nucleotide
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Nucleotides are organic molecules that serve as the monomer units for forming the nucleic acid polymers DNA and RNA, both of which are essential biomolecules in all life-forms on Earth. Nucleotides are the blocks of nucleic acids, they are composed of three subunit molecules, a nitrogenous base, a five-carbon sugar, and at least one phosphate group. They are also known as phosphate nucleotides, a nucleoside is a nitrogenuous base and a 5-carbon sugar. Thus a nucleoside plus a group yields a nucleotide. Nucleotides also play a role in life-form metabolism at the fundamental. In addition, nucleotides participate in signaling, and are incorporated into important cofactors of enzymatic reactions. In experimental biochemistry, nucleotides can be radiolabeled with radionuclides to yield radionucleotides, a nucleotide is composed of three distinctive chemical sub-units, a five-carbon sugar molecule, a nitrogenous base—which two together are called a nucleoside—and one phosphate group. With all three joined, a nucleotide is also termed a nucleoside monophosphate, thus, the terms nucleoside diphosphate or nucleoside triphosphate may also indicate nucleotides. Nucleotides contain either a purine or a pyrimidine base—i. e, the nitrogenous base molecule, also known as a nucleobase—and are termed ribonucleotides if the sugar is ribose, or deoxyribonucleotides if the sugar is deoxyribose. These chain-joins of sugar and phosphate molecules create a backbone strand for a single- or double helix, in any one strand, the chemical orientation of the chain-joins runs from the 5-end to the 3-end —referring to the five carbon sites on sugar molecules in adjacent nucleotides. Unlike in nucleic acid nucleotides, singular cyclic nucleotides are formed when the group is bound twice to the same sugar molecule. These individual nucleotides function in cell metabolism rather than the nucleic acid structures of long-chain molecules, nucleic acids then are polymeric macromolecules assembled from nucleotides, the monomer-units of nucleic acids. The purine bases adenine and guanine and pyrimidine base cytosine occur in both DNA and RNA, while the pyrimidine bases thymine and uracil in just one, adenine forms a base pair with thymine with two hydrogen bonds, while guanine pairs with cytosine with three hydrogen bonds. Nucleotides can be synthesized by a variety of both in vitro and in vivo. In vivo, nucleotides can be synthesized de novo or recycled through salvage pathways, the components used in de novo nucleotide synthesis are derived from biosynthetic precursors of carbohydrate and amino acid metabolism, and from ammonia and carbon dioxide. The liver is the organ of de novo synthesis of all four nucleotides. De novo synthesis of pyrimidines and purines follows two different pathways, purines, however, are first synthesized from the sugar template onto which the ring synthesis occurs. For reference, the syntheses of the purine and pyrimidine nucleotides are carried out by several enzymes in the cytoplasm of the cell, nucleotides undergo breakdown such that useful parts can be reused in synthesis reactions to create new nucleotides
11.
Complementarity (molecular biology)
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In molecular biology, complementarity describes a relationship between two structures each following the lock-and-key principle. This complementary base pairing allows cells to copy information from one generation to another and even find, the degree of complementarity between two nucleic acid strands may vary, from complete complementarity to no complementarity and determines the stability of the sequences to be together. Furthermore, various DNA repair functions as well as functions are based on base pair complementarity. In biotechnology, the principle of base pair complementarity allows the generation of DNA hybrids between RNA and DNA, and opens the door to modern tools such as cDNA libraries, complementarity is achieved by distinct interactions between nucleobases, adenine, thymine, guanine and cytosine. Adenine and guanine are purines, while thymine, cytosine and uracil are pyrimidines, both types of molecules complement each other and can only base pair with the opposing type of nucleobase. In nucleic acid, nucleobases are held together by hydrogen bonding, the base complement A=T shares two hydrogen bonds, while the base pair G≡C has three hydrogen bonds. All other configurations between nucleobases would hinder double helix formation, DNA strands are oriented in opposite directions, they are said to be antiparallel. A complementary strand of DNA or RNA may be constructed based on nucleobase complementarity, each base pair, A=T vs. G≡C, takes up roughly the same space, thereby enabling a twisted DNA double helix formation without any spatial distortions. Hydrogen bonding between the nucleobases also stabilizes the DNA double helix, complementarity of DNA strands in a double helix make it possible to use one strand as a template to construct the other. This principle plays an important role in DNA replication, setting the foundation of heredity by explaining how genetic information can be passed down to the next generation, complementarity is also utilized in DNA transcription, which generates an RNA strand from a DNA template. DNA repair mechanisms such as proof reading are complementarity based and allow for error correction during DNA replication by removing mismatched nucleobases, nucleic acids strands may also form hybrids in which single stranded DNA may readily anneal with complementary DNA or RNA. This principle is the basis of commonly performed laboratory techniques such as the chain reaction. Two strands of complementary sequence are referred to as sense and anti-sense, the sense strand is, generally, the transcribed sequence of DNA or the RNA that was generated in transcription. While the anti-sense strand is the strand that is complementary to the sense sequence, self-complementarity refers to the fact that a sequence of DNA or RNA may fold back on itself, creating a double-strand like structure. Depending on how close together the parts of the sequence are that are self-complementary, RNA is more likely to form these kinds of structures due to base pair binding not seen in DNA, such as guanine binding with uracil. Complementarity can be found between short nucleic acid stretches and a region or an transcribed gene, and results in base pairing. These short nucleic acid sequences are found in nature and have regulatory functions such as gene silencing. Antisense transcripts are stretches of non coding mRNA that are complementary to the coding sequence, genome wide studies have shown that RNA antisense transcripts occur commonly within nature
12.
Antiparallel (biochemistry)
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In biochemistry, two biopolymers are antiparallel if they run parallel to each other but with opposite alignments. An example is the two strands of a DNA double helix, which run in opposite directions alongside each other. Nucleic acid molecules have an end and a hydroxyl end. This notation follows from organic chemistry nomenclature, and can be used to define the movement of such as DNA polymerases relative to the DNA strand in a non-arbitrary manner. G-quadruplexes, also known as G4 DNA are secondary structures found in nucleic acids that are rich in guanine and these structures are normally located at the ends of the chromosomes know as the telomeric regions. The G-quadruplex can either be parallel or antiparallel depending on the loop configuration, if all the DNA strands run in the same direction, it is termed to be a parallel quadruplex, and is known as a strand-reversal/propeller, connecting adjacent parallel strands. The structure of these G-quadruplexes can be determined by a cation, in DNA, the 5 carbon is located at the top of the leading strand, and the 3 carbon is located at the lower section of the lagging strand. The nucleic acid sequences are complementary and parallel, but they go in opposite directions, the antiparallel structure of DNA is important in DNA replication because it replicates the leading strand one way and the lagging strand the other way. During DNA replication the leading strand is replicated continuously whereas the lagging strand is replicated in segments known as Okazaki fragments, polypeptides have an N-terminus and a C-terminus, which refer to the ends of the polymer in a way that reflects the direction in which the polymer was synthesized. Many proteins may adopt a beta sheet as part of their secondary structure, in beta sheets, sections of a single polypeptide may run side-by-side and antiparallel to each other, to allow for hydrogen bonding between their backbone chains. Beta sheets can also be either a parallel or anti-parallel secondary structure, however, an anti-parallel beta sheet is significantly more stable than a parallel structure due to their well aligned H-bonds, which are at a nighty degree angle
13.
Primary transcript
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A primary transcript is the single-stranded ribonucleic acid product synthesized by transcription of DNA, and processed to yield various mature RNA products such as mRNAs, tRNAs, and rRNAs. The primary transcripts designated to be mRNAs are modified in preparation for translation, for example, a precursor messenger RNA is a type of primary transcript that becomes a messenger RNA after processing. There are several steps contributing to the production of primary transcripts, all these steps involve a series of interactions to initiate and complete the transcription of DNA in the nucleus of eukaryotes. Certain factors play key roles in the activation and inhibition of transcription, transcription produces primary transcripts that are further modified by several processes. These processes include the 5 cap, 3-polyadenylation, and alternative splicing, in particular, alternative splicing directly contributes to the diversity of mRNA found in cells. The modifications of primary transcripts have been studied in research seeking greater knowledge of the role. Experimental studies based on changes to primary transcripts the processes before. The steps contributing to the production of primary transcripts involve a series of interactions that initiate transcription of DNA within a cells nucleus. Based on the needs of a cell, certain DNA sequences are transcribed to produce a variety of RNA products to be translated into functional proteins for cellular use. One strand of the DNA template is used for transcription of the primary transcript mRNA. This DNA strand is bound by an RNA polymerase at the region of the DNA. Studies of primary transcripts produced by RNA polymerase II reveal that a primary transcript is 7,000 nucleotides in length, with some growing as long as 20,000 nucleotides in length. The inclusion of both exon and intron sequences within primary transcripts explains the difference between larger primary transcripts and smaller, mature mRNA ready for translation into protein. A number of factors contribute to the activation and inhibition of transcription, activation of RNA polymerase activity to produce primary transcripts is often controlled by sequences of DNA called enhancers. Inhibition of RNA polymerase activity can also be regulated by DNA sequences called silencers, like enhancers, silencers may be located at locations farther up or downstream from the genes they regulate. These DNA sequences bind to factors that contribute to the destabilization of the initiation complex required to activate RNA polymerase, histone modification by transcription factors is another key regulatory factor for transcription by RNA polymerase. In general, factors that lead to histone acetylation activate transcription factors that lead to histone deacetylation inhibit transcription. Acetylation of histones induces repulsion between negative components within nucleosomes, allowing for RNA polymerase access, deacetylation of histones stabilizes tightly coiled nucleosomes, inhibiting RNA polymerase access
14.
Transcription factor
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In molecular biology, a transcription factor is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. In turn, this helps to regulate the expression of genes near that sequence, transcription factors work alone or with other proteins in a complex, by promoting, or blocking the recruitment of RNA polymerase to specific genes. A defining feature of transcription factors is that they contain at least one DNA-binding domain, transcription factors are usually classified into different families based on their DBDs. Transcription factors are essential for the regulation of expression and are, as a consequence. The number of factors found within an organism increases with genome size. Therefore, approximately 10% of genes in the code for transcription factors. Hence, the use of a subset of the approximately 2000 human transcription factors easily accounts for the unique regulation of each gene in the human genome during development. Transcription factors bind to either enhancer or promoter regions of DNA adjacent to the genes that they regulate, depending on the transcription factor, the transcription of the adjacent gene is either up- or down-regulated. Transcription factors use a variety of mechanisms for the regulation of gene expression and these mechanisms include, stabilize or block the binding of RNA polymerase to DNA catalyze the acetylation or deacetylation of histone proteins. The transcription factor can either do this directly or recruit other proteins with this catalytic activity and they bind to the DNA and help initiate a program of increased or decreased gene transcription. As such, they are vital for important cellular processes. Many of these GTFs do not actually bind DNA, but rather are part of the large transcription preinitiation complex that interacts with RNA polymerase directly, the most common GTFs are TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. The preinitiation complex binds to regions of DNA upstream to the gene that they regulate. Other transcription factors regulate the expression of various genes by binding to enhancer regions of DNA adjacent to regulated genes. These transcription factors are critical to making sure that genes are expressed in the cell at the right time and in the right amount. Many transcription factors in multicellular organisms are involved in development, the Hox transcription factor family, for example, is important for proper body pattern formation in organisms as diverse as fruit flies to humans. Another example is the transcription factor encoded by the Sex-determining Region Y gene, cells can communicate with each other by releasing molecules that produce signaling cascades within another receptive cell. If the signal requires upregulation or downregulation of genes in the recipient cell, the estrogen receptor then goes to the cells nucleus and binds to its DNA-binding sites, changing the transcriptional regulation of the associated genes
15.
Promoter (genetics)
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In genetics, a promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites of genes, on the same strand, promoters can be about 100–1000 base pairs long. For transcription to take place, the enzyme that synthesizes RNA, known as RNA polymerase and these transcription factors have specific activator or repressor sequences of corresponding nucleotides that attach to specific promoters and regulate gene expression. In eukaryotes The process is complicated, and at least seven different factors are necessary for the binding of an RNA polymerase II to the promoter. Promoters represent critical elements that can work in concert with other regions to direct the level of transcription of a given gene. A promoter is induced in response to changes in abundance or conformation of proteins in a cell. In the cell nucleus, it seems that promoters are distributed preferentially at the edge of the chromosomal territories, furthermore, in humans, promoters show certain structural features characteristic for each chromosome. g. The sequence at -10 has the consensus sequence TATAAT, the sequence at -35 has the consensus sequence TTGACA. The above consensus sequences, while conserved on average, are not found intact in most promoters, on average, only 3 to 4 of the 6 base pairs in each consensus sequence are found in any given promoter. The optimal spacing between the -35 and -10 sequences is 17 bp, some promoters contain one or more upstream promoter element subsites. It should be noted that the above promoter sequences are recognized only by RNA polymerase holoenzyme containing sigma-70, RNA polymerase holoenzymes containing other sigma factors recognize different core promoter sequences. Gene promoters are typically located upstream of the gene and can have regulatory elements several kilobases away from the start site. In eukaryotes, the complex can cause the DNA to bend back on itself. The TATA element and BRE typically are located close to the start site. Eukaryotic promoter regulatory sequences typically bind proteins called transcription factors that are involved in the formation of the transcriptional complex, an example is the E-box, which binds transcription factors in the basic helix-loop-helix family. Some promoters that are targeted by multiple transcription factors might achieve a hyperactive state, bidirectional promoters are short intergenic regions of DNA between the 5 ends of the genes in a bidirectional gene pair. A “bidirectional gene pair” refers to two adjacent genes coded on opposite strands, with their 5 ends oriented toward one another, the two genes are often functionally related, and modification of their shared promoter region allows them to be co-regulated and thus co-expressed. Bidirectional promoters are a feature of mammalian genomes
16.
Hydrogen bond
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Hydrogen bonds can occur between molecules or within different parts of a single molecule. Depending on geometry and environment, the hydrogen bond free energy content is between 1 and 5 kcal/mol and this makes it stronger than a van der Waals interaction, but weaker than covalent or ionic bonds. This type of bond can occur in molecules such as water and in organic molecules like DNA. Intermolecular hydrogen bonding is responsible for the boiling point of water compared to the other group 16 hydrides that have much weaker hydrogen bonds. Intramolecular hydrogen bonding is responsible for the secondary and tertiary structures of proteins. It also plays an important role in the structure of polymers, in 2011, an IUPAC Task Group recommended a modern evidence-based definition of hydrogen bonding, which was published in the IUPAC journal Pure and Applied Chemistry. An accompanying detailed technical report provides the rationale behind the new definition, a hydrogen atom attached to a relatively electronegative atom will play the role of the hydrogen bond donor. This electronegative atom is usually fluorine, oxygen, or nitrogen, a hydrogen attached to carbon can also participate in hydrogen bonding when the carbon atom is bound to electronegative atoms, as is the case in chloroform, CHCl3. An example of a hydrogen donor is the hydrogen from the hydroxyl group of ethanol. In a hydrogen bond, the electronegative atom not covalently attached to the hydrogen is named proton acceptor, because of the small size of hydrogen relative to other atoms and molecules, the resulting charge, though only partial, represents a large charge density. A hydrogen bond results when this positive charge density attracts a lone pair of electrons on another heteroatom. The hydrogen bond is described as an electrostatic dipole-dipole interaction. These covalent features are more substantial when acceptors bind hydrogens from more electronegative donors, the partially covalent nature of a hydrogen bond raises the following questions, To which molecule or atom does the hydrogen nucleus belong. And Which should be labeled donor and which acceptor, liquids that display hydrogen bonding are called associated liquids. Hydrogen bonds can vary in strength from weak to extremely strong. For example, the central interresidue N−H···N hydrogen bond between guanine and cytosine is much stronger in comparison to the N−H···N bond between the adenine-thymine pair, the length of hydrogen bonds depends on bond strength, temperature, and pressure. The bond strength itself is dependent on temperature, pressure, bond angle, the typical length of a hydrogen bond in water is 197 pm. The ideal bond angle depends on the nature of the hydrogen bond donor, moore and Winmill used the hydrogen bond to account for the fact that trimethylammonium hydroxide is a weaker base than tetramethylammonium hydroxide
17.
Cell nucleus
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In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. Eukaryotes usually have a nucleus, but a few cell types, such as mammalian red blood cells, have no nuclei. Cell nuclei contain most of the genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones. The genes within these chromosomes are the nuclear genome and are structured in such a way to promote cell function. The nucleus maintains the integrity of genes and controls the activities of the cell by regulating gene expression—the nucleus is, therefore, because the nuclear membrane is impermeable to large molecules, nuclear pores are required to regulate nuclear transport of molecules across the envelope. The pores cross both nuclear membranes, providing a channel through which larger molecules must be transported by carrier proteins while allowing free movement of small molecules. Movement of large molecules such as proteins and RNA through the pores is required for gene expression and the maintenance of chromosomes. The best-known of these is the nucleolus, which is involved in the assembly of ribosomes. After being produced in the nucleolus, ribosomes are exported to the cytoplasm where they translate mRNA, the nucleus was the first organelle to be discovered. What is most likely the oldest preserved drawing dates back to the early microscopist Antonie van Leeuwenhoek and he observed a lumen, the nucleus, in the red blood cells of salmon. Unlike mammalian red blood cells, those of other vertebrates still contain nuclei, the nucleus was also described by Franz Bauer in 1804 and in more detail in 1831 by Scottish botanist Robert Brown in a talk at the Linnean Society of London. Brown was studying orchids under microscope when he observed an opaque area and he did not suggest a potential function. In 1838, Matthias Schleiden proposed that the plays a role in generating cells. He believed that he had observed new cells assembling around cytoblasts, Franz Meyen was a strong opponent of this view, having already described cells multiplying by division and believing that many cells would have no nuclei. The function of the nucleus remained unclear, between 1877 and 1878, Oscar Hertwig published several studies on the fertilization of sea urchin eggs, showing that the nucleus of the sperm enters the oocyte and fuses with its nucleus. This was the first time it was suggested that an individual develops from a nucleated cell, therefore, the necessity of the sperm nucleus for fertilization was discussed for quite some time. However, Hertwig confirmed his observation in other groups, including amphibians. Eduard Strasburger produced the results for plants in 1884
18.
Polyadenylation
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Polyadenylation is the addition of a poly tail to a messenger RNA. The poly tail consists of multiple adenosine monophosphates, in other words, in eukaryotes, polyadenylation is part of the process that produces mature messenger RNA for translation. It, therefore, forms part of the process of gene expression. The process of polyadenylation begins as the transcription of a gene terminates, the 3-most segment of the newly made pre-mRNA is first cleaved off by a set of proteins, these proteins then synthesize the poly tail at the RNAs 3 end. In some genes these proteins add a poly tail at one of possible sites. Therefore, polyadenylation can produce more than one transcript from a single gene, the poly tail is important for the nuclear export, translation, and stability of mRNA. The tail is shortened over time, and, when it is short enough, however, in a few cell types, mRNAs with short poly tails are stored for later activation by re-polyadenylation in the cytosol. In contrast, when polyadenylation occurs in bacteria, it promotes RNA degradation, for further information, see RNA and Messenger RNA RNAs are a type of large biological molecules, whose individual building blocks are called nucleotides. The name poly tail reflects the way RNA nucleotides are abbreviated, RNAs are produced from a DNA template. By convention, RNA sequences are written in a 5 to 3 direction, the 5 end is the part of the RNA molecule that is transcribed first, and the 3 end is transcribed last. The 3 end is also where the tail is found on polyadenylated RNAs. Messenger RNA is RNA that has a region that acts as a template for protein synthesis. The rest of the mRNA, the regions, tune how active the mRNA is. There are also many RNAs that are not translated, called non-coding RNAs, like the untranslated regions, many of these non-coding RNAs have regulatory roles. In nuclear polyadenylation, a tail is added to an RNA at the end of transcription. On mRNAs, the poly tail protects the mRNA molecule from enzymatic degradation in the cytoplasm and aids in transcription termination, export of the mRNA from the nucleus, almost all eukaryotic mRNAs are polyadenylated, with the exception of animal replication-dependent histone mRNAs. Many eukaryotic non-coding RNAs are always polyadenylated at the end of transcription, there are small RNAs where the poly tail is seen only in intermediary forms and not in the mature RNA as the ends are removed during processing, the notable ones being microRNAs. The processive polyadenylation complex in the nucleus of eukaryotes works on products of RNA polymerase II, here, a multi-protein complex cleaves the 3-most part of a newly produced RNA and polyadenylates the end produced by this cleavage
19.
Five-prime cap
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In molecular biology, the five-prime cap is a specially altered nucleotide on the 5′ end of some primary transcripts such as precursor messenger RNA. This process, known as mRNA capping, is regulated and vital in the creation of stable. Mitochondrial and chloroplast mRNA are not capped, in eukaryotes, the 5′ cap, found on the 5′ end of an mRNA molecule, consists of a guanine nucleotide connected to mRNA via an unusual 5′ to 5′ triphosphate linkage. This guanosine is methylated on the 7 position directly after capping in vivo by a methyltransferase and it is referred to as a 7-methylguanylate cap, abbreviated m7G. In multicellular eukaryotes and some viruses, further modifications exist, including the methylation of the 2′ hydroxy-groups of the first 2 ribose sugars of the 5′ end of the mRNA. Cap-1 has a methylated 2-hydroxy group on the first ribose sugar, the 5′ cap is chemically similar to the 3′ end of an RNA molecule. This provides significant resistance to 5′ exonucleases, small nuclear RNAs contain unique 5-caps. Sm-class snRNAs are found with 5-trimethylguanosine caps, while Lsm-class snRNAs are found with 5-monomethylphosphate caps, in bacteria, and potentially also in higher organisms, some RNAs are capped with NAD+, NADH, or 3-dephospho-coenzyme A. In all organisms, mRNA molecules can be decapped in a known as messenger RNA decapping. The starting point for capping with 7-methylguanylate is the unaltered 5′ end of an RNA molecule and this features a final nucleotide followed by three phosphate groups attached to the 5′ carbon. The capping process is initiated before the completion of transcription, as the nascent pre-mRNA is being synthesized, the mechanism of capping with NAD+, NADH, or 3-dephospho-coenzyme A is different. Both bacterial RNA polymerase and eukaryotic RNA polymerase II are able to carry out this ab initio capping mechanism, for capping with 7-methylguanylate, the capping enzyme complex binds to RNA polymerase II before transcription starts. As soon as the 5′ end of the new transcript emerges from RNA polymerase II, the enzymes for capping can only bind to RNA polymerase II, ensuring specificity to only these transcripts, which are almost entirely mRNA. Capping with NAD+, NADH, or 3-dephospho-coenzyme A is targeted by promoter sequence, the 5′ cap has four main functions, Regulation of nuclear export, Prevention of degradation by exonucleases, Promotion of translation, Promotion of 5′ proximal intron excision. Nuclear export of RNA is regulated by the cap binding complex, the CBC is then recognized by the nuclear pore complex and exported. Once in the cytoplasm after the round of translation, the CBC is replaced by the translation factors eIF4E. This complex is recognized by other translation initiation machinery including the ribosome. Capping with 7-methylguanylate prevents 5′ degradation in two ways, first, degradation of the mRNA by 5′ exonucleases is prevented by functionally looking like a 3′ end
20.
RNA splicing
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In molecular biology, splicing is the editing of the nascent precursor messenger RNA transcript. After splicing, introns are removed and exons are joined together, for nuclear-encoded genes, splicing takes place within the nucleus either co-transcriptionally or immediately after transcription. For those eukaryotic genes contain introns, splicing is usually required in order to create an mRNA molecule that can be translated into protein. For many eukaryotic introns, splicing is carried out in a series of reactions which are catalyzed by the spliceosome, self-splicing introns, or ribozymes capable of catalyzing their own excision from their parent RNA molecule, also exist. Several methods of RNA splicing occur in nature, the type of splicing depends on the structure of the spliced intron, the word intron is derived from the term intervening sequence, that is, a segment of DNA that intervenes between two exons of a gene. The term intron refers to both the DNA sequence within a gene and the sequence in the unprocessed RNA transcript. As part of the RNA processing pathway, introns are removed by RNA splicing either shortly after or concurrent with transcription, introns are found in the genes of most organisms and many viruses. They can be located in a range of genes, including those that generate proteins, ribosomal RNA. Spliceosomal introns often reside within the sequence of eukaryotic protein-coding genes, within the intron, a donor site, a branch site and an acceptor site are required for splicing. The splice donor site includes an almost invariant sequence GU at the 5 end of the intron, within a larger, the splice acceptor site at the 3 end of the intron terminates the intron with an almost invariant AG sequence. Upstream from the AG there is a high in pyrimidines. Further upstream from the tract is the branchpoint, which includes an adenine nucleotide involved in lariat formation. The consensus sequence for an intron is, G-G--G-U-R-A-G-U, also, point mutations in the underlying DNA or errors during transcription can activate a cryptic splice site in part of the transcript that usually is not spliced. This results in a mature messenger RNA with a section of an exon. In this way, a point mutation, which might otherwise affect only a single amino acid, splicing is catalyzed by the spliceosome, a large RNA-protein complex composed of five small nuclear ribonucleoproteins. Assembly and activity of the spliceosome occurs during transcription of the pre-mRNA, the RNA components of snRNPs interact with the intron and are involved in catalysis. Two types of spliceosomes have been identified which contain different snRNPs, the major spliceosome splices introns containing GU at the 5 splice site and AG at the 3 splice site. It is composed of the U1, U2, U4, U5, in addition, a number of proteins including U2 small nuclear RNA auxiliary factor 1, U2AF2 and SF1 are required for the assembly of the spliceosome
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Cytoplasm
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In cell biology, the cytoplasm is the material within a living cell, excluding the cell nucleus. It comprises cytosol and the organelles – the cells internal sub-structures, all of the contents of the cells of prokaryotic organisms are contained within the cytoplasm. Within the cells of organisms the contents of the cell nucleus are separated from the cytoplasm. The cytoplasm is about 80% water and usually colorless, the submicroscopic ground cell substance or cytoplasmatic matrix which remains after exclusion the cell organelles and particles is groundplasm. It is within the cytoplasm that most cellular activities occur, such as many metabolic pathways including glycolysis, the concentrated inner area is called the endoplasm and the outer layer is called the cell cortex or the ectoplasm. Movement of calcium ions in and out of the cytoplasm is an activity for metabolic processes. In plants, movement of the cytoplasm around vacuoles is known as cytoplasmic streaming, the term was introduced by Rudolf von Kölliker in 1863, originally as a synonym for protoplasm, but later it has come to mean the cell substance and organelles outside the nucleus. There has been disagreement on the definition of cytoplasm, as some authors prefer to exclude from it some organelles. The physical properties of the cytoplasm have been contested in recent years and it remains uncertain how the varied components of the cytoplasm interact to allow movement of particles and organelles while maintaining the cell’s structure. The flow of cytoplasmic components plays an important role in cellular functions which are dependent on the permeability of the cytoplasm. An example of function is cell signalling, a process which is dependent on the manner in which signaling molecules are allowed to diffuse across the cell. While small signaling molecules like calcium ions are able to diffuse with ease, larger molecules, the irregular dynamics of such particles have given rise to various theories on the nature of the cytoplasm. There has long been evidence that the cytoplasm behaves like a sol-gel and it is thought that the component molecules and structures of the cytoplasm behave at times like a disordered colloidal solution and at other times like an integrated network, forming a solid mass. Recently it has proposed that the cytoplasm behaves like a glass-forming liquid approaching the glass transition. A cells ability to vitrify in the absence of activity, as in dormant periods. There has been examining the motion of cytoplasmic particles independent of the nature of the cytoplasm. In such an approach, the aggregate random forces within the cell caused by motor proteins explain the non-Brownian motion of cytoplasmic constituents. The three major elements of the cytoplasm are the cytosol, organelles and inclusions, the cytosol is the portion of the cytoplasm not contained within membrane-bound organelles
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Nuclear pore
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Nuclear pores are large protein complexes that cross the nuclear envelope, which is the double membrane surrounding the eukaryotic cell nucleus. There are about an average of 2000 nuclear pore complexes, in the envelope of a vertebrate cell, but it varies depending on cell type. The proteins that make up the pore complex are known as nucleoporins. About half of the nucleoporins typically contain solenoid protein domains—either an alpha solenoid or a beta-propeller fold, each NPC contains at least 456 individual protein molecules and is composed of 30 distinct proteins. The other half show structural characteristics typical of natively unfolded or intrinsically disordered proteins and these disordered proteins are the FG nucleoporins, so called because their amino-acid sequence contains many phenylalanine—glycine repeats. Nuclear pore complexes allow the transport of molecules across the nuclear envelope and this transport includes RNA and ribosomal proteins moving from nucleus to the cytoplasm and proteins, carbohydrates, signaling molecules and lipids moving into the nucleus. It is notable that the pore complex can actively conduct 1000 translocations per complex per second. Nucleoporin-mediated transport is not directly energy requiring, but depends on concentrations gradients associated with the RAN cycle, each of the eight protein subunits surrounding the actual pore projects a spoke-shaped protein over the pore channel. The center of the pore often appears to contain a plug-like structure and it is yet unknown whether this corresponds to an actual plug or is merely cargo caught in transit. The entire nuclear pore complex has a diameter of about 120 nanometers in vertebrates, the molecular mass of the mammalian NPC is about 124 megadaltons and it contains approximately 30 different protein components, each in multiple copies. In contrast, the yeast Saccharomyces cerevisiae is smaller, weighing only 66 MDa, small particles are able to pass through the nuclear pore complex by passive diffusion. Larger particles are able to diffuse passively through the large diameter of the pore. Other nuclar transport receptors include NTF2 and some NTF2-like proteins, several NLS sequences are known, generally containing a conserved sequence with basic residues such as PKKKRKV. Any material with an NLS will be taken up by importins to the nucleus, the classical scheme of NLS-protein importation begins with Importin-α first binding to the NLS sequence, which then acts as a bridge for Importin-β to attach. The importinβ—importinα—cargo complex is directed towards the nuclear pore and diffuses through it. Once the complex is in the nucleus, RanGTP binds to Importin-β, then the cellular apoptosis susceptibility protein, an exportin which in the nucleus is bound to RanGTP, displaces Importin-α from the cargo. The NLS-protein is thus free in the nucleoplasm, although cargo passes through the pore with the assistance of chaperone proteins, the translocation through the pore itself is not energy-dependent. However, the import cycle needs the hydrolysis of 2 GTPs and is thus energy-dependent and has to be considered as active transport
23.
Gene
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A gene is a locus of DNA which is made up of nucleotides and is the molecular unit of heredity. The transmission of genes to an offspring is the basis of the inheritance of phenotypic traits. These genes make up different DNA sequences called genotypes, genotypes along with environmental and developmental factors determine what the phenotypes will be. Most biological traits are under the influence of polygenes as well as gene–environment interactions, genes can acquire mutations in their sequence, leading to different variants, known as alleles, in the population. These alleles encode slightly different versions of a protein, which cause different phenotypical traits, usage of the term having a gene typically refers to containing a different allele of the same, shared gene. Genes evolve due to natural selection or survival of the fittest of the alleles, the concept of a gene continues to be refined as new phenomena are discovered. For example, regulatory regions of a gene can be far removed from its coding regions, some viruses store their genome in RNA instead of DNA and some gene products are functional non-coding RNAs. The existence of discrete inheritable units was first suggested by Gregor Mendel, from 1857 to 1864, in Brno, he studied inheritance patterns in 8000 common edible pea plants, tracking distinct traits from parent to offspring. He described these mathematically as 2n combinations where n is the number of differing characteristics in the original peas, although he did not use the term gene, he explained his results in terms of discrete inherited units that give rise to observable physical characteristics. This description prefigured the distinction between genotype and phenotype, charles Darwin developed a theory of inheritance he termed pangenesis, from Greek pan and genesis / genos. Darwin used the term gemmule to describe hypothetical particles that would mix during reproduction, de Vries called these units pangenes, after Darwins 1868 pangenesis theory. In 1909 the Danish botanist Wilhelm Johannsen shortened the name to gene, advances in understanding genes and inheritance continued throughout the 20th century. Deoxyribonucleic acid was shown to be the repository of genetic information by experiments in the 1940s to 1950s. In the early 1950s the prevailing view was that the genes in a chromosome acted like discrete entities, indivisible by recombination, collectively, this body of research established the central dogma of molecular biology, which states that proteins are translated from RNA, which is transcribed from DNA. This dogma has since shown to have exceptions, such as reverse transcription in retroviruses. The modern study of genetics at the level of DNA is known as molecular genetics, in 1972, Walter Fiers and his team at the University of Ghent were the first to determine the sequence of a gene, the gene for Bacteriophage MS2 coat protein. The subsequent development of chain-termination DNA sequencing in 1977 by Frederick Sanger improved the efficiency of sequencing, an automated version of the Sanger method was used in early phases of the Human Genome Project. The theories developed in the 1930s and 1940s to integrate molecular genetics with Darwinian evolution are called the evolutionary synthesis
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Protein
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Proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, a linear chain of amino acid residues is called a polypeptide. A protein contains at least one long polypeptide, short polypeptides, containing less than 20–30 residues, are rarely considered to be proteins and are commonly called peptides, or sometimes oligopeptides. The individual amino acid residues are bonded together by peptide bonds, the sequence of amino acid residues in a protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the code specifies 20 standard amino acids, however. Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors, proteins can also work together to achieve a particular function, and they often associate to form stable protein complexes. Once formed, proteins only exist for a period of time and are then degraded and recycled by the cells machinery through the process of protein turnover. A proteins lifespan is measured in terms of its half-life and covers a wide range and they can exist for minutes or years with an average lifespan of 1–2 days in mammalian cells. Abnormal and or misfolded proteins are degraded more rapidly due to being targeted for destruction or due to being unstable. Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of organisms, many proteins are enzymes that catalyse biochemical reactions and are vital to metabolism. Proteins also have structural or mechanical functions, such as actin and myosin in muscle and the proteins in the cytoskeleton, other proteins are important in cell signaling, immune responses, cell adhesion, and the cell cycle. In animals, proteins are needed in the diet to provide the essential amino acids that cannot be synthesized, digestion breaks the proteins down for use in the metabolism. Methods commonly used to study structure and function include immunohistochemistry, site-directed mutagenesis, X-ray crystallography, nuclear magnetic resonance. Most proteins consist of linear polymers built from series of up to 20 different L-α-amino acids, all proteinogenic amino acids possess common structural features, including an α-carbon to which an amino group, a carboxyl group, and a variable side chain are bonded. Only proline differs from this structure as it contains an unusual ring to the N-end amine group. The amino acids in a chain are linked by peptide bonds. Once linked in the chain, an individual amino acid is called a residue, and the linked series of carbon, nitrogen. The peptide bond has two forms that contribute some double-bond character and inhibit rotation around its axis, so that the alpha carbons are roughly coplanar
25.
Translation (biology)
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In molecular biology and genetics, translation is the process in which ribosomes in a cells cytoplasm create proteins, following transcription of DNA to RNA in the cells nucleus. The entire process is a part of gene expression, in translation, messenger RNA is decoded by a ribosome, outside the nucleus, to produce a specific amino acid chain, or polypeptide. The polypeptide later folds into a protein and performs its functions in the cell. The ribosome facilitates decoding by inducing the binding of complementary tRNA anticodon sequences to mRNA codons, the tRNAs carry specific amino acids that are chained together into a polypeptide as the mRNA passes through and is read by the ribosome. Translation proceeds in three phases, Initiation, The ribosome assembles around the target mRNA, the first tRNA is attached at the start codon. Elongation, The tRNA transfers an amino acid to the corresponding to the next codon. The ribosome then moves to the next mRNA codon to continue the process, termination, When a stop codon is reached, the ribosome releases the polypeptide. In bacteria, translation occurs in the cytoplasm, where the large, in eukaryotes, translation occurs in the cytosol or across the membrane of the endoplasmic reticulum in a process called vectorial synthesis. Many types of transcribed RNA, such as transfer RNA, ribosomal RNA, a number of antibiotics act by inhibiting translation. These include anisomycin, cycloheximide, chloramphenicol, tetracycline, streptomycin, erythromycin, prokaryotic ribosomes have a different structure from that of eukaryotic ribosomes, and thus antibiotics can specifically target bacterial infections without any harm to a eukaryotic hosts cells. The basic process of production is addition of one amino acid at a time to the end of a protein. This operation is performed by a ribosome, a ribosome is made up of two subunits, a small subunit and a large subunit. These subunits come together before translation of mRNA into a protein to provide a location for translation to be carried out, the choice of amino acid type to add is determined by an mRNA molecule. Each amino acid added is matched to a three nucleotide subsequence of the mRNA, for each such triplet possible, the corresponding amino acid is accepted. The successive amino acids added to the chain are matched to successive nucleotide triplets in the mRNA, in this way the sequence of nucleotides in the template mRNA chain determines the sequence of amino acids in the generated amino acid chain. Addition of an amino acid occurs at the C-terminus of the peptide, the mRNA carries genetic information encoded as a ribonucleotide sequence from the chromosomes to the ribosomes. The ribonucleotides are read by translational machinery in a sequence of nucleotide triplets called codons, each of those triplets codes for a specific amino acid. The ribosome molecules translate this code to a sequence of amino acids
26.
Non-coding RNA
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A non-coding RNA is an RNA molecule that is not translated into a protein. Less-frequently used synonyms are non-protein-coding RNA, non-messenger RNA, or functional RNA, the DNA sequence from which a functional non-coding RNA is transcribed is often called an RNA gene. The number of ncRNAs encoded within the genome is unknown, however. But see Many of the newly identified ncRNAs have not been validated for their function and it is also likely that many ncRNAs are non functional, and are the product of spurious transcription. Nucleic acids were first discovered in 1868 by Friedrich Miescher and by 1939 RNA had been implicated in protein synthesis. Two decades later, Francis Crick predicted a functional RNA component which mediated translation, the first non-coding RNA to be characterised was an alanine tRNA found in bakers yeast, its structure was published in 1965. To produce a purified alanine tRNA sample, Robert W. Holley et al. used 140kg of commercial bakers yeast to give just 1g of purified tRNAAla for analysis, the 80 nucleotide tRNA was sequenced by first being digested with Pancreatic ribonuclease and then with takadiastase ribonuclease Tl. Chromatography and identification of the 5 and 3 ends then helped arrange the fragments to establish the RNA sequence, of the three structures originally proposed for this tRNA, the cloverleaf structure was independently proposed in several following publications. The cloverleaf secondary structure was finalised following X-ray crystallography analysis performed by two independent research groups in 1974, ribosomal RNA was next to be discovered, followed by URNA in the early 1980s. Since then, the discovery of new non-coding RNAs has continued with snoRNAs, Xist, CRISPR, recent notable additions include riboswitches and miRNA, the discovery of the RNAi mechanism associated with the latter earned Craig C. Mello and Andrew Fire the 2006 Nobel Prize in Physiology or Medicine, noncoding RNAs belong to several groups and are involved in many cellular processes. These range from ncRNAs of central importance that are conserved across all or most cellular life through to more transient ncRNAs specific to one or a few related species. Many of the conserved, essential and abundant ncRNAs are involved in translation, ribonucleoprotein particles called ribosomes are the factories where translation takes place in the cell. The ribosome consists of more than 60% ribosomal RNA, these are made up of 3 ncRNAs in prokaryotes and 4 ncRNAs in eukaryotes, ribosomal RNAs catalyse the translation of nucleotide sequences to protein. Another set of ncRNAs, Transfer RNAs, form an adaptor molecule between mRNA and protein, the H/ACA box and C/D box snoRNAs are ncRNAs found in archaea and eukaryotes. RNase MRP is restricted to eukaryotes, both groups of ncRNA are involved in the maturation of rRNA. The snoRNAs guide covalent modifications of rRNA, tRNA and snRNAs, the ubiquitous ncRNA, RNase P, is an evolutionary relative of RNase MRP. RNase P matures tRNA sequences by generating mature 5-ends of tRNAs through cleaving the 5-leader elements of precursor-tRNAs, another ubiquitous RNP called SRP recognizes and transports specific nascent proteins to the endoplasmic reticulum in eukaryotes and the plasma membrane in prokaryotes
27.
MicroRNA
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A microRNA is a small non-coding RNA molecule found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression. The human genome may encode over 1000 miRNAs, which are abundant in mammalian cell types and appear to target about 60% of the genes of humans. MiRNAs are well conserved in plants and animals, and are thought to be a vital and evolutionarily ancient component of gene regulation. Plant miRNAs usually have near-perfect pairing with their targets, which induces gene repression through cleavage of the target transcripts. Combinatorial regulation is a feature of miRNA regulation in animals, a given miRNA may have hundreds of different mRNA targets, and a given target might be regulated by multiple miRNAs. The first miRNA was discovered in the early 1990s, however, miRNAs were not recognized as a distinct class of biological regulators until the early 2000s. MiRNA research revealed different sets of miRNAs expressed in different cell types and tissues and multiple roles for miRNAs in plant and animal development, aberrant miRNA expression are implicated in disease states. For example, an analysis of the highly conserved in vertebrates shows that each has, on average. Likewise, experiments show that a single species can reduce the stability of hundreds of unique messenger RNAs. Other experiments show that a single species may repress the production of hundreds of proteins. The first human disease associated with deregulation of miRNAs was chronic lymphocytic leukemia and these groups published back-to-back papers on the lin-4 gene, which was known to control the timing of C. elegans larval development by repressing the lin-14 gene. This complementarity was proposed to inhibit the translation of the lin-14 mRNA into the LIN-14 protein, at the time, the lin-4 small RNA was thought to be a nematode idiosyncrasy. In 2000 a second small RNA was characterized, let-7 RNA, a year later, the lin-4 and let-7 RNAs were found to be part of a large class of small RNAs present in C. elegans, Drosophila and human cells. The many RNAs of this class resembled the lin-4 and let-7 RNAs and this suggested that most might function in other types of regulatory pathways. At this point, researchers started using the term “microRNA” to refer to this class of small regulatory RNAs, the first human disease associated with deregulaton of miRNAs was chronic lymphocytic leukemia. Under a standard system, names are assigned to experimentally confirmed miRNAs before publication. The prefix miR is followed by a dash and a number, for example, miR-124 was named and likely discovered prior to miR-456. A capitalized miR- refers to the form of the miRNA, while the uncapitalized mir- refers to the pre-miRNA and the pri-miRNA
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Ribosomal RNA
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Ribosomal ribonucleic acid is the RNA component of the ribosome, and is essential for protein synthesis in all living organisms. It constitutes the predominant material within the ribosome, which is approximately 60% rRNA and 40% protein by weight, ribosomes contain two major rRNAs and 50 or more proteins. The ribosomal RNAs form two subunits, the large subunit and small subunit, the LSU rRNA acts as a ribozyme, catalyzing peptide bond formation. RRNA sequences are used for working out evolutionary relationships among organisms. The ribosomal RNAs complex with proteins to form two subunits, the large subunit and small subunit. During translation, mRNA is sandwiched between the small and large subunits, and the ribosome catalyzes the formation of a bond between the two amino acids that are contained in the rRNA. A ribosome also has three binding sites called A, P, and E, the A site in the ribosome binds to an aminoacyl-tRNA. This reaction is catalyzed by peptidyl transferase, the tRNA that was holding onto the last amino acid is moved to the E site, and what used to be the aminoacyl-tRNA is the peptidyl-tRNA. A single mRNA can be translated simultaneously by multiple ribosomes, the sedimentation rate of each subunit is affected by its shape, as well as by its mass. Thent units can be added as these represent the number of units in the linear rRNA polymers. In prokaryotes a small 30S ribosomal subunit contains the 16S ribosomal RNA, the large 50S ribosomal subunit contains two rRNA species. Bacterial 16S ribosomal RNA, 23S ribosomal RNA, and 5S rRNA genes are organized as a co-transcribed operon. There may be one or more copies of the operon dispersed in the genome, archaea contains either a single rDNA operon or multiple copies of the operon. The 3 end of the 16S ribosomal RNA binds to a sequence on the 5 end of mRNA called the Shine-Dalgarno sequence, in contrast, eukaryotes generally have many copies of the rRNA genes organized in tandem repeats, in humans approximately 300–400 repeats are present in five clusters. Because of their structure and transcription behaviour, rRNA gene clusters are commonly called ribosomal DNA. The 18S rRNA in most eukaryotes is in the small subunit. Mammalian cells have 2 mitochondrial rRNA molecules and 4 types of cytoplasmic rRNA, the 28S,5. 8S, and 18S rRNAs are encoded by a single transcription unit separated by 2 internally transcribed spacers. The 45S rDNA is organized into 5 clusters on chromosomes 13,14,15,21 and these are transcribed by RNA polymerase I
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Transfer RNA
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A transfer RNA is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length, that serves as the physical link between the mRNA and the amino acid sequence of proteins. It does this by carrying an amino acid to the protein synthetic machinery of a cell as directed by a sequence in a messenger RNA. As such, tRNAs are a component of translation, the biological synthesis of new proteins in accordance with the genetic code. The mRNA encodes a protein as a series of contiguous codons, one end of the tRNA matches the genetic code in a three-nucleotide sequence called the anticodon. The anticodon forms three base pairs with a codon in mRNA during protein biosynthesis, on the other end of the tRNA is a covalent attachment to the amino acid that corresponds to the anticodon sequence. Each type of molecule can be attached to only one type of amino acid. Because the genetic code contains multiple codons that specify the amino acid. The covalent attachment to the tRNA 3’ end is catalyzed by enzymes called aminoacyl tRNA synthetases, a large number of the individual nucleotides in a tRNA molecule may be chemically modified, often by methylation or deamidation. These unusual bases sometimes affect the interaction with ribosomes and sometimes occur in the anticodon to alter base-pairing properties. The structure of tRNA can be decomposed into its structure, its secondary structure. The cloverleaf structure becomes the 3D L-shaped structure through coaxial stacking of the helices, the lengths of each arm, as well as the loop diameter, in a tRNA molecule vary from species to species. The tRNA structure consists of the following, A 5-terminal phosphate group, the acceptor stem is a 7- to 9-base pair stem made by the base pairing of the 5-terminal nucleotide with the 3-terminal nucleotide. The acceptor stem may contain non-Watson-Crick base pairs, the CCA tail is a cytosine-cytosine-adenine sequence at the 3 end of the tRNA molecule. The amino acid loaded onto the tRNA by aminoacyl tRNA synthetases and this sequence is important for the recognition of tRNA by enzymes and critical in translation. In prokaryotes, the CCA sequence is transcribed in some tRNA sequences, in most prokaryotic tRNAs and eukaryotic tRNAs, the CCA sequence is added during processing and therefore does not appear in the tRNA gene. The D arm is a 4- to 6-bp stem ending in a loop that often contains dihydrouridine, the anticodon arm is a 5-bp stem whose loop contains the anticodon. The tRNA 5-to-3 primary structure contains the anticodon but in reverse order, the T arm is a 4- to 5- bp stem containing the sequence TΨC where Ψ is pseudouridine, a modified uridine. Bases that have modified, especially by methylation, occur in several positions throughout the tRNA
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Ribozyme
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Ribozymes are RNA molecules that are capable of catalyzing specific biochemical reactions, similar to the action of protein enzymes. The most common activities of natural or in vitro-evolved ribozymes are the cleavage or ligation of RNA and DNA, within the ribosome, ribozymes function as part of the large subunit ribosomal RNA to link amino acids during protein synthesis. They also participate in a variety of RNA processing reactions, including RNA splicing, viral replication, examples of ribozymes include the hammerhead ribozyme, the VS ribozyme, Leadzyme and the hairpin ribozyme. Mutagenesis and selection has been performed resulting in isolation of improved variants of the Round-18 polymerase ribozyme from 2001, b6.61 is able to add up to 20 nucleotides to a primer template in 24 hours, until it decomposes by cleavage of its phosphodiester bonds. The tC19Z ribozyme can add up to 95 nucleotides with a fidelity of 0.0083 mutations/nucleotide, before the discovery of ribozymes, enzymes, which are defined as catalytic proteins, were the only known biological catalysts. In 1967, Carl Woese, Francis Crick, and Leslie Orgel were the first to suggest that RNA could act as a catalyst and this idea was based upon the discovery that RNA can form complex secondary structures. In 1989, Thomas R. Cech and Sidney Altman shared the Nobel Prize in chemistry for their discovery of properties of RNA. The term ribozyme was first introduced by Kelly Kruger et al. in 1982 in a paper published in Cell and it had been a firmly established belief in biology that catalysis was reserved for proteins. The concept of acids as catalysts circumvents this problem. RNA, in essence, can be both the chicken and the egg, in the 1980s Thomas Cech, at the University of Colorado at Boulder, was studying the excision of introns in a ribosomal RNA gene in Tetrahymena thermophila. While trying to purify the enzyme responsible for the splicing reaction, as much as they tried, Cech and his colleagues could not identify any protein associated with the splicing reaction. After much work, Cech proposed that the intron sequence portion of the RNA could break, much to their surprise, they found that RNase-P contained RNA in addition to protein and that RNA was an essential component of the active enzyme. This was such an idea that they had difficulty publishing their findings. Since Cechs and Altmans discovery, other investigators have discovered other examples of self-cleaving RNA or catalytic RNA molecules, many ribozymes have either a hairpin – or hammerhead – shaped active center and a unique secondary structure that allows them to cleave other RNA molecules at specific sequences. It is now possible to make ribozymes that will specifically cleave any RNA molecule and these RNA catalysts may have pharmaceutical applications. For example, a ribozyme has been designed to cleave the RNA of HIV, if such a ribozyme were made by a cell, all incoming virus particles would have their RNA genome cleaved by the ribozyme, which would prevent infection. Despite having only four choices for each unit, compared to 20 amino acid side chains found in proteins, ribozymes have diverse structures. In many cases they are able to mimic the mechanism used by their protein counterparts, in comparison, RNase A, a protein that catalyzes the same reaction, uses a coordinating histidine and lysine to act as a base to attack the phosphate backbone
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Cell (biology)
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The cell is the basic structural, functional, and biological unit of all known living organisms. A cell is the smallest unit of life that can replicate independently, the study of cells is called cell biology. Cells consist of cytoplasm enclosed within a membrane, which contains many such as proteins. Organisms can be classified as unicellular or multicellular, while the number of cells in plants and animals varies from species to species, humans contain more than 10 trillion cells. Most plant and animal cells are only under a microscope. The cell was discovered by Robert Hooke in 1665, who named the unit for its resemblance to cells inhabited by Christian monks in a monastery. Cells emerged on Earth at least 3.5 billion years ago, Cells are of two types, eukaryotic, which contain a nucleus, and prokaryotic, which do not. Prokaryotes are single-celled organisms, while eukaryotes can be either single-celled or multicellular, prokaryotic cells were the first form of life on Earth, characterised by having vital biological processes including cell signaling and being self-sustaining. They are simpler and smaller than eukaryotic cells, and lack membrane-bound organelles such as the nucleus, prokaryotes include two of the domains of life, bacteria and archaea. The DNA of a prokaryotic cell consists of a chromosome that is in direct contact with the cytoplasm. The nuclear region in the cytoplasm is called the nucleoid, most prokaryotes are the smallest of all organisms ranging from 0.5 to 2.0 µm in diameter. Though most prokaryotes have both a cell membrane and a wall, there are exceptions such as Mycoplasma and Thermoplasma which only possess the cell membrane layer. The envelope gives rigidity to the cell and separates the interior of the cell from its environment, the cell wall consists of peptidoglycan in bacteria, and acts as an additional barrier against exterior forces. It also prevents the cell from expanding and bursting from osmotic pressure due to a hypotonic environment, some eukaryotic cells also have a cell wall. Inside the cell is the region that contains the genome, ribosomes. The genetic material is found in the cytoplasm. Prokaryotes can carry extrachromosomal DNA elements called plasmids, which are usually circular, linear bacterial plasmids have been identified in several species of spirochete bacteria, including members of the genus Borrelia notably Borrelia burgdorferi, which causes Lyme disease. Though not forming a nucleus, the DNA is condensed in a nucleoid, plasmids encode additional genes, such as antibiotic resistance genes
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Virology
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Virology is the study of viruses – submicroscopic, parasitic particles of genetic material contained in a protein coat – and virus-like agents. Virology is considered to be a subfield of microbiology or of medicine, a major branch of virology is virus classification. Viruses can be classified according to the host cell they infect, animal viruses, plant viruses, fungal viruses, another classification uses the geometrical shape of their capsid or the viruss structure. Viruses range in size from about 30 nm to about 450 nm, the shape and structure of viruses has been studied by electron microscopy, NMR spectroscopy, and X-ray crystallography. The latest report by the International Committee on Taxonomy of Viruses lists 5450 viruses, virologists also study subviral particles, infectious entities notably smaller and simpler than viruses, viroids, satellites, and prions. Taxa in virology are not necessarily monophyletic, as the relationships of the various virus groups remain unclear. Viruses arose from mobile genetic elements of cells that became encapsulated in protein capsids, acquired the ability to free from the host cell. Of particular interest here is mimivirus, a giant virus that infects amoebae, is it a simplified version of a parasitic prokaryote, or did it originate as a simpler virus that acquired genes from its host. The evolution of viruses, which occurs in concert with the evolution of their hosts, is studied in the field of viral evolution. While viruses reproduce and evolve, they do not engage in metabolism, do not move, the often-debated question of whether they are alive or not is a matter of definition that does not affect the biological reality of viruses. Herpes simplex causes cold sores and genital herpes and is under investigation as a factor in Alzheimers. Some viruses, known as oncoviruses, contribute to the development of forms of cancer. The best studied example is the association between Human papillomavirus and cervical cancer, almost all cases of cancer are caused by certain strains of this sexually transmitted virus. Another example is the association of infection with hepatitis B and hepatitis C viruses, the study of the manner in which viruses cause disease is viral pathogenesis. The degree to which a virus disease is its virulence. When the immune system of a vertebrate encounters a virus, it may produce specific antibodies which bind to the virus, antibody presence in blood serum is often used to determine whether a person has been exposed to a given virus in the past, with tests such as ELISA. Vaccinations protect against viral diseases, in part, by eliciting the production of antibodies, monoclonal antibodies, specific to the virus, are also used for detection, as in fluorescence microscopy. This mechanism is jump-started by certain vaccinations, RNA interference, an important cellular mechanism found in plants, animals and many other eukaryotes, most likely evolved as a defense against viruses
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Transcription (biology)
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Transcription is the first step of gene expression, in which a particular segment of DNA is copied into RNA by the enzyme RNA polymerase. Both DNA and RNA are nucleic acids, which use base pairs of nucleotides as a complementary language, during transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA strand called a primary transcript. Transcription proceeds in the general steps, RNA polymerase, together with one or more general transcription factors. RNA polymerase creates a bubble, which separates the two strands of the DNA helix. This is done by breaking the bonds between complementary DNA nucleotides. RNA sugar-phosphate backbone forms with assistance from RNA polymerase to form an RNA strand, hydrogen bonds of the RNA–DNA helix break, freeing the newly synthesized RNA strand. If the cell has a nucleus, the RNA may be further processed and this may include polyadenylation, capping, and splicing. The RNA may remain in the nucleus or exit to the cytoplasm through the pore complex. The stretch of DNA transcribed into an RNA molecule is called a transcription unit, if the gene encodes a protein, the transcription produces messenger RNA, the mRNA, in turn, serves as a template for the proteins synthesis through translation. Alternatively, the gene may encode for either non-coding RNA, ribosomal RNA, transfer RNA. Overall, RNA helps synthesize, regulate, and process proteins, in virology, the term may also be used when referring to mRNA synthesis from an RNA molecule. For instance, the genome of a negative-sense single-stranded RNA virus may be template for a positive-sense single-stranded RNA and this is because the positive-sense strand contains the information needed to translate the viral proteins for viral replication afterwards. This process is catalyzed by a viral RNA replicase, the regulatory sequence before the coding sequence is called the five prime untranslated region, the sequence after the coding sequence is called the three prime untranslated region. As opposed to DNA replication, transcription results in an RNA complement that includes the nucleotide uracil in all instances where thymine would have occurred in a DNA complement, only one of the two DNA strands serve as a template for transcription. The antisense strand of DNA is read by RNA polymerase from the 3 end to the 5 end during transcription. The complementary RNA is created in the direction, in the 5 →3 direction. This directionality is because RNA polymerase can only add nucleotides to the 3 end of the growing mRNA chain and this use of only the 3 →5 DNA strand eliminates the need for the Okazaki fragments that are seen in DNA replication. This also removes the need for an RNA primer to initiate RNA synthesis, the non-template strand of DNA is called the coding strand, because its sequence is the same as the newly created RNA transcript
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Genome
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In modern molecular biology and genetics, a genome is the genetic material of an organism. The genome includes both the genes, the noncoding DNA and the material of the mitochondria and chloroplasts. The term genome was created in 1920 by Hans Winkler, professor of botany at the University of Hamburg, the Oxford Dictionary suggests the name is a blend of the words gene and chromosome. However, see omics for a thorough discussion. A few related -ome words already existed—such as biome, rhizome, Some organisms have multiple copies of chromosomes, diploid, triploid, tetraploid and so on. In classical genetics, in a sexually reproducing organism the gamete has half the number of chromosomes of the somatic cell, the halving of the genetic material in gametes is accomplished by the segregation of homologous chromosomes during meiosis. Additionally, the genome can comprise non-chromosomal genetic elements such as viruses, plasmids, even in species that exist in only one sex, what is described as a genome sequence may be a composite read from the chromosomes of various individuals. Colloquially, the genetic makeup is sometimes used to signify the genome of a particular individual or organism. Both the number of pairs and the number of genes vary widely from one species to another. The only exception in humans is found in red blood cells which become enucleated during development. In 1976, Walter Fiers at the University of Ghent was the first to establish the complete sequence of a viral RNA-genome. The next year Fred Sanger completed the first DNA-genome sequence, Phage Φ-X174, the first genome sequence for an archaeon, Methanococcus jannaschii, was completed in 1996, again by The Institute for Genomic Research. The development of new technologies has made it easier and cheaper to do sequencing. The US National Institutes of Health maintains one of several databases of genomic information. Among the thousands of completed genome sequencing projects include those for rice, a mouse, the plant Arabidopsis thaliana, the fish. In December 2013, scientists first sequenced the genome of a Neanderthal. The genome was extracted from the toe bone of a 130, new sequencing technologies, such as massive parallel sequencing have also opened up the prospect of personal genome sequencing as a diagnostic tool, as pioneered by Manteia Predictive Medicine. A major step toward that goal was the completion in 2007 of the genome of James D. Watson
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Viral replication
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Viral replication is the formation of biological viruses during the infection process in the target host cells. Viruses must first get into the cell before viral replication can occur, from the perspective of the virus, the purpose of viral replication is to allow production and survival of its kind. By generating abundant copies of its genome and packaging these copies into viruses, replication between viruses is greatly varied and depends on the type of genes involved in them. Most DNA viruses assemble in the nucleus while most RNA viruses develop solely in cytoplasm, viruses multiply only in living cells. The host cell must provide the energy and synthetic machinery and the low molecular-weight precursors for the synthesis of viral proteins and nucleic acids. The virus replication occurs in seven stages, namely, Adsorption, Entry, Uncoating, Transcription / mRNA production, Synthesis of virus components, Virion assembly, the virus attaches itself to the Cell Membrane of the host cell. It then injects its DNA or RNA into the host to initiate infection, the cell membrane of the host cell invaginates the virus particle, enclosing it in a pinocytotic vacoule. This protects the cell from antibodies like in the case of the HIV virus, Cell enzymes strip off the virus protein coat. This releases or renders accessible the virus nucleic acid or genome, for some RNA viruses, the infecting RNA produces messenger RNA. This is translation of the genome into protein produces, for others with negative stranded RNA and DNA, viruses are produced by transcription then translation. The mRNA is used to instruct the host cell to make virus components, the virus takes advantage of the existing cell structures to replicate itself. The following components are manufactured by the virus through the hosts existing organelles, Viral protein synthesis, structural, the proteins which make up the virus particle are manufactured and assembled. Non – structural, not found in particle, mainly enzymes for virus genome replication, Viral nucleic acid synthesis new virus genome are synthesized, templates are either the parental genome or with single stranded nucleic acid genomes, newly formed complementary strands. By a virus called polymerate or replicate in some DNA viruses by a cell enzyme and this is done in rapidly dividing cells. A virion is simply an active or intact virus particle, in this stage, newly synthesized genome, and proteins are assembled to form new virus particles. This may take place in the nucleus, cytoplasm, or at plasma membrane for most developed viruses. The viruses, now being mature are released by either sudden rupture of the cell, the new viruses may invade or attack other cells, or remain dormant in the cell. Viruses are classed into 7 types of genes, each of which has its own families of viruses, david Baltimore, a Nobel Prize-winning biologist, devised a system called the Baltimore Classification System to classify different viruses based on their unique replication strategy
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RNA-dependent RNA polymerase
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RNA-dependent RNA polymerase, or RNA replicase, is an enzyme that catalyzes the replication of RNA from an RNA template. This is in contrast to a typical DNA-dependent RNA polymerase, which catalyzes the transcription of RNA from a DNA template, RNA-dependent RNA polymerase is an essential protein encoded in the genomes of all RNA-containing viruses with no DNA stage i. e. only RNA viruses. It catalyses synthesis of the RNA strand complementary to a given RNA template, the RNA replication process is a two-step mechanism. The de novo initiation consists in the addition of a nucleoside triphosphate to the 3-OH of the first initiating NTP, during the following so-called elongation phase, this nucleotidyl transfer reaction is repeated with subsequent NTPs to generate the complementary RNA product. This lack of sensitivity suggested that there is an enzyme that could copy RNA from an RNA template. The most famous example of RdRP is that of the polio virus, the viral genome is composed of RNA, which enters the cell through receptor-mediated endocytosis. From there, the RNA is able to act as a template for complementary RNA synthesis, immediately. The complementary strand is then, itself, able to act as a template for the production of new viral genomes that are packaged and released from the cell ready to infect more host cells. The advantage of this method of replication is that there is no DNA stage, replication is quick, the disadvantage is that there is no back-up DNA copy. Many RdRPs are associated tightly with membranes and are, therefore, the best-known RdRPs are polioviral 3Dpol, vesicular stomatitis virus L, and hepatitis C virus NS5B protein. Many eukaryotes also have RdRPs involved in RNA interference, these amplify microRNAs and small temporal RNAs, in fact these same RdRPs that are used in the defense mechanisms can be usurped by RNA viruses for their benefit. RdRps are highly conserved throughout viruses and is related to telomerase. The similarity has led to speculation that viral RdRps are ancestral to human telomerase, only the palm subdomain, composed of a four-stranded antiparallel beta-sheet with two alpha-helices, is well conserved among all of these enzymes. In RdRp, the palm subdomain comprises three well-conserved motifs, motif A and motif C are spatially juxtaposed, the Asp residues of these motifs are implied in the binding of Mg2+ and/or Mn2+. The Asn residue of motif B is involved in selection of ribonucleoside triphosphates over dNTPs and, thus, the domain organization and the 3D structure of the catalytic centre of a wide range of RdPps, even those with a low overall sequence homology, are conserved. The catalytic centre is formed by several motifs containing a number of conserved amino acid residues. e, orthomyxoviruses, Arenaviruses, Bunyaviruses, Hantaviruses, Nairoviruses, Phleboviruses, Tenuiviruses and Tospoviruses Birnaviridae family of dsRNA viruses. Flaviviruses produce a polyprotein from the ssRNA genome, the polyprotein is cleaved to a number of products, one of which is NS5. Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity and this RNA-directed RNA polymerase possesses a number of short regions and motifs homologous to other RNA-directed RNA polymerases
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Five prime untranslated region
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The 5′ untranslated region is the region of an mRNA that is directly upstream from the initiation codon. This region is important for the regulation of translation of a transcript by differing mechanisms in viruses, prokaryotes and eukaryotes, while called untranslated, the 5′ UTR or a portion of it is sometimes translated into a protein product. This product can then regulate the translation of the coding sequence of the mRNA. In many other organisms, however, the 5′ UTR is completely untranslated, the 5′ UTR has been found to interact with proteins relating to metabolism, and proteins translate sequences within the 5′ UTR. In addition, this region has involved in transcription regulation. Regulatory elements within 5′ UTRs have also linked to mRNA export. The 5′ UTR begins at the start site and ends one nucleotide before the initiation sequence of the coding region. In prokaryotes, the length of the 5′ UTR tends to be 3-10 nucleotides long, for example, the ste11 transcript in Schizosaccharomyces pombe has a 2273 nucleotide 5′ UTR while the lac operon in Escherichia coli only has 7 nucleotides in its 5′ UTR. The differing sizes are likely due to the complexity of the regulation which the 5′ UTR holds. The elements of a eukaryotic and prokaryotic 5′ UTR differ greatly, the prokaryotic 5′ UTR contains a ribosome binding site, also known as the Shine Dalgarno sequence, which is usually 3-10 base pairs upstream from the initiation codon. In contrast, the eukaryotic 5′ UTR contains the Kozak consensus sequence, unlike prokaryotes, 5′ UTRs can harbor introns in eukaryotes. In humans, ~35% of all genes harbor introns within the 5′ UTR, as the 5′ UTR has a high GC content, secondary structures often occur within it. Hairpin loops are one secondary structure that can be located within the 5′ UTR. These secondary structures also impact the regulation of translation, in prokaryotes, the initiation of translation occurs when IF-3, along with the 30S ribosomal subunit, bind to the Shine-Dalgarno sequence of the 5′ UTR. This then recruits other proteins, such as the 50S ribosomal subunit. Each of these steps regulates the initiation of translation, the regulation of translation in eukaryotes is more complex than in prokaryotes. Initially, the complex is recruited to the 5′ cap. Both eIF4E and eIF4G bind the 5′ UTR, which limit the rate at which translational initiation can occur, however, this is not the only regulatory step of translation that involves the 5′ UTR
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Three prime untranslated region
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In molecular genetics, the three prime untranslated region is the section of messenger RNA that immediately follows the translation termination codon. An mRNA molecule is transcribed from the DNA sequence and is translated into protein. Several regions of the molecule are not translated into protein including the 5 cap,5 untranslated region,3 untranslated region. The 3-UTR often contains regulatory regions that post-transcriptionally influence gene expression, regulatory regions within the 3-untranslated region can influence polyadenylation, translation efficiency, localization, and stability of the mRNA. The 3-UTR contains both binding sites for proteins as well as microRNAs. By binding to sites within the 3-UTR, miRNAs can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript. The 3-UTR also has silencer regions which bind to repressor proteins, many 3-UTRs also contain AU-rich elements. Proteins bind AREs to affect the stability or decay rate of transcripts in a manner or affect translation initiation. Furthermore, the 3-UTR contains the sequence AAUAAA that directs addition of several hundred adenine residues called the tail to the end of the mRNA transcript. Poly binding protein binds to this tail, contributing to regulation of translation, stability. For example, poly tail bound PABP interacts with associated with the 5 end of the transcript. The 3-UTR can also contain sequences that attract proteins to associate the mRNA with the cytoskeleton, transport it to or from the cell nucleus, or perform other types of localization. In addition to sequences within the 3-UTR, the characteristics of the region, including its length and secondary structure. These diverse mechanisms of gene regulation ensure that the genes are expressed in the correct cells at the appropriate times. The 3-UTR of mRNA has a variety of regulatory functions that are controlled by the physical characteristics of the region. One such characteristic is the length of the 3-UTR, which in the genome has considerable variation. This region of the mRNA transcript can range from 60 nucleotides to about 4000, on average the length for the 3-UTR in humans is approximately 800 nucleotides, while the average length of 5-UTRs is only about 200 nucleotides. The length of the 3-UTR is significant since longer 3-UTRs are associated with levels of gene expression
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DNA replication
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In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. This process occurs in all living organisms and is the basis for biological inheritance, DNA is made up of a double helix of two complementary strands. During replication, these strands are separated, each strand of the original DNA molecule then serves as a template for the production of its counterpart, a process referred to as semiconservative replication. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication, in a cell, DNA replication begins at specific locations, or origins of replication, in the genome. Unwinding of DNA at the origin and synthesis of new results in replication forks growing bi-directionally from the origin. A number of proteins are associated with the fork to help in the initiation and continuation of DNA synthesis. Most prominently, DNA polymerase synthesizes the new strands by adding nucleotides that complement each strand, DNA replication occurs during the S-stage of interphase. DNA replication can also be performed in vitro, DNA polymerases isolated from cells and artificial DNA primers can be used to initiate DNA synthesis at known sequences in a template DNA molecule. The polymerase chain reaction, a laboratory technique, cyclically applies such artificial synthesis to amplify a specific target DNA fragment from a pool of DNA. DNA usually exists as a structure, with both strands coiled together to form the characteristic double-helix. Each single strand of DNA is a chain of four types of nucleotides, nucleotides in DNA contain a deoxyribose sugar, a phosphate, and a nucleobase. These nucleotides form phosphodiester bonds, creating the phosphate-deoxyribose backbone of the DNA double helix with the bases pointing inward. Nucleotides are matched between strands through hydrogen bonds to form base pairs, adenine pairs with thymine, and guanine pairs with cytosine. DNA strands have a directionality, and the different ends of a single strand are called the 3 end and the 5 end. By convention, if the sequence of a single strand of DNA is given. The strands of the helix are anti-parallel with one being 5 to 3. These terms refer to the atom in deoxyribose to which the next phosphate in the chain attaches. Directionality has consequences in DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3 end of a DNA strand, the pairing of complementary bases in DNA means that the information contained within each strand is redundant
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Uracil
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Uracil /ˈjʊərəsɪl/ is one of the four nucleobases in the nucleic acid of RNA that are represented by the letters A, G, C and U. The others are adenine, cytosine, and guanine, in RNA, uracil binds to adenine via two hydrogen bonds. In DNA, the uracil nucleobase is replaced by thymine, Uracil is a demethylated form of thymine. Uracil is a common and naturally occurring pyrimidine derivative, the name uracil was coined in 1885 by the German chemist Robert Behrend, who was attempting to synthesize derivatives of uric acid. Originally discovered in 1900 by Alberto Ascoli, it was isolated by hydrolysis of yeast nuclein, it was found in bovine thymus and spleen, herring sperm. It is a planar, unsaturated compound that has the ability to absorb light, based on 12C/13C isotopic ratios of organic compounds found in the Murchison meteorite, it is believed that uracil, xanthine and related molecules can also be formed extraterrestrially. In 2012, an analysis of data from the Cassini mission orbiting in the Saturn system showed that Titans surface composition may include uracil, in RNA, uracil base-pairs with adenine and replaces thymine during DNA transcription. In DNA, the substitution of thymine for uracil may have increased DNA stability. Uracil pairs with adenine through hydrogen bonding, when base pairing with adenine, uracil acts as both a hydrogen bond acceptor and a hydrogen bond donor. In RNA, uracil binds with a sugar to form the ribonucleoside uridine. When a phosphate attaches to uridine, uridine 5-monophosphate is produced, Uracil undergoes amide-imidic acid tautomeric shifts because any nuclear instability the molecule may have from the lack of formal aromaticity is compensated by the cyclic-amidic stability. The amide tautomer is referred to as the structure, while the imidic acid tautomer is referred to as the lactim structure. These tautomeric forms are predominant at pH7, the lactam structure is the most common form of uracil. Uracil also recycles itself to form nucleotides by undergoing a series of phosphoribosyltransferase reactions, degradation of uracil produces the substrates aspartate, carbon dioxide, and ammonia. C4H4N2O2 → H3NCH2CH2COO− + NH4+ + CO2 Oxidative degradation of uracil produces urea and maleic acid in the presence of H2O2 and Fe2+ or in the presence of diatomic oxygen, the first site of ionization of uracil is not known. The negative charge is placed on the anion and produces a pKa of less than or equal to 12. The basic pKa = -3.4, while the acidic pKa =9.389, in the gas phase, uracil has 4 sites that are more acidic than water. Uracil is rarely found in DNA, and this may have been a change to increase genetic stability
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Sense (molecular biology)
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In molecular biology and genetics, sense is a concept used to compare the polarity of nucleic acid molecules, such as DNA or RNA, to other nucleic acid molecules. Depending on the context within molecular biology, sense may have different meanings. Molecular biologists call a single strand of DNA sense if an RNA version of the sequence is translated or translatable into protein. Its complementary strand is called antisense, the two complementary strands of double-stranded DNA are usually differentiated as the sense strand and the antisense strand. However, the DNA sense strand itself is not used to make protein by the cell. It is the DNA antisense strand which serves as the source for the code, because, with bases complementary to the DNA sense strand. Since transcription results in an RNA product complementary to the DNA template strand, the mRNA is what is used for translation. Hence, a base triplet 3-TAC-5 in the DNA antisense strand can be used as a template which will result in an 5-AUG-3 base triplet in mRNA. The DNA sense strand will have the triplet ATG, which looks just like AUG, the DNA sense strand is called a sense strand not because it will be used to make protein, but because it has a sequence that looks like the protein codon sequence. In biology and research, short antisense molecules can interact with complementary strands of nucleic acids, see the section on antisense oligonucleotides below. For a cell to use information, one strand of the DNA serves as a template for the synthesis of a complementary strand of RNA. The template DNA strand is called the transcribed strand with antisense sequence, because the DNA is double-stranded, the strand complementary to the antisense sequence is called the non-transcribed strand and has the same sense sequence as the mRNA transcript. A sequence 5 CGCTAT3 is equivalent to a sequence written 3 TATCGC5 as long as the 5 and 3 ends are noted, if the ends are not labeled, convention is to assume that the sequence is written from left to right in the 5 to 3 direction. Watson strand refers to 5 to 3 top strand, whereas Crick strand refers to 5 to 3 bottom strand, both Watson and Crick strands can be either sense or antisense strands depending on the gene whose sequences are displayed in the genome sequence database. YKL074C defines the 74th ORF to the left of the centromere of chromosome XI, another confusing term referring to Plus and Minus strand is also widely used. Whether the strand is sense or antisense, the query sequence in NCBI BLAST alignment is Plus strand. A single-stranded genome that contains both positive-sense and negative-sense is said to be ambisense, Antisense RNA is an RNA transcript that is complementary to endogenous mRNA. In other words, it is a non-coding strand complementary to the sequence of RNA
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Okazaki fragments
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Okazaki fragments are short, newly synthesized DNA fragments that are formed on the lagging template strand during DNA replication. They are complementary to the template strand, together forming short double-stranded DNA sections. Okazaki fragments are between 1000 and 2000 nucleotides long in prokaryotes and are roughly 100 to 200 nucleotides long in eukaryotes and this is because the enzymes that synthesise the new DNA can only work in one direction along the parent DNA molecule and the two strands are anti-parallel. On the leading strand this route is continuous, but on the lagging strand it is discontinuous, DNA is synthesised from 5 to 3, so when copying the 3 to 5 strand, replication is continuous. Phosphodiester links form between the 3 to 5 and nucleotides can be added with the aid of the enzyme DNA polymerase for the leading strand. However, in order to synthesise the lagging strand synthesis must occur in small sections and these new stretches of DNA are called Okazaki fragments and each one requires its own RNA primer. A series of experiments eventually lead to the discovery of Okazaki fragments, the experiments were conducted during the 1960s with Reiji Okazaki, Tsuneko Okazaki, Kiwako Sakabe and their colleagues during their research on DNA replication of Escherichia coli. In 1966, Kiwako Sakabe and Reiji Okazaki first showed that DNA replication was a process involving fragments. The fragments were further investigated by the researchers and their colleagues through their research including the study on bacteriophage DNA replication in Escherichia coli, the work of Kiwako Sakabe and Reiji Okazaki provided experimental evidence supporting the hypothesis that DNA replication is a discontinuous process. Previously, it was accepted that replication was continuous in both the 3 to 5 and 5 to 3 directions. 3 and 5 are specifically numbered carbons on the ring in nucleic acids. The team hypothesized that if discontinuous replication was used, short strands of DNA, synthesized at the replicating point, to distinguish the method of replication used by DNA experimentally, the team pulse-labeled newly replicated areas of Escherichia coli chromosomes, denatured, and extracted the DNA. A large amount of short units meant that the replication method was likely discontinuous. The hypothesis was supported by the discovery of polynucleotide ligase. In 1968, Reiji and Tsuneko Okazaki gathered additional evidence of nascent DNA strands, E. coli were infected with bacteriophage T4 that produce temperature-sensitive polynucleotide ligase. The cells infected with the T4 phages accumulated a large amount of short, newly synthesized DNA chains, as predicted in the hypothesis and this experiment further supported the Okazakis hypothesis of discontinuous replication and linkage by polynucleotide ligase. It disproved the notion that short chains were produced during the process as well. The Okazakis experiments provided extensive information on the process of DNA
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Primer (molecular biology)
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A primer is a short strand of RNA or DNA that serves as a starting point for DNA synthesis. It is required for DNA replication because the enzymes that catalyze this process, DNA polymerases, the polymerase starts replication at the 3-end of the primer, and copies the opposite strand. In vivo DNA replication utilizes short strands of RNA called RNA primers to initiate DNA synthesis on both the leading and lagging strands — DNA primers are not seen in vivo in humans and these RNA primers can be made de novo. On the other hand, many of the in vitro laboratory techniques that involve DNA polymerase in biochemistry and molecular biology, in experiments, it is often important to use a primer with a similar Tm to the template strand it will be hybridizing to. These primers are usually short, chemically synthesized oligonucleotides, with a length of about twenty bases and they are hybridized to a target DNA, which is then copied by the polymerase. The lagging strand of DNA is that strand of the DNA double helix that is orientated in a 5 to 3 manner, therefore, its complement must be synthesized in a 3→5 manner. Because DNA polymerase III cannot synthesize in the 3→5 direction, the strand is synthesized in short segments known as Okazaki fragments. Along the lagging strands template, primase builds RNA primers in short bursts, DNA polymerases are then able to use the free 3-OH groups on the RNA primers to synthesize DNA in the 5→3 direction. The RNA fragments are removed by DNA polymerase I for prokaryotes or DNA polymerase δ for eukaryotes. DNA ligase then joins the deoxyribonucleotides together, completing the synthesis of the lagging strand and this mechanism is a potential explanation of how the HIV virus can transform its genome into double-stranded DNA from the RNA-DNA formed after reverse transcription of its RNA. DNA sequencing is used to determine the nucleotides in a DNA strand, the Sanger chain termination method of sequencing uses a primer to start the chain reaction. In PCR, primers are used to determine the DNA fragment to be amplified by the PCR process, the length of primers is usually not more than 30 nucleotides, and they need to match the beginning and the end of the DNA fragment to be amplified. They direct replication towards each other – the extension of one primer by polymerase then becomes the template for the other, leading to an exponential increase in the target segment. It is worth noting that primers are not always for DNA synthesis, pairs of primers should have similar melting temperatures since annealing in a PCR occurs for both simultaneously. Primer sequences need to be chosen to select for a region of DNA. A commonly used method is BLAST search whereby all the regions to which a primer may bind can be seen. Both the nucleotide sequence as well as the primer itself can be BLAST searched, the free NCBI tool Primer-BLAST integrates primer design and BLAST search into one application, as do commercial software products such as ePrime and Beacon Designer. Computer simulations of theoretical PCR results may be performed to assist in primer design, many online tools are freely available for primer design, some of which focus on specific applications of PCR
. PMID 19443781.
