Reverse transcriptase

Reverse transcriptase is an RNA-dependent polymerase that was discovered in 1970 in many retroviruses such as the human immunodeficiency virus (HIV) and the avian myeloblastosis virus (AMV).

Reverse transcriptase catalyzes the conversion of RNA molecules as a template into a DNA double helix and is a very useful tool for research in the field of molecular biology. Reverse transcriptases are often used to produce complementary DNA libraries (cDNA) from various expressed mRNAs and, in combination with the polymerase chain reaction, known as RT-PCR, are also used to quantify the level of mRNA synthesis. The reverse transcriptase contains three enzymatic activities: (1) RNA-dependent DNA polymerase, (2) RNase H and (3) DNA-dependent DNA polymerase. First, RNA-dependent DNA polymerase synthesizes a DNA strand that is complementary to the RNA template. Subsequently, RNase H removes the RNA strand from the RNA-DNA hybrid double helix. The DNA-dependent DNA polymerase then completes the double-stranded DNA synthesis.

In contrast to other DNA polymerases, the reverse transcriptase (RT) does not have a correction function and therefore exhibits high error rates during DNA synthesis, up to one error in 2000 base integrations. The high error rates of viral reverse transcriptases provide a selective advantage for their survival in the host system.

Reverse transcriptase and retroviruses: Retroviruses consist of an RNA genome contained in a protein envelope, which in turn is enclosed in a lipid envelope. The retrovirus genome usually consists of three genes: the group-specific antigen gene (gag), the polymerase gene (pol) and the envelope gene (env). The pol gene encodes the three enzymes - protease, reverse transcriptase and integrase - that catalyze the steps of retroviral infection. Once a retrovirus is inside a host cell (a process mediated by a protease), it hijacks the host's genetic transcription machinery to construct a DNA provirus. This process, the conversion of retroviral RNA into proviral DNA, is catalyzed by reverse transcriptase and is necessary for the insertion of the proviral DNA into the host DNA - a step that is initiated by the integrase enzyme.

1. Retroviral DNA synthesis is absolutely dependent on the two distinct enzymatic activities of reverse transcriptase (RT): a DNA polymerase that can use either RNA or DNA as a template, and a nuclease called ribonuclease H (RNase H) that is specific for the RNA strand of RNA:DNA duplexes. Although a role of other proteins cannot be excluded and it is likely that certain viral proteins (e.g. nucleocapsid, NC) increase the efficiency of reverse transcription, all enzymatic functions required to complete the series of steps in the generation of a retroviral DNA can be attributed to either DNA polymerase or RNase H of RT. It is assumed that the process of retroviral DNA synthesis follows the scheme shown in Figure 2:
2. Minus-strand DNA synthesis is initiated with the 3' end of a partially unfolded transfer RNA annealed to the primer binding site (PBS) in the genomic RNA as a primer. Minus-strand DNA synthesis proceeds until the 5' end of the genomic RNA is reached, resulting in a discrete-length DNA intermediate called minus-strand strong-stop DNA (-sssDNA). Since the binding site for the tRNA primer is located near the 5' end of the viral RNA, the -sssDNA is relatively short, on the order of 100-150 bases
3. After RNase H-mediated degradation of the RNA strand of the RNA:-sssDNA duplex, the first strand transfer causes the -sssDNA to be annealed to the 3' end of a viral genomic RNA. This transfer is mediated by identical sequences, known as repeated (R) sequences, located at the 5' and 3' ends of the RNA genome. The 3' end of the -sssDNA has been copied from the R sequences at the 5' end of the viral genome and therefore contains sequences that are complementary to R. After the RNA template has been removed, the -sssDNA can anneal to the R sequences at the 3' end of the RNA genome. The annealing reaction appears to be facilitated by the NC.
4. Once the -sssDNA has been transferred to the 3'-R segment of the viral RNA, minus-strand DNA synthesis resumes, accompanied by digestion of the template strand by RNase H. However, this digestion is not complete. However, this degradation is not complete.
5. The RNA genome contains a short polypurine tract (PPT), which is relatively resistant to degradation by RNase H. A defined RNA segment derived from the PPT primers serves as a starting point for plus-strand DNA synthesis. Plus-strand synthesis is halted after a portion of the primer tRNA is reverse transcribed, resulting in DNA termed plus-strand strong-stop DNA (+sssDNA). Although all strains of retroviruses generate a defined plus-strand primer from the PPT, some viruses generate additional plus-strand primers from the RNA genome.
6. RNase H removes the primer tRNA and exposes sequences in the +sssDNA that are complementary to sequences at or near the 3' end of the plus-strand DNA.
7. Annealing of the complementary PBS segments in the +sssDNA and the minus strand DNA represents the second strand transfer.
8. The plus and minus strand syntheses are then completed, with the plus and minus strands of DNA each serving as a template for the other strand.

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