Prokaryotic transcription

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Prokaryotic transcription is the process in which messenger RNA transcripts of genetic material in prokaryotes are produced, to be translated for the production of proteins. Prokaryotic transcription occurs in the cytoplasm alongside translation. Unlike in eukaryotes, prokaryotic transcription and translation can occur simultaneously. This is impossible in eukaryotes, where transcription occurs in a membrane-bound nucleus while translation occurs outside the nucleus in the cytoplasm. In prokaryotes genetic material is not enclosed in a membrane-enclosed nucleus and has access to ribosomes in the cytoplasm. [1] Transcription is known to be controlled by a variety of regulators in prokaryotes. Many of these transcription factors are homodimers containing helix-turn-helix DNA-binding motifs.[2]
Contents
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1 Initiation 2 Elongation 3 Termination 4 References 5 External links

[edit]Initiation
The following steps occur, in order, for transcription initiation:

RNA polymerase (RNAP) binds to one of several specificity factors, , to form a holoenzyme. In this form, it can recognize and bind to specific promoter regions in the DNA. The -35 region and the -10 ("Pribnow box") region comprise the basic prokaryotic promoter, and |T| stands for the terminator. The DNA on the template strand between the +1 site and the terminator is transcribed into RNA, which is then translated into protein.At this stage, the DNA is double-stranded ("closed"). This holoenzyme/wound-DNA structure is referred to as the closed complex.

The DNA is unwound and becomes single-stranded ("open") in the vicinity of the initiation site (defined as +1). This holoenzyme/unwound-DNA structure is called the open complex.

The RNA polymerase transcribes the DNA (the beta subunit initiates the synthesis), but produces about 10 abortive (short, non-productive) transcripts which are unable to leave the RNA polymerase because the exit channel is blocked by the -factor.

The -factor eventually dissociates from the holoenzyme, and elongation proceeds.

[edit]Elongation
Promoters can differ in "strength"; that is, how actively they promote transcription of their adjacent DNA sequence. Promoter strength is in many (but not all) cases, a matter of how tightly RNA polymerase and its associated accessory proteins bind to their respective DNA sequences. The more similar the sequences are to a consensus sequence, the stronger the binding is. Additional transcription regulation comes from transcription factors that can affect the stability of the holoenzyme structure at initiation. Most transcripts originate using adenosine-5'-triphosphate (ATP) and, to a lesser extent, guanosine-5'triphosphate (GTP) (purine nucleoside triphosphates) at the +1 site. Uridine-5'-triphosphate (UTP) and cytidine-5'-triphosphate (CTP) (pyrimidine nucleoside triphosphates) are disfavoured at the initiation site.

[edit]Termination
Two termination mechanisms are well known:

Intrinsic termination (also called Rho-independent transcription termination) involves terminator sequences within the RNA that signal the RNA polymerase to stop. The terminator sequence is usually a palindromic sequence that forms a stem-loop hairpin structure that leads to the dissociation of the RNAP from the DNA template.

Rho-dependent termination uses a termination factor called factor(rho factor) which is a protein to stop RNA synthesis at specific sites. This protein binds at a rho utilisation site on the nascent RNA strand and runs along the mRNA towards the RNAP. A stem loop structure upstream of the terminator region pauses the RNAP, when -factor reaches the RNAP, it causes RNAP to dissociate from the DNA, terminating transcription.

[edit]References

o fully understand transcription, students need to appreciate the dynamic nature of this process. Transcription "works" because nucleotides diffuse into position from the cytoplasm, the DNA helix unwinds and then rewinds, and the RNA polymerase travels rapidly along a DNA template strand for hundreds or thousands of bases to produce a very long RNA molecule in its wake. This simple, 40-s animation is presented at three levels of magnification to emphasize the movements of all the players. At the beginning of the animation, an RNA polymerase (RNAP) is shown binding to the promoter region of a double helix of DNA. The sigma subunit of RNAP helps to locate the promoter, after which it is no longer needed. Then, as we look "inside" the RNAP, we can see that the double helix begins to unwind. A growing strand of RNA (in red) begins to form along one of the DNA strands. As the strand lengthens, the RNAP continues to travel along the DNA, unwinding it to expose more DNA bases. Behind the RNAP the DNA helix reforms, displacing the RNA strand. Finally, we zoom in further to the active site of the RNAP molecule. Here, we can see how an RNA nucleotide triphosphate (in this case, an "A," for a ribose triphosphate with an adenine base) diffuses into position and binds to its complement a "T" on the DNA strand (note that the blue deoxyriboses on the DNA strand have the opposite orientation to the red ribose residues on the the RNA strand). Once hydrogen bonding has positioned the "A" and the "T" together, the RNAP is able to catalyze covalent bonding of the "A" to the 3-OH on the growing RNA strand. This bond formation is powered by the cleavage of the diphosphate from the RNA

nucleotide. The process is quickly repeated as another RNA nucleotide triphosphate, "C," is added to the RNA chain. Moments later, a DNA nucleotidea "T"diffuses into position. While this nucleotide can hydrogen bond with the "A" on the DNA template strand, the RNAP discriminates between RNA and DNA nucleotides, and no reaction is catalyzed. Ultimately, the deoxyribonucleotide diffuses away, allowing RNA strand synthesis to continue when another ribonucleotide triphosphatea "U"hydrogen bonds with the A on the DNA template strand.

Concept 2: Transcription and Translation in Cells

In a prokaryotic cell, transcription and translation are coupled; that is, translation begins while the mRNA is still being synthesized. In a eukaryotic cell, transcription occurs in the nucleus, and translation occurs in the cytoplasm. Prokaryotic Cell

Because there is no nucleus to separate the processes of transcription and translation, when bacterial genes are transcribed, their transcripts can immediately be translated. Eukaryotic Cell

Transcription and translation are spatially and temporally separated in eukaryotic cells; that is, transcription occurs in the nucleus to produce a pre-mRNA molecule. The pre-mRNA is typically processed to produce the mature mRNA, which exits the nucleus and is translated in the cytoplasm.

Concept 3: Different Genes for Different RNAs

There are 4 types of RNA, each encoded by its own type of gene. The genomic DNA contains all the information for the structure and function of an organism. In any cell, only some of the genes are expressed, that is, transcribed into RNA.

There are 4 types of RNA, each encoded by its own type of gene: mRNA - Messenger RNA: Encodes amino acid sequence of a polypeptide. tRNA - Transfer RNA: Brings amino acids to ribosomes during translation. rRNA - Ribosomal RNA: With ribosomal proteins, makes up the ribosomes, the organelles that translate the mRNA. snRNA - Small nuclear RNA: With proteins, forms complexes that are used in RNA processing in eukaryotes. (Not found in prokaryotes.)

Concept 4: Basic Structure of a Protein-Coding Gene

A protein-coding gene consists of a promoter followed by the coding sequence for the protein and then a terminator.

The promoter is a base-pair sequence that specifies where transcription begins. The coding sequence is a base-pair sequence that includes coding information for the polypeptide chain specified by the gene. The terminator is a sequence that specifies the end of the mRNA transcript.

Concept 6: The Transcription Process

RNA synthesis involves separation of the DNA strands and synthesis of an RNA molecule in the 5' to 3' direction by RNA polymerase, using one of the DNA strands as a template. In complementary base pairing, A, T, G, and C on the template DNA strand specify U, A, C, and G, respectively, on the RNA strand being synthesized.

Concept 7: Complete Transcription of an RNA Molecule

Transcription begins at the promoter, proceeds through the coding region, and ends at the terminator.

Concept 8: mRNA in Prokaryotes

The sequence of a prokaryotic protein-coding gene is colinear with the translated mRNA; that is, the transcript of the gene is the molecule that is translated into the polypeptide.

mRNA in Eukaryotes
The sequence of a eukaryotic protein-coding gene is typically not colinear with the translated mRNA; that is, the transcript of the gene is a molecule that must be processed to remove extra sequences (introns) before it is translated into the polypeptide.

Most eukaryotic protein-coding genes contain segments called introns, which break up the amino acid coding sequence into segments called exons. The transcript of these genes is the pre-mRNA (precursor-mRNA). The pre-mRNA is processed in the nucleus to remove the introns and splice the exons together into a translatable mRNA. That mRNA exits the nucleus and is translated in the cytoplasm.

Pre-mRNA Processing (Splicing)

Eukaryotic pre-mRNAs typically include introns. Introns are removed by RNA processing in which the intron is looped out and cut away from the exons by snRNPs, and the exons are spliced together to produce the translatable mRNA.

The steps of pre-mRNA splicing (intron removal) are as follows: The intron loops out as snRNPs (small nuclear ribonucleoprotein particles, complexes of snRNAs and proteins) bind to form the spliceosome. The intron is excised, and the exons are then spliced together. The resulting mature mRNA may then exit the nucleus and be translated in the cytoplasm.

Transcription in Prokaryotes
The most detailed molecular information about the transcription cycle is available in bacterial systems. The synthesis of RNA is initiated at the promoter sequence by the enzyme RNA polymerase. A single RNA polymerase type is responsible for the synthesis of messenger, transfer, and ribosomal RNAs. When isolated from bacteria, prokaryotic RNA polymerase has two forms: The coreenzyme and the holoenzyme. The core enzyme is a tetramer whose composition is given as 2 (two alpha subunits, one beta subunit, and one betaprime subunit). Core RNA polymerase is capable of faithfully copying DNA into RNA but does not initiate at the correct site in a gene. That is, it does not recognize the promoter specifically. Correct promoter recognition is the function of the holoenzyme form of RNA polymerase.

Figure 1
The RNA polymerase holoenzyme contains another subunit, s( sigma), in addition to the subunits found in the core enzyme. Holoenzyme, 2, is capable of correct initiation at the promoter region of a gene. Sigma thus must be involved in promoter recognition. Sigma subunits are related but distinct in different forms of RNA polymerase holoenzyme. These specialized subunits direct RNA polymerase to promoter sequences for different classes of genes. For example, bacteria exposed to high temperatures synthesize a set of protective proteins called heat-shock proteins. The genes for the heat-shock proteins have special promoter sequences that are recognized by an RNA polymerase holoenzyme with a specific subunit. The discussed here is the major of the common bacterium E. coli, about which most is known.

Promoter recognition
RNA polymerase holoenzyme starts by recognizing the promoter of a gene. The promoter isn't copied into RNA, but it is, nonetheless, an important piece of genetic information. The information in a promoter was determined by lining up a large number of promoters and counting how many times a particular base appeared at a given position in the various promoter sequences. The consensus sequence is given by the statistically most probable base at each pointthe bases that appear most often in the promoter collection. Very few, if any, naturally occurring promoters match the consensus sequence exactly, but the strength of a promoter (how actively RNA polymerase initiates at it) correlates well with the degree of consensus match. For example, the promoters of genes for ribosomal RNA match the consensus well, while the promoters for the mRNA encoding some regulatory proteins match the consensus poorly. This correlates with the relative amounts of each gene product that are needed at any one time: many ribosomes, and only a few regulatory proteins. The consensus sequence for an E. coli promoter has two conserved regions near positions -35 and -10 relative to the transcription start site. That is, the templatedirected synthesis of RNA begins 35 base pairs downstream of the first consensus region and ten base pairs downstream of the second. The -35 consensus is: TTG ACA. The -10 consensus is: TATAA T. A couple of important points exist about the consensus. First, not all bases in the consensus are conserved to the same amount. The bases marked with bold type and underlined are more conserved than the others, and the -10 region is more conserved overall than is the -35 region. Secondly, the promoter sequence is asymmetrical; that is, it reads differently in one direction than in the other. (Compare this to the recognition sequence for the restriction enzyme BamHI, GGATCC.) This asymmetry means that RNA polymerase gets directional

information from the promoter in addition to information about the starting point for transcription.

The transcription process

RNA polymerase only goes one direction from a promoter and only one strand of DNA is used as a template at any one time. To provide this template strand, the initiation of transcription involves a short unwinding of the DNA double helix. This is accomplished in a two-step fashion. First, RNA polymerase binds to the promoter to form the closed complex, which is relatively weak. Then, the double-stranded DNA goes through a conformational change to form the much stronger open complex through opening of the base pairs at the -10 sequence, as shown in Figure 2 .

Figure 2
The initiator nucleotide binds to the complex and the first phosphodiester bonds are made, accompanied by release of . The remaining core polymerase is now in the elongation mode. Several experimental observations support the picture presented in the next figure, namely the fact that less than one exists in the cell per core enzyme in each cell.

Figure 3
Elongation is the function of the RNA polymerase core enzyme. RNA polymerase moves along the template, locally unzipping the DNA double helix. This allows a transient base pairing between the incoming nucleotide and newly-synthesized RNA and the DNA template strand. As it is made, the RNA transcript forms secondary structure through intra-strand base pairing. The average speed of transcription is about 40 nucleotides per second, much slower than DNA polymerase. Other protein factors may bind to polymerase and alter the rate of transcription and some specific sequences are transcribed more slowly than others are. Eventually, RNA polymerase must come to the end of the region to be transcribed. Termination of transcription in vitro is classified as to its dependence on the protein factor, rho (). Rho-independent terminators have a characteristic structure, which features (a) A strong G-C rich stem and loop, (b) a sequence of 46 U residues in the RNA, which are transcribed from a corresponding stretch of As in the template. Rho-factor-dependent terminators are less well defined, as shown in Figure 4 .

Ribosomal RNA
Ribosomal RNA is essential for protein synthesis. In fact, RNA is thought to be the catalytically active part of the very large complex of proteins and RNAs that synthesize proteins. Ribosomes and ribosomal RNAs are heterogeneous, with different sized rRNAs found in the small and large subunits of the ribosome. Ribosomes can be separated into two subunits. Each subunit contains both protein and RNA. Although they vary widely in size, ribosomal RNAs have common secondary structures. The larger size of the eukaryotic RNAs is due to their having extra structural domains inserted into the midst of the smaller ones, rather than by a totally new folding pattern. Antibiotics are natural products, usually from soil bacteria and molds, which interfere with the growth of other bacteria. Often these antibiotics act on ribosomal RNA targets. For example, streptomycin, which has been used to treat tuberculosis, binds to a single region of bacterial 16S RNA, interfering with protein synthesis. The drug doesn't disrupt protein synthesis in humans, which allows for streptomycin's relatively high therapeutic indexthe ratio of harmful to helpful doses of the drug. Conversely, bacteria can become resistant to antibiotics by changes in their rRNA, either by a change in the nucleotide sequence of the ribosomal RNA or by methylation of the rRNA.

Transcription (genetics)
From Wikipedia, the free encyclopedia

Transcription is the process of creating a complementary RNA copy of a sequence of DNA.[1] Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementarylanguage that can be converted back and forth from DNA to RNA by the action of the correct enzymes. During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA strand. As opposed to DNA replication, transcription results in an RNA complement that includes uracil (U) in all instances where thymine (T) would have occurred in a DNA complement. Also unlike DNA replication where DNA is synthesised, transcription does not involve an RNA primer to initiate RNA synthesis. Transcription is explained easily in 4 or 5 steps, each moving like a wave along the DNA. 1. RNA polymerase moves the transcription bubble, a stretch of unpaired nucleotides, by breaking the hydrogen bonds between complementary nucleotides. 2. RNA polymerase adds matching RNA nucleotides that are paired with complementary DNA bases. 3. RNA sugar-phosphate backbone forms with assistance from RNA polymerase. 4. Hydrogen bonds of the untwisted RNA + DNA helix break, freeing the newly synthesized RNA strand. 5. If the cell has a nucleus, the RNA is further processed (addition of a 3' poly-A tail and a 5' cap) and exits through to the cytoplasm through the nuclear pore complex. Transcription is the first step leading to gene expression. The stretch of DNA transcribed into an RNA molecule is called a transcription unit and encodes at least one gene. If the gene transcribed encodes a protein, the result of transcription is messenger RNA (mRNA), which will then be used to create that protein via the process of translation. Alternatively, the transcribed gene may encode for either non-coding RNA genes (such as microRNA, lincRNA, etc.) or ribosomal RNA (rRNA) or transfer RNA (tRNA), other components of the protein-assembly process, or otherribozymes.[2] A DNA transcription unit encoding for a protein contains not only the sequence that will eventually be directly translated into the protein (the coding sequence) but also regulatory sequences that direct and regulate the synthesis of that protein. The regulatory sequence before (upstream from) the coding sequence is called the five prime untranslated region (5'UTR), and the sequence following (downstream from) the coding sequence is called the three prime untranslated region (3'UTR).[2] Transcription has some proofreading mechanisms, but they are fewer and less effective than the controls for copying DNA; therefore, transcription has a lower copying fidelity than DNA replication. [3] As in DNA replication, DNA is read from 3' 5' during transcription. Meanwhile, the complementary RNA is created from the 5' 3' direction. This means its 5' end is created first in base pairing. Although DNA is arranged as two antiparallel strands in a double helix, only one of the two DNA strands, called the template strand, is used for transcription. This is because RNA is only single-stranded, as opposed to doublestranded DNA. The other DNA strand is called the coding (lagging) strand, because its sequence is the

same as the newly created RNA transcript (except for the substitution of uracil for thymine). The use of only the 3' 5' strand eliminates the need for the Okazaki fragments seen in DNA replication.[2] Transcription is divided into 5 stages: pre-initiation, initiation, promoter clearance, elongation and termination.[2]
Contents
[hide]

1 Major steps

o o o o o

1.1 Pre-initiation 1.2 Initiation 1.3 Promoter clearance 1.4 Elongation 1.5 Termination

2 Measuring and detecting transcription 3 Transcription factories 4 History 5 Reverse transcription 6 Inhibitors 7 See also 8 References 9 External links

[edit]Major

steps

[edit]Pre-initiation
In eukaryotes, RNA polymerase, and therefore the initiation of transcription, requires the presence of a core promoter sequence in the DNA. Promoters are regions of DNA that promote transcription and, in eukaryotes, are found at -30, -75, and -90 base pairs upstream from the transcription start site (abbreviated to TSS). Core promoters are sequences within the promoter that are essential for transcription initiation. RNA polymerase is able to bind to core promoters in the presence of various specific transcription factors.[citation needed] The most characterized type of core promoter in eukaryotes is a short DNA sequence known as a TATA box, found 25-30 base pairs upstream from the TSS.[citation needed] The TATA box, as a core promoter, is the binding site for a transcription factor known as TATA-binding protein (TBP), which is itself a subunit of another transcription factor, called Transcription Factor II D (TFIID). After TFIID binds to the TATA box via the TBP, five more transcription factors and RNA polymerase combine around the TATA box in a series of stages to form a preinitiation complex. One transcription factor, Transcription factor II H, has two

components with helicase activity and so is involved in the separating of opposing strands of doublestranded DNA to form the initial transcription bubble. However, only a low, or basal, rate of transcription is driven by the preinitiation complex alone. Other proteins known as activators and repressors, along with any associatedcoactivators or corepressors, are responsible for modulating transcription rate.[citation needed] Thus, preinitiation complex contains:[citation needed] 1. Core Promoter Sequence 2. Transcription Factors 3. RNA Polymerase 4. Activators and Repressors. The transcription preinitiation inarchaea is, in essence, homologous to that of eukaryotes, but is much less complex.[4] The archaeal preinitiation complex assembles at a TATA-box binding site; however, in archaea, this complex is composed of only RNA polymerase II, TBP, and TFB (the archaeal homologue of eukaryotic transcription factor II B (TFIIB)).[5][6]

[edit]Initiation

Simple diagram of transcription initiation. RNAP = RNA polymerase

In bacteria, transcription begins with the binding of RNA polymerase to the promoter in DNA. RNA polymerase is a core enzyme consisting of five subunits: 2 subunits, 1 subunit, 1 ' subunit, and 1 subunit. At the start of initiation, the core enzyme is associated with a sigma factor that aids in finding the appropriate -35 and -10 base pairs downstream ofpromoter sequences.[7] When the sigma factor and RNA polymerase combine, they form a holoenzyme. Transcription initiation is more complex in eukaryotes. Eukaryotic RNA polymerase does not directly recognize the core promoter sequences. Instead, a collection of proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription. Only after certain transcription factors are attached to the promoter does the RNA polymerase bind to it. The completed assembly of transcription factors and RNA polymerase bind to the promoter, forming a transcription initiation complex. Transcription in the archaea domain is similar to transcription in eukaryotes.[8]

[edit]Promoter

clearance

After the first bond is synthesized, the RNA polymerase must clear the promoter. During this time there is a tendency to release the RNA transcript and produce truncated transcripts. This is called abortive initiation and is common for both eukaryotes and prokaryotes.[9] Abortive initiation continues to occur until the factor rearranges, resulting in the transcription elongation complex (which gives a 35 bp moving footprint). The factor is released before 80 nucleotides of mRNA are synthesized.[10] Once the transcript reaches approximately 23 nucleotides, it no longer slips and elongation can occur. This, like most of the

remainder of transcription, is an energy-dependent process, consuming adenosine triphosphate (ATP).[citation needed] Promoter clearance coincides with phosphorylation of serine 5 on the carboxy terminal domain of RNAP II in eukaryotes, which is phosphorylated by TFIIH.[citation needed]

[edit]Elongation

Simple diagram of transcription elongation

One strand of the DNA, the template strand (or noncoding strand), is used as a template for RNA synthesis. As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy. Although RNA polymerase traverses the template strand from 3' 5', the coding (non-template) strand and newly-formed RNA can also be used as reference points, so transcription can be described as occurring 5' 3'. This produces an RNA molecule from 5' 3', an exact copy of the coding strand (except thatthymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one less oxygen atom) in its sugar-phosphate backbone).[citation needed] Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases on a single DNA template and multiple rounds of transcription (amplification of particular mRNA), so many mRNA molecules can be rapidly produced from a single copy of a gene.[citation needed] Elongation also involves a proofreading mechanism that can replace incorrectly incorporated bases. In eukaryotes, this may correspond with short pauses during transcription that allow appropriate RNA editing factors to bind. These pauses may be intrinsic to the RNA polymerase or due to chromatin structure. [citation
needed]

[edit]Termination
Bacteria use two different strategies for transcription termination.1.Rho-independent transcription 2.Rhodependent transcription. In Rho-independent transcription termination,also called intrinsic termination, RNA transcription stops when the newly synthesized RNA molecule forms a G-C-rich hairpin loop followed by a run of Us. When the hairpin forms, the mechanical stress breaks the weak rU-dA bonds, now filling the DNA-RNA hybrid. This pulls the poly-U transcript out of the active site of the RNA polymerase, in effect, terminating transcription. In the "Rho-dependent" type of termination, a protein factor called "Rho" destabilizes the interaction between the template and the mRNA, thus releasing the newly synthesized mRNA from the elongation complex.[11]

Transcription termination in eukaryotes is less understood but involves cleavage of the new transcript followed by template-independent addition of As at its new 3' end, in a process calledpolyadenylation.[12]

[edit]Measuring

and detecting transcription

Electron micrograph of the ribosomal transcription process. The forming mRNAstrands are visible as branches from the main DNA strand.[citation needed]

Transcription can be measured and detected in a variety of ways:[citation needed]

Nuclear Run-on assay: measures the relative abundance of newly formed transcripts RNase protection assay and ChIP-Chip of RNAP: detect active transcription sites RT-PCR: measures the absolute abundance of total or nuclear RNA levels, which may however differ from transcription rates

DNA microarrays: measures the relative abundance of the global total or nuclear RNA levels; however, these may differ from transcription rates

In situ hybridization: detects the presence of a transcript MS2 tagging: by incorporating RNA stem loops, such as MS2, into a gene, these become incorporated into newly synthesized RNA. The stem loops can then be detected using a fusion of GFP and the MS2 coat protein, which has a high affinity, sequence-specific interaction with the MS2 stem loops. The recruitment of GFP to the site of transcription is visualised as a single fluorescent spot. This remarkable new approach has revealed that transcription occurs in discontinuous bursts, or pulses (see Transcriptional bursting). With the notable exception of in situ techniques, most other methods provide cell population averages, and are not capable of detecting this fundamental property of genes.[13]

Northern blot: the traditional method, and until the advent of RNA-Seq, the most quantitative

RNA-Seq: applies next-generation sequencing techniques to sequence whole transcriptomes, which allows the measurement of relative abundance of RNA, as well as the detection of additional variations such as fusion genes, post-translational edits and novel splice sites

[edit]Transcription

factories

Main article: Transcription factories Active transcription units are clustered in the nucleus, in discrete sites called transcription factories or euchromatin. Such sites can be visualized by allowing engaged polymerases to extend their transcripts in tagged precursors (Br-UTP or Br-U) and immuno-labeling the tagged nascent RNA. Transcription factories can also be localized using fluorescence in situ hybridization or marked by antibodies directed against polymerases. There are ~10,000 factories in the nucleoplasm of a HeLa cell, among which are ~8,000 polymerase II factories and ~2,000 polymerase III factories. Each polymerase II factory contains ~8 polymerases. As most active transcription units are associated with only one polymerase, each factory usually contains ~8 different transcription units. These units might be associated through promoters and/or enhancers, with loops forming a cloud around the factor.[citation needed]

[edit]History
A molecule that allows the genetic material to be realized as a protein was first hypothesized by Franois Jacob and Jacques Monod. Severo Ochoa won a Nobel Prize in Physiology or Medicinefor developing a process of RNA synthesis in 1959. RNA synthesis by RNA polymerase was established in vitro by several laboratories by 1965; however, the RNA synthesized by these enzymes had properties that suggested the existence of an additional factor needed to terminate transcription correctly.[citation needed] In 1972, Walter Fiers became the first person to actually prove the existence of the terminating enzyme. Roger D. Kornberg won the 2006 Nobel Prize in Chemistry "for his studies of the molecular basis of eukaryotic transcription".[14]

[edit]Reverse

transcription

Scheme of reverse transcription

Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA. HIV has an RNA genome that is duplicated into DNA. The resulting DNA can be merged with the DNA genome of the host cell. The main enzyme responsible for synthesis of DNA from an RNA template is called reverse transcriptase. In the case of HIV, reverse transcriptase is responsible for synthesizing acomplementary DNA strand (cDNA) to the viral RNA genome. An associated enzyme, ribonuclease H, digests the RNA strand, and reverse transcriptase synthesises a complementary strand of DNA to form a double helix DNA structure. This cDNA is integrated into the host cell's genome via another enzyme (integrase) causing the host cell to generate viral proteins that reassemble into new viral particles. In HIV, subsequent to this, the host cell undergoes programmed cell death, apoptosis of T cells.[15] However, in other retroviruses, the host cell remains intact as the virus buds out of the cell. Some eukaryotic cells contain an enzyme with reverse transcription activity called telomerase. Telomerase is a reverse transcriptase that lengthens the ends of linear chromosomes. Telomerase carries an RNA template from which it synthesizes DNA repeating sequence, or "junk" DNA. This repeated sequence of DNA is important because, every time a linear chromosome is duplicated, it is shortened in length. With "junk" DNA at the ends of chromosomes, the shortening eliminates some of the non-essential, repeated sequence rather than the protein-encoding DNA sequence farther away from the chromosome end. Telomerase is often activated in cancer cells to enable cancer cells to duplicate their genomes indefinitely without losing important protein-coding DNA sequence. Activation of telomerase could be part of the process that allows cancer cells to become immortal. However, the true in vivo significance of telomerase has still not beenempirically proven.[citation needed]

[edit]Inhibitors
Transcription inhibitors can be used as antibiotics against, for example, pathogenic bacteria (antibacterials) and fungi (antifungals). An example of such an antibacterial is rifampicin, which inhibitsprokaryotic DNA transcription into mRNA by inhibiting DNA-dependent RNA polymerase by binding its beta-subunit. 8Hydroxyquinoline is an antifungal transcription inhibitor.[16]

[edit]See

also