Reverse transcription
Overview
Reverse transcription is the process of making a double stranded DNA (deoxyribonucleic acid) molecule from a single stranded RNA (ribonucleic acid) template. It is called reverse transcription as it acts in the opposite or reverse direction to transcription. This idea was very unpopular at first as it contradicted the Central Dogma of Molecular Biology which states DNA is transcribed into RNA which is translated into proteins. However, in 1970 when the scientists Howard Temin and David Baltimore both independently discovered the enzyme responsible for reverse transcription, named reverse transcriptase, the possibility that genetic information could be passed on in this manner was finally accepted.This was the beginning of a resurgence of interest in Jean-Baptiste de Lamarck, a French scientist who in 1809 developed the first theory of evolution, pre-dating Charles Darwin by fifty years. Lamarck proposed that characteristics acquired during a lifetime could be communicated back to the germline (DNA) and passed on the succeeding generations. Reverse transcription was a critical precursor to wider acceptance of Lamarck's theory.
Reverse transcription in Class VI viruses
Class VI viruses ssRNA-RT, also called the retroviruses are RNA reverse transcribing viruses with a DNA intermediate. Their genomes consist of two molecules of positive sense single stranded RNA with a 5' cap and 3' polyadenylated tail. Examples of retroviruses include the Human Immunodeficiency Virus HIV and Human T-Lymphotropic virus HTLV. Once the viruses have entered the cell and been uncoated the genome is reverse transcribed into double stranded DNA which can be incorporated into the host cell and subsequently expressed. Reverse transcription by the enzyme reverse transcriptase occurs in a series of steps:
- A specific cellular tRNA acts as a primer and hybridizes to a complementary part of the virus genome called the primer binding site or PBS
- Complementary DNA then binds to the U5 (non-coding region) and R region (a direct repeat found at both ends of the RNA molecule) of the viral RNA
- A domain on the reverse transcriptase enzyme called RNAse H degrades the 5’ end of the RNA which removes the U5 and R region
- The primer then ‘jumps’ to the 3’ end of the viral genome and the newly synthesised DNA strands hybridizes to the complementary R region on the RNA
- The first strand of complementary DNA (cDNA) is extended and the majority of viral RNA is degraded by RNAse H
- Once the strand is completed, second strand synthesis is initiated from the viral RNA
- There is then another ‘jump’ where the PBS from the second strand hybridizes with the complementary PBS on the first strand
- Both strands are extended further and can be incorporated into the hosts genome by the enzyme integrase
References
- Alan J. Cann, Principles of Molecular Viriology (1997) Secong Edition, Academic Press, ISBN 0-12-158532-8