Reverse transcriptase template specificity

Proteins never serve as templates for RNA. However, RNA chains can in rare circumstances reverse the flow of information from RNA to DNA. An example of this is the infecting RNA of retroviruses that serves as a template for the synthesis of a single-stranded complementary DNA (cDNA) chain that functions as a template for a complementary DNA chain. The resulting double-stranded DNA then acts as a template for the synthesis of the original viral RNA chain. The virus-specific enzyme, reverse transcriptase, catalyzes the synthesis of DNA on the RNA template. Little if any reverse transcriptase activity is present in normal cells, so that very little DNA is formed on RNA templates. 

Telomeres are nucleoprotein structures located at the ends of eucaryotic chromosomes. They protect chromosome ends from fusion and degradation and ensure complete replication of chromosomal DNA. In human somatic cells, telomeres have 1.000 to 3,000 repeats. They gradually shorten with every cell division. This shortening i.s thought to limit their proliferative capacity. Cancer cells, in contrast, can maintain their telomere length and thus become immortalized. They do this by reactivating telomerase, a specific reverse transcriptase with an endogenous RNA template. ... 

These studies demonstrated that (1) even a relatively minor chemical modification e.g., introduction of a 5-mercapto group into the cytosine base of 1 out of 100 nucleotide units) can convert a functional DNA or RNA template into a potent, competitive inhibitory analogue (antitemplate) which will bind to the template site of a polymerase either reversibly or irreversibly, and (2) even such antitemplates that are not made to be specific structural analogues of a natural template e.g., MPC) can differentiate between various polymerases. However, there are already some indications that antitemplates more closely related to the natural template of a given polymerase are more effective inhibitors of the latter, and it is expected that modified nucleic acids of viruses and tumors will show even much greater selectivities toward the corresponding reverse transcriptases in the presence of their endogenous templates. 

The 3 -ends are labeled, after specific restriction enzymes have produced a protruding 5 -end, by a fill-in reaction that allows only one label to be introduced (e.g., labeling with dATP with an overhang with 2 Ts should be avoided). Reverse transcriptase or Sequenase are the preferred enzymes for this fill-in although the Klenow fragment has been widely used. The 3 -> 5 exonuclease activity of Klenow, however, may remove the protruding template end. 

Once the gpl20 envelope protein of HIV binds to the CD4 antigen and chemokine receptor on the T cell, the viral envelope fuses with the host cell s plasma membrane. The two RNA strands are released into the cytoplasm. Reverse transcriptase, a heterodimer with several enzymatic activities, then catalyzes the synthesis of an ssDNA using the vRNA as a template. The heterodimer s RNase activity then degrades the vRNA. The same protein produces a double-stranded vDNA by forming a complementary strand of the ssDNA. Viral integrase integrates the vDNA into a host cell chromosome. The proviral DNA remains latent until the specific infected T cell is activated by an immune response. The proviral DNA can then direct the cell to synthesize viral components. Newly synthesized viruses bud from the infected cell. 

To analyze RNA by PCR an additional step has to be introduced to transcribe RNA into cDNA, which is a more stable single-strand DNA complementary to the targeted RNA. This can be achieved by the reverse transcription (RT) of the template RNA using a reverse transcriptase enzyme. RT and PCR can be carried out either sequentially in the same tube (one-step RT-PCR) or separately (two-step RT-PCR). One-step RT-PCR requires gene-specific primers for the reverse transcription reaction, whereas in two-step RT-PCR random primers can be used. 

RACE, also described as one-sided PCR or anchored PCR, is a powerfiil techniqne using messenger RNA as template to amplify the nucleic acid sequences at either the 3 (3 -RACE) or the 5 (5 -RACE) end of the mRNA [10], For studies of mRNA polyadenylation, 3 -RACE is the method of interest. In 3 -RACE, cDNA copies of a population of mRNAs are produced using oligo-dT as a primer for reverse transcriptase, and sequences that correspond to the 3 -ends (including the poly(A) sites) of specific... 

Enzymatic Synthesis. DNA and RNA are biosynthesized by polymerase enzymes. RNA is synthesized from a DNA strand with the assistance of an RNA polymerase enzyme. Inversely, DNA can be synthesized from an RNA strand with a reverse transcriptase (RT) enzyme. This mechanism is important because of retroviruses (such as HIV) in which RT makes up part of the viral replication cycle. (HIV-RT is an important target for anti-HIV therapy.) In any enzymatic synthesis of DNA or RNA, it is critical to the life of the organism that these enzymes synthesize their respective polynucleotides in a specific sequence of monomer units. The enzymes are able to accomplish this by utilizing a template strand to direct the synthesis. By reading the sequence of nucleobases on the template, the enzymes select the complementary monomer for incorporation into the propagating strand (Fig. 9). It was widely accepted that the polymerase enzymes accomplish this by matching the nucleotide to the template on the basis of complementary... 

Primarily, because it has both sensitivity and specificity, PCR technology has revolutionized many aspects of biomedical research. Several modifications of the basic methodology have provided additional powerful tools. For example, a trace amount of RNA can be quantitated by reverse transcriptase PCR (RTPCR), where a reverse transcriptase synthesizes the complementary DNA strand of the RNA, which then serves as the template for regular PCR. 

HEPT does not compete with pH-A/c]thymidine for phosphorylation by thymidine kinase derived from MT-4 cells. 2) The synthetic triphosphate of HEPT does not inhibit HIV-1 RT (reverse transcriptase) at concentrations much higher than that of its ECso, irrespective of the template-primer used [either poly(rA)-oligo(dT) or poly(c)-oligo(dG)]. Thus, HEPT can be considered a highly unique and specific lead for anti-HIV-1 agents. This prompted us to carry out synthesis of its analogues with a directed aim to improve its activity. 

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