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Transcription Factor oligodeoxynucleotide decoys (ODN decoys). Use our GeneDetect® transcription factor decoys to inhibit specific transcription factors in cell culture. Complete list of Transcription Factor decoy Products. Introduction

Transcription factor ODN decoy approach

Advantages and disadvantages of the ODN decoy approach for studying cellular gene expression

ODN Decoys available from

How are these decoys used?




Cells can respond to stimuli (normal or pathological) by changing the levels of expression of specific genes. The cellular proteins that regulate changes in gene expression are called transcription factors. Transcription factors are generally nuclear and can either be constitutively expressed within the cell (present under basal conditions, for example CREB) or themselves inducible (for example AP-1). These transcription factor proteins bind specific sequences found in the promoter regions of genes (target genes) whose expression they then regulate (switch on or off). These binding sequences are generally 6-10 base pairs in length and are occasionally found in multiple copies within the promoter regions of target genes. Although the transcription factor protein-DNA interaction is sequence-specific, the binding site for one given transcription factor may vary by several base pairs within different target genes. Therefore when we describe the specific DNA binding sequence for a transcription factor we refer to the non-variable part of the binding sequence, that is, the transcription factor consensus sequence. For example, the AP-1 transcription factor made up of Fos and Jun proteins binds to the TGACTCA consensus sequence. In comparison the consensus sequence for the Smad transcription factor family which mediate TGF-b, activin and BMP induced changes in gene expression is CAGACA. Fig. 1. Transcription factor ODN decoy approach. The basic theory behind the transcription factor ODN decoy approach involves flooding the cell with competing synthetic, transcription factor-specific consensus sequences. These synthetic decoys "compete" for binding of the transcription factor with consensus sequences in target genes. If delivered into the cell in sufficient concentrations these "decoys" thus have the potential to attenuate the binding of the transcription factor to promoter regions of target genes and thus attenuate the function of the transcription factor to regulate the expression of its target gene(s). Transfected at high concentrations these decoys have been reported in the literature to completely block transcription factor function. Clearly they represent powerful research tools for studying gene regulation both in vitro and also more recently in vivo (for Reviews see Moshita et al., 1998, Mann and Dzau, 2000). Fig. 2. Advantages and disadvantages of the ODN decoy approach for studying cellular gene expression. Advantages. # ODN decoys offer a means of specifically inhibiting transcription factor function in living cells. # Inexpensive compared to other more classical methods of investigating gene expression such as chloramphenicol acetyltransferase and luciferase constructs in promoter-reporter gene transfection experiments. # Allows for investigation of both endogenous and pathological gene regulation # Proven to be highly effective and selective within in vitro experiments. # Easy to use. Disadvantages. # An emerging technology that has not yet been fully characterized # Issues of decoy synthesis. High levels of purity and stability required. # Transfection issues. Which method is best. How to optimize transfection. # Issue of controls. What controls are needed. ODN Decoys available from Click here for a full listing. We have designed ODN decoys to over 45 common transcription factors. Our decoys are double-stranded synthetic phosphorothioate deoyxynucleotides which range in length from 20-28 base pairs. The transcription factor consensus sequence occurs within the middle of the decoy sequence and is flanked by carefully selected base-pairs that allow for "optimized" transcription factor binding. These ODN decoys are also available labeled so that you are able to optimize your specific transfection technique by imaging the passage of the decoy into the cell (for example by fluorescence microscopy). Our ODN decoys are purified by HPLC and assessed by gel electrophoresis to ensure that >99% of decoy supplied represents full length, double stranded, functional decoy. As a control, matching mutant decoys are available for each transcription factor. Mutant decoys have the same flanking sequences but contain a disrupted consensus sequence in comparison with the (wild type) ODN decoy. How are these decoys used?The majority of experiments to date have used transcription factor ODN decoys to examine gene regulation in cultured primary cells and cell lines. The most important variables involved in determining whether or not your ODN decoy performs its required function include (a) the combination of cell type/cell density and transfection reagent used (b) the time cells are incubated with ODN decoy and (c) the concentration of ODN decoy used. A. The combination of cell type/cell density and transfection reagent used. While some investigators have achieved success by simply adding naked ODN decoys directly into the cell culture media, the most common method of in vitro ODN decoy transfection is to mix the ODN decoy with a cationic lipid to form a liposome complex before adding the ODN decoy/liposome mixture directly to the media. To aid transfection therefore we recommend mixing of your ODN decoy with an effective liposome-based carrier substance. One transfection reagent we have had good success with in our laboratories is the OligofectAMINE reagent. This is a proprietary formulation available from Invitrogen that is designed to optimize transfection of phosphorothioate ODNs into eukaryotic cells. Stable complexes are formed between the lipid and the ODN permitting efficient delivery of the ODN into mammalian cells. This product represents an improvement over the Lipofectin reagent in respect to transfection of ODNs. Please follow the manufacturers guidelines for use of this product. Product sheets are available via their website. Other transfection reagents we have had previous success with include FuGene 6 from Roche Diagnostics and Superfect Transfection Reagent from Qiagen. Obviously certain cell types are more susceptible to transfection than others and certain liposome "carriers" perform better with certain cell types. Therefore an amount of trial and error may be required to optimize transfection under your specific conditions. It is therefore helpful to have a way of measuring the kinetics and efficiency of transfection of your ODN decoy. One way of doing this is to use either biotin or fluorescently labeled ODN decoys. After incubation of ODN decoys with your cells you can assess transfection efficiency by fluorescent microscopy or biotin detection. With successful transfection you should expect to see a strong nuclear signal with weaker but noticeable signal in the cytoplasm in 60-90% of your cells. We have noticed that the transfection efficiency of ODN decoys and indeed ODNs in general (for example antisense ODNs) is much more sensitive to cell density than that of plasmid DNA. Therefore we recommend that a standard seeding protocol be maintained from experiment to experiment and that cell density be varied, if required, to optimize transfection efficiency. B. The time cells are incubated with ODN decoy. The time of incubation of cells with ODN decoys is critical. While there is no standard time of incubation due to the many other variables that can affect the incubation time required (including but not limited to ODN decoy concentration, cell type and transfection reagent used) an incubation time of 8hrs (minimum) to 24-28 hrs (maximum without re-addition of ODN decoy) is suggested. Significant ODN decoy degradation has been reported to occur after incubation periods of longer than 24 hrs. Obviously frequent re-addition of ODN decoy could be used to provide continuous blockade of transcription factor functionality beyond 24 hrs if required. C. The concentration of ODN decoy used. Within the recent literature ODN decoy concentrations of up to 5mM appear to be well tolerated and highly effective in most cell types with little or no observable effect on cell viability. With the newer transfection reagents (such as OligofectAMINE) a final ODN decoy concentration within the range of 0.1-2µM will be sufficient to block transcription factor activity without inducing non-specific cellular toxicity. Controls. To confirm that the effects of the ODN decoy are due to a consensus sequence-specific inhibition of transcription factor functionality rather than a non-specific effect of the ODN decoy on cell viability or functioning we recommend using our matching mutant ODN decoys as controls in each experiment. Mutant decoys have the same flanking sequences but contain a disrupted consensus sequence that does not bind transcription factor. References Morishita, R., Higaki, J., Tomita N. and Ogihara T. (1998) Application of transcription factor "decoy" strategy as means of gene therapy and study of gene expression in cardiovascular disease. Circ Res 82, 1023-1028. Mann, M.J. and Dzau, V.J. (2000) Therapeutic applications of transcription factor decoy oligonucleotides. J. Clin. Invest. 106, 1071-1075. Products | Accounts | FAQ | Contact | Search | Home Terms of Use | Privacy Policy | Shopping Basket | Quotes | CheckoutCopyright © 2000-2007 Limited

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Q: What is decoy oligonucleotides?
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How do you locate genes on the chromosomes of humans?

Traditionally, radiolabeled oligonucleotides were used to locate genes on chromosomes. In this method, oligonucleotide sequences that are complimentary to a section of the gene being located are labelled with a radioactive isotope - P32. Following annealing, the gene location is detected through auto-radiography. These days, fluorescent labels are used. These labels are more versatile and easy to detect. They also overcome the risks of using radioactivity.

What is MLPA?

MLPA introductionMLPA (Multiplex Ligation-dependent Probe Amplification) is a multiplex PCR method detecting abnormal copy numbers of up to 50 different genomic DNA or RNA sequences, which is able to distinguish sequences differing in only one nucleotide (1). The MLPA technique is easy to use and can be performed in many laboratories, as it only requires a thermocycler and capillary electrophoresis equipment. Up to 96 samples can be handled simultaneously, with results being available within 24 hours. Although for most hereditary conditions, (partial) gene deletions or duplications account for less than 10 % of all disease-causing mutations, for many other disorders this is 10 to 30 % (2-8) or even higher still (9, 10). The inclusion of MLPA in clinical settings can therefore significantly increase the detection rate of many genetic disorders.Advantages of MLPAUsing MLPA for copy number detection offers many advantages over other techniques. First of all, methods which were primarily developed for detecting point mutations, such as sequencing and DHPLC, generally fail to detect copy numbers changes. Southern blot analysis, on the other hand, will reveal many aberrations but will not always detect small deletions and is not ideal as a routine technique. Although well-characterised deletions and amplifications can be detected by PCR, the exact breakpoint site of most deletions is unknown. Furthermore, when comparing MLPA to FISH, MLPA not only has the advantage of being a multiplex technique, but also one in which very small (50-70 NT) sequences are targeted, enabling MLPA to identify the frequent, single gene aberrations which are too small to be detected by FISH. Moreover, MLPA can be used on purified DNA. Finally, as compared to array CGH, MLPA is a low cost and technically uncomplicated method. Although MLPA is not suitable for genome-wide research screening, it is a good alternative to array-based techniques for many routine applications. The over 300 probe sets now commercially available are dedicated to applications ranging from the relatively common (Duchenne, DiGeorge syndrome, SMA) to the very rare (hereditary pancreatitis, Antithrombin deficiency, Birt-Hogg-Dube syndrome).MLPA reactionTypical for MLPA is that it is not target sequences that are amplified, but MLPA probes that hybridise to the target sequence. In contrast to a standard multiplex PCR, a single pair PCR primers is used for MLPA amplification. The resulting amplification products of a SALSA MLPA kits range between 130 and 480 NT in length and can be analysed by capillary electrophoresis. Comparing the peak pattern obtained to that of reference samples indicates which sequences show aberrant copy numbers.The MLPA reaction can be divided in five major steps: 1) DNA denaturation and hybridisation of MLPA probes; 2) ligation reaction; 3) PCR reaction; 4) separation of amplification products by electrophoresis; and 5) data analysis. During the first step, the DNA is denatured and incubated overnight with a mixture of MLPA probes. MLPA probes consist of two separate oligonucleotides, each containing one of the PCR primer sequences. The two probe oligonucleotides hybridise to immediately adjacent target sequences. Only when the two probe oligonucleotides are both hybridised to their adjacent targets can they be ligated during the ligation reaction. Because only ligated probes will be exponentially amplified during the subsequent PCR reaction, the number of probe ligation products is a measure for the number of target sequences in the sample. The amplification products are separated using capillary electrophoresis. Probe oligonucleotides that are not ligated only contain one primer sequence. As a consequence, they cannot be amplified exponentially and will not generate a signal. The removal of unbound probes is therefore unnecessary in MLPA and makes the MLPA method easy to perform.

Why Viral infection cannot be cured easily?

Viruses are unique in that they aren't alive; that is, they don't feed and reproduce in a manner characteristic to other living things. Rather, a virus is a microscopic set of gene code that makes copies of itself inside bodily cells, causing them to function improperly. To go about ridding a virus, therefore, you cannot attack the virus by killing it, as you do with living (bacterial or parasitic) infections. The life cycle of a virus is as follows: * Attachment to a host cell. * Release of viral genes and possibly enzymes into the host cell. * Replication of viral components using host-cell machinery. * Assembly of viral components into complete viral particles. * Release of viral particles to infect new host cells. Viruses sneak into the body, sabotage cells into making copies of them. Viruses are made of the same kinds of materials all cells are: Fat, Protein, DNA. Any means of destroying a virus would have to be very calculated (as it could harm the host if not careful). Antiviral drugs inhibit the development of viruses. One method antiviral drugs work is to prevent viruses from attaching to cells. A recent type of antiviral is called "antisense" molecule, it is more or less a decoy that virus cells attach to instead of host cells.

What are the steps in the process of transcription?

Transcription is a hugely complicated process and can be described in several levels of detail. Below is probably too much detail for most people, so here is a shorter summary first:RNA polymerase II from yeast (eukaryotic) is a 12 subunit polymerase that assembles with a number of transcription factors (transcription factors TFII D, A, B, F, E, H and J, in that order) to form the preinitiation complex for the production of a complementary mRNA copy of the DNA antisense strand. Other polymerases have other functions: RNAP I produces rRNA and RNAP III produces tRNA. Initiation occurs at specific sites on the DNA where there are promoter regions such as the TATA box (which is recognised by TFIID's TATA binding protein) and one or more other up or downstream promoter elements. TFIID can have associated factors for recognition of promoter regions other than the TATA box. Elongation occurs when the polymerase matches base pairs of DNA to free ribonucleotides and catalyses the formation of a phosphodiester bond between it and the end of the RNA chain (if there is one) being synthesises. It occurs in the 3'->5' direction. An important obstacle to overcome is "abortive transcripts" where short (up to nine nucleotides long) RNA strands are forced to dissociate due to sterics clashes between the "B finger" of polymerase and the transcribed DNA-RNA hybrid, as well as the difficulty with which very short oligonucleotides (the new RNA strand) can be held in position in the polymerase. The B finger fills the space where the hybrid duplex passes, which is also a binding site for TFIIB, so that past 5 nucleotides long the transcript duplex must compete for space. An abortive transcript is formed when TFIIB remains bound, whereas release from the initiation site will occur if TFIIB is displaced. Abortive transcript frequency is reduced by burying the sites of the first few nucleotides deep in the protein, with various short range interactions, but also one long range electrostatic interaction with an arginine residue. Mutation of that residue resulted in an increase in abortive transcript frequency. If TFIIB is successfully displaced (so no more abortive transcripts are formed), elongation occurs until termination at a site many base pairs later than the end of the gene. Regulation can be achieved through a number of mechanisms, making use of regulators, enhancers, repressors, alteration of nucleosome structure and different transcription factors. Eukaryotes are capable of post translational regulation by regulating the processes of polyadenylation, splicing and capping.More detailed response: RNA synthesis occurs in a process known as transcription. It is a vital process within cells, as it is required for protein synthesis and ribosome synthesis, as well as in the creation of smaller RNA strands for various functions, such as snRNA for splicing. Genes are organized in operons on prokaryotes, in a highly logical way, so that all related genes are transcribed together. In contrast, eukaryotic gene organization has related genes frequently separated physically (even on different chromosomes), and there are sections of DNA within the gene coding region not used in translation (suggesting the existence of clipping methods - introns and exons in splicing). There is only one RNA polymerase involved in RNA synthesis in E. coli (prokaryotic) cells. On the other hand, there are three different RNA polymerases (I, II and III) used in eukaryotic cells for the same process. For the production of tRNA, RNase P is also required to cleave off the extra nucleotides on the tRNA precursor. Again there are differences in this for eukaryotic and prokaryotic cells, since eukaryotic RNase P requires many more associated proteins. The part of the RNase P molecule that is not the associated proteins (eukaryotes), or the single polypeptide chain for prokaryotes, is actually an RNA chain itself. Other RNases are also used to cleave transcribed RNA strands, though this is much more common in eukaryotes, since prokaryotes begin translation while transcription is still occurring (since it takes place in the cytoplasm rather than the nucleus). For example, RNase III in prokaryotes, which can cleave double stranded regions of RNA (many are known to process precursors to rRNA). In eukaryotes, another protein may be used in transcription - the TATA binding protein - for initiation of transcription, as it helps to melt the TATA region of DNA (sequence TATA followed by a number of A bases), and bends the DNA. It is required by all three RNA polymerases in eukaryotes, but primarily for RNA polymerase II. In a similar way, the sigma factor in prokaryotes is a protein required as a subunit of RNA polymerase (for a holoenzyme) for initiation of transcription, though the sigma factor dissociates from the enzyme soon after the elongation stage of transcription begins. The sigma factors, and its alternative versions, are important in regulating gene expression under different conditions, such as RpoH, which is used in 'heat shock' to transcribe genes required to combat the cause of the heat shock. RNA polymerase, in prokaryotes, requires ATP, CTP, GTP and UTP (short term life of RNA so thymine not important) to work, as well as the Mg2+ divalent cation and the DNA template (with a promoter region and sigma factor). The E. coli polymerase consists of two alpha subunits (40 kDal), a beta subunit (155 kDal), a beta prime subunit (160 kDal) and the sigma factor (often 85 kDal). In most cases of transcription in E. coli, the sigma factor will bind to the -10 and -35 sequences of DNA (relative to the start of the gene being transcribed). The sigma factor (and holoenzyme) is able to find these sites as the entire holoenzyme slides along the DNA molecule, making and breaking weak hydrogen bonds with exposed bases until the promoter sequence is found (which is often TTGACA on the -35 sequence and TATAAT on the -10 sequence). RNA polymerase then unwinds a section of DNA to allow for transcription to begin (about 12-17 base pairs) in a transcription bubble. At either end of the transcription bubble, positive (3' end) or negative (5' end) supercoiling occurs to stabilize the DNA strand after helix twists are removed by RNA polymerase. No primers are required for RNA polymerase to create RNA chains from the DNA template, and like DNA synthesis, transcription occurs in the 5' to 3' direction (as shown experimentally by labeling). RNA polymerase will then synthesise RNA by allowing complementary base pairs to pair with the template strand of the DNA molecule and catalyzing the hydrolysis of the 3' OH group and the phosphate groups of the next ribonucleotide to be added. The newly synthesised RNA forms a hybrid with DNA for eight base pairs (almost a complete turn), before structures in the RNA polymerase enzymes force it to separate. As RNA polymerase moves along the DNA chain, transcription cofactor proteins are able to assist with proof reading abilities of the enzyme. It can pause and backtrack when errors occur, and can also initiate DNA repair pathways if base pairs are mismatched in the DNA (the Mfd cofactor). A termination sequence causes RNA polymerase to stop transcribing DNA into RNA. In intrinsic termination, a G and C rich sequence followed by a number of U residues causes a hairpin bend and a double strand to form, which makes the RNA polymerase pause. The U-A bonds binding the RNA strand to the DNA strand are very weak, and so this can then break off easily from the template and then enzyme. The transcription bubble can then close when the DNA reforms its double helix. Alternatively, rho dependent termination involves another protein. When some RNA molecules were produced in vitro, the RNA strands were longer than those produced in vivo, since the rho protein was not added. The rho protein destabilizes the interaction (and thus unwinds, as helicase does) between the template and the RNA strand, causing it to separate as with rho-independent (intrinsic) termination. Though the eukaryotic method of transcription is similar to this, there are some key differences. One such difference is the location of transcription, since prokaryotic transcription takes place in the cytoplasm (and translation frequently occurs instantly on the transcript), whereas eukaryotic transcription takes place in the nucleus, before the products are edited and transported to the cytoplasm. Perhaps most importantly, eukaryotes use three different RNA polymerases. RNA polymerase I synthesises the precursor to rRNA, polymerase II synthesises precursors to mRNA, and III synthesises tRNA precursors (and small RNA sequences, including the 5S subunit of ribosomes). RNA Pol I is made of twelve subunits, and is the simplest and fastest acting transcription enzyme, since little regulation is required of ribosome synthesis, due to its constant demand in high concentration. A ribosomal initiator element, and an upstream promoter element, bind proteins that help to recruit RNA polymerase I. RNA polymerase III have promoters within the transcribed sequence (downstream). RNA Pol II also contains twelve subunits, but this time requires transcription factors to bind to it to promote transcription of genes, since it is used to create a vast number of different mRNA strands, so strict regulation is required. Upstream of the transcription sequence can be a TATA box and Inr (initiator element), or an Inr and DPE (downstream promoter element). In both cases, there will be enhancer elements many bases upstream from the start site of transcription, to enhance the transcription of a region (enhancers are only active in the cells the genes are required in). General transcription factors are required to activate RNA polymerase II, which are DNA binding proteins. The most common promoter region in eukaryotic DNA is the TATA box aforementioned. Transcription factors bind to these promoter regions, such as TFII. Initially, TFIID binds to the TATA box of the DNA strand, if present, since the TBP is present in the TFIID complex. The shape of TBP causes large changes in the shape of the DNA molecule, unwinding it greatly. This allows TFIIA and then TFIIB (with TFIID) to bind to the promoter region, followed by TFIIF (similar to sigma factor in prokaryotes) with RNA polymerase II, then TFIIE, which enables the polymerase to move along the DNA easily, and TFIIH, which contains a helicase to unwind the DNA and an enzyme region to phosphorylate (initiating elongation) a region of the polymerase II enzyme known as the C terminal domain (which is unique to this form of polymerase II). This forms a complex known as the basal transcription apparatus. Additional transcription factors are used at other promoter sites to selectively stimulate gene transcription and increase the rate of mRNA synthesis. Transcription factors binding to regulatory sequences can also have negative effects, inhibiting transcription. Whereas in E. coli, gene repressor proteins directly compete with RNA polymerase, eukaryotic repressor proteins compete indirectly, though the mechanism is not well understood. It is possible that the activator is blocked, or the activator binds to the respressor, or the repressor affects the TATA sequence so that the activator can't. Prokaryotic regulation makes use of the sigma factor, as discussed previously, and operons. These operons regulate gene expression due to repressor proteins, which bind to the promoter sequence and prevent the sigma factor from binding to it. In certain conditions, this process will be reversed to allow transcription to take place. An example of this is the lac operon, in which absence of lactose allows the repressor to bind, whereas lactose can bind to the repressor and remove it from the promoter region when present, so that it can be metabolized by E. coli. Sometimes a second protein, acting as a sensor, is required in regulation in this way, which then activates the repressor protein or activator protein. In E. coli, and many prokaryotes, mRNA molecules are generally not altered after transcription. However, tRNA and rRNA is (in fact, four of these all come from one single RNA transcript). RNase P in E. coli cleaves tRNA molecules after transcription. Enzymes may modify the RNA produced, by addition of nucleotides or groups (such as methylation). In contrast, (almost) all eukaryotic RNA transcripts are processed in some way. Since RNA polymerase only produces one RNA strand, this must be processed to produce the subunits of ribosomal RNA. First, nucleotides are modified by snoRNPs, then cleaved into the three separate RNA strands required. This generally takes place in the nucleolus. RNA polymerase III produces tRNA, which are processed as in prokaryotes. RNase P cleaves a section off, then a CCA is added. However, unlike prokaryotes, tRNA is often base modified, or spliced (endonuclease and ligase) to remove introns. Once again RNA polymerase II is the complicated one. In eukaryotes, the mRNA produced at first may contain coding regions for a huge number of different genes. By splicing this 'pre-mRNA' with spliceosomes to remove introns, the desired product can be formed. Spliceosomes are snRNPs, and splice at splice sites which are recognized by a specific base sequence. Splicing is catalysed by small RNA sequences present in the nucleus. The upstream exon is first cleaved from the upstream intron, leaving a 3' OH terminus which can attack the phosphodiester bond between the exon and intron downstream of this. The mRNA in eukaryotes may also be capped, by the addition of a 5' to 5' phosphodiester bond and methylated to help stabilize the mRNA molecules. The 3' end is also modified by cleaving the pre-mRNA at an AAUAAA sequence by endonuclease, then addition of poly(A) occurs by polymerase to add a poly(A) tail to the mRNA, as this enhances translation. RNA editing can alter the base sequence of the RNA after transcription, such as apoliprotein B's RNA, which is changed from a cytidine residue to uridine, to change the codon from Gln to stop, leading to a shorter polypeptide for a different function.Mitochondrial and chloroplast DNA are transcribed in ways specific to these organelles, as they contain their own polymerases. Mitochondrial RNA polymerase resembles bacteriophage RNA polymerase, and the sigma factor of eukaryotic polymerase. Chloroplast RNA polymerase is homologous to prokaryotic polymerase without the sigma factor.Inhibition of transcription is an important method of destroying prokaryotic pathogens. Highly specific inhibitors can be used as antibiotics, such as rifampicin and actinomycin. Rifampicin blocks the path the RNA/DNA hybrid passes through in transcription, preventing initiation. Many viruses rely on the opposite to transcription - reverse transcription - to replicate, by invading cells and inserting their DNA from reverse transcription of the RNA they carry. This DNA is then transcribed by the cell to produce more viruses. Mutation of any part of transcription, including the post transcriptional processing, could lead to serious diseases in affected cells.It is possible to go into yet more detail, by examining the structure of the polymerase enzyme and its complex with transcription factors, DNA and RNA. For this level of detail, look up the original publications of the structures on a journal database.

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