Reverse Transcriptase

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Reverse transcriptase Summary

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Reverse Transcriptase

Reverse transcriptase is the replication enzyme of retroviruses. Because it polymerizes DNA precursors, reverse transcriptase is a DNA polymerase. However, whereas cellular DNA polymerases use DNA as a template for making new DNAs, reverse transcriptase uses the single-stranded RNA in retroviruses as the template for synthesizing viral DNA. This unusual process of making DNA from RNA is called "reverse transcription" because it reverses the flow of genetic information (from DNA to RNA, rather than from RNA to DNA found in transcription). Because reverse transcriptase is essential for retroviruses such as HIV-1 (the virus that causes AIDS), it is the target of many antiretroviral therapeutics. Reverse transcriptase is also a molecular tool used in the cloning of genes and the analysis of gene expression.

Discovery

Retroviruses were originally known as RNA tumor viruses because they have RNA, not DNA, genomes, and because they were the first viruses recognized to cause certain cancers in animals. At the middle of the twentieth century, Howard Temin was interested in understanding how RNA tumor viruses cause cancer. One finding that interested him was the genetic-like stability of the uncontrolled cell growth caused by these viruses. It was known then that certain bacterial viruses, called phages, could integrate their DNA into their hosts' chromosomes and persist as stable genetic elements known as prophages. By analogy, Temin proposed the provirus hypothesis, which suggests that RNA tumor viruses can cause permanent alterations to cells by integrating into host chromosomes. In order for this to occur, Temin suggested that virion RNAs were first converted into DNAs, which could then become integrated.

The chemistry of using RNA as a template for DNA seemed possible. However, reverse transcription was at odds with the then-popular central dogma of molecular biology, advanced by Francis Crick, which maintained that genetic information flowed unidirectionally from DNA to RNA to protein. RNA tumor viruses were RNA viruses, so it was assumed that their replication involved RNA polymerases, as had been demonstrated for other RNA viruses, and not a DNA polymerase. Because his proposal of a reverseflow of genetic information from RNA to DNA seemed heretical, and because the experimental techniques needed to test this idea were not yet developed, Temin and his hypothesis were rebuffed for many years.

Reverse transcriptase uses a single-stranded RNA template to create a double-stranded DNA.

The biochemical proof for reverse transcription finally arrived in 1970 when two separate research teams, one led by Temin and the other by David Baltimore, simultaneously discovered the elusive RNA-copying DNA polymerase in purified virions. In 1975 Temin and Baltimore shared the Nobel Prize in physiology or medicine for their discovery of reverse transcriptase.

Laboratory Uses of Reverse Transcriptase

Reverse transcriptase went on to play a critical role in the molecular revolution of the late 1970s and 1980s, especially in the fields of gene discovery and biotechnology. Genes can often be discovered most easily by isolating and analyzing the messenger RNA (mRNA) production in a cell. Reverse transcriptase allowed the synthesis of cDNA, or complementary copies of messenger RNAs. The cDNA can then be expressed in a model organism such as Escherichia coli, and the protein it codes for can then be made in abundance. The cloning of cDNA was instrumental to gene discovery in the later part of the twentieth century. Using cDNA copies of genes is necessary when bacteria are used to produce human protein-based pharmaceuticals. This is because bacteria lack the machinery necessary to recognize unspliced genes, but bacteria can use cDNAs to direct the synthesis of human or other higher organism proteins.

Even though the human genome sequence was reported in 2001, copying RNAs with reverse transcriptase remains important. One reason for this is that some human diseases result from mutations in genes whose products act to adjust the sequences of RNAs after transcription but before protein synthesis. Thus, even though prototype human sequences are available, it appears likely that molecular diagnostics will include screening cDNA copies of individual people's RNAs. Other uses of cDNA include generating probes to screen microarrays to assess variation in gene expression and regulation.

Reverse Transcriptase and Aids

Soon after AIDS was recognized in the early 1980s, Luc Montagneer of France and, subsequently, the American Robert Gallo determined that the causative agent was a retrovirus. Like other retroviruses, HIV-1 contains reverse transcriptase and must generate DNA. Differences between reversetranscriptase and cellular DNA polymerases in the sorts of DNA precursors (nucleosides) that they can utilize have been exploited to develop drugs that are selectively toxic to HIV-1.

AZT becomes incorporated into the DNA chain formed by reverse transcriptase, but the N3 groups block further replication.

AZT becomes incorporated into the DNA chain formed by reverse transcriptase, but the N₃ groups block further replication.

Azidothymidine (AZT) is an example of a nucleoside analog DNA precursor that can serve as a reverse transcriptase "suicide inhibitor," because AZT incorporation into viral DNA prevents later steps in viral replication. However, the effectiveness of these sorts of drugs is limited by several factors. AZT is occasionally incorporated into cellular DNA, which contributes to the toxicity some patients experience when treated with reverse transcriptase inhibitors. Additionally, reverse transcriptase inhibitor resistance often develops during antiretroviral therapy. This resistance results from reverse transcriptase's high error rate, which generates a remarkable amount of genetic variation within HIV populations. If some viral genetic variants are less sensitive to antivirals than other variants, the resistant mutants will replicate during antiviral therapy. Despite these complications, reverse transcriptase inhibitors remain important components of the combined antiviral regimen that has dramatically lengthened the lives of many HIV-infected patients since the mid-1990s.

Reverse Transcription and the Human Genome

When reverse transcriptase was first described, it was believed to be a peculiarity of retroviruses. However, researchers now know that reverse transcription also occurs during the replication of the DNA virus hepatitis B, and that RNA-copying DNA polymerases function within human cells. One of these host reverse transcriptases is telomerase, an enzyme that helps maintain chromosome ends.

Other human reverse transcriptases are parts of endogenous retroviruses and retroelements, such as those that encoded the majority of the repetitive "junk" DNA in human chromosomes. Many of these retroelements integrated their DNAs into our chromosomes so long ago that they predate human speciation. Because of this, molecular phylogeneticists can use sites of retroelement insertions to determine the lineages and ancestral relationships of species. Thus, while retroviruses, in the form of HIV-1, represent one of the newest diseases of humans, the prevalence of other retrovirus-like elements in our genomes demonstrates the long-standing relationship of humans with reverse transcribing elements.