Reverse Transcriptase

Reverse transcriptase catalyzes the formation of double-stranded deoxyri­bonucleic acid (DNA) from a single-stranded ribonucleic acid (RNA) genome. It is called “reverse” transcriptase because it reverses the usual direction of in­formation flow, from DNA to RNA. Reverse transcriptase is characteristic of retroviruses, including HIV (human immunodeficiency virus), the virus re­sponsible for AIDS (acquired immunodeficiency syndrome).

All retroviruses encode a polymerase enzyme in their pol gene that is both necessary and sufficient for the replication of their RNA genomes. The enzyme was first detected in virus particles in 1970 by Howard Temin and David Baltimore. These investigators permeablized the virus membrane with non-ionic detergents, which allowed them to introduce deoxynucleotides. They detected the synthesis of DNA that was dependent upon the RNA genome. This was a novel reaction because, at this time, polymerases were only known to use DNA for the synthesis of RNA in a process known as transcription. Thus this new process of using RNA as a template/primer for the synthesis of DNA was named reverse transcription. For their dis­covery, Temin and Baltimore shared the Nobel Prize.

Reverse transcriptase (RT) has several enzymatic activities, includ­ing an RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, RNase H (a ribonuclease that degrades RNA in RNA-DNA hy­brid structure), and the ability to unwind DNA-DNA and RNA-DNA duplexes. Each of these activities is required during the process of reverse transcription to convert the single-stranded RNA genome into a double DNA copy, which in turn becomes integrated into the host chromosome of the infected cell catalyzed by a second pol gene-encoded enzyme, called integrase.

Purified RT has become a very useful as a tool for modern molecular biology, especially coupled to polymerase chain reaction (PCR) techniques. It provides the ability to reverse transcribe any RNA with the appropriate complementary primer into a DNA copy that can then be amplified many times by a thermal stable DNA polymerase during the PCR reaction. The combination of the two techniques has allowed scientists to clone actively expressed genes in cells from their mRNAs (messenger RNAs).

 References

Coffin, John M., Stephen H. Hughes, and Harold E. Varmus, eds. Retroviruses. Plain- view, NY: Cold Spring Harbor Laboratory Press, 1997.

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