reverse transcriptase: Definition and Much More from Answers.com

Any of the deoxyribonucleic acid (DNA) polymerases present in particles of retroviruses which are able to carry out DNA synthesis using an RNA template. This reaction is called reverse transcription since it is the opposite of the usual transcription reaction, which involves RNA synthesis using a DNA template. See also Retrovirus.

The transfer of genetic information from RNA to DNA in retrovirus replication was proposed in 1964 by H. M. Temin in the DNA provirus hypothesis for the replication of Rous sarcoma virus, an avian retrovirus which causes tumors in chickens and transformation of cells in culture, and reverse transcriptase has since been purified from virions of many retroviruses. The avian, murine, and human retrovirus DNA polymerases have been extensively studied.

Studies indicate that reverse transcriptase is widely distributed in living organisms and that all reverse transcriptases are evolutionarily related. For example, the organization of the nucleotide sequence of integrated retroviral DNA has a remarkable resemblance to the structure of bacterial transposable elements, in particular, transposons.

Reverse transcriptase genes are present in the eukaryotic organisms in retrotransposons and in retroposons or long interspersed (LINE) elements. Both of these types of elements can transpose in cells. See also Deoxyribonucleic acid (DNA); Ribonucleic acid (RNA); Transposons.

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 reverse flow 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.

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 reverse transcriptase 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.

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.

Bibliography

Kazazian, Haig H., Jr. "L1 Retrotransposons Shape the Mammalian Genome." Science 289, no. 5482 (2000): 1152-1153.

Varmus, H. "Reverse Transcription." Scientific American 257, no. 3 (1987): 56-59.

—Alice Telesnitsky

Reverse Transcriptase
3D model of HIV reverse transcriptase
Other names:	Deoxynucleoside-triphosphate: DNA deoxynucleotidyltransferase (RNA-directed) RNA-directed DNA polymerase DNA nucleotidyltransferase (RNA-directed) Revertase
Database Links
EC number:	2.7.7.49

In biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA. Normal transcription involves the synthesis of RNA from DNA, hence reverse transcription is the reverse of this.

Reverse transcriptase was discovered by Howard Temin at the University of Wisconsin-Madison, and independently by David Baltimore in 1970. The two shared the 1975 Nobel Prize in Physiology or Medicine with Renato Dulbecco for their discovery.

Commonly used examples of reverse transcriptases include:

• HIV-1 reverse transcriptase from the human immunodeficiency virus type 1 (PDB 1HMV)
• M-MLV reverse transcriptase from the Moloney murine leukemia virus
• AMV reverse transcriptase from the avian myeloblastosis virus
• Telomerase reverse transcriptase that maintains the telomeres of eukaryotic chromosomes

Function

Viruses

The enzyme is encoded and used by reverse-transcribing viruses, which use the enzyme during the process of replication. Reverse-transcribing RNA viruses, such as retroviruses, use the enzyme to reverse-transcribe their RNA genomes into DNA, which is then integrated into the host genome and replicated along with it. Reverse-transcribing DNA viruses, such as the hepadnaviruses, transcribe their genomes into an RNA intermediate and then, using reverse transcriptase, back into DNA.

Eukaryotes

Self-replicating stretches of eukaryotic genomes known as retrotransposons utilise reverse transcriptase to move from one position in the genome to another via a RNA intermediate. They are found abundantly in the genomes of plants and animals. Telomerase is another reverse transcriptase found in many eukaryotes, including humans, which carries its own RNA template; this RNA is used as a template for DNA replication^[1].

Prokaryotes

Reverse transcriptases are also found in bacterial retrons, distinct sequences which code for reverse transcriptase, and are used in the synthesis of msDNA.

Structure

Reverse transcriptase enzymes include an RNA-dependent DNA polymerase and a DNA-dependent DNA polymerase, which work together to perform transcription. In addition to the transcription function, retroviral reverse transcriptases have a domain belonging to the RNase H family which is vital to their replication.

Replication fidelity

Reverse transcriptase has a high error rate when transcribing RNA into DNA as unlike DNA polymerases it has no proofreading ability. This high error rate allows mutations to accumulate at an accelerated rate relative to proofread forms of replication. The commercially available reverse transcriptases produced by Promega are quoted by their manuals as having error rates in the range of 1 in 17,000 bases for AMV and 1 in 30,000 bases for M-MLV^[2]

Applications

The molecular structure of zidovudine (Retrovir®), a drug used to inhibit HIV reverse transcriptase

Antiviral drugs

As HIV uses reverse transcriptase to copy its genetic material and generate new viruses (part of a retrovirus proliferation circle), specific drugs have been designed to disrupt the process and thereby suppress its growth. Collectively, these drugs are known as reverse transcriptase inhibitors and include the nucleoside and nucleotide analogues zidovudine (Retrovir®), lamivudine (Epivir®) and tenofovir (Viread®), as well as non-nucleoside inhibitors, such as nevirapine (Viramune®).

Molecular biology

Reverse transcriptase is commonly used in research to apply the polymerase chain reaction technique to RNA in a technique called reverse transcription polymerase chain reaction (RT-PCR). The classical PCR technique can only be applied to DNA strands, but with the help of reverse transcriptase, RNA can be transcribed into DNA, thus making PCR analysis of RNA molecules possible. Reverse transcriptase is also used to create cDNA libraries from mRNA. The commercial availability of reverse transcriptase greatly improved knowledge in the area of molecular biology as, along with other enzymes, it allowed scientists to clone, sequence and characterise DNA.

See also

• cDNA library
• DNA polymerase
• msDNA
• Reverse transcribing virus
• RNA polymerase
• Telomerase
• Retrotransposon marker

External links

• animation of reverse transcriptase action and three reverse transcriptase inhibitors
• Molecule of the month (September 2002) at the Protein Data Bank
• BRENDA database entry - highly detailed information from a protein database
• MeSH RNA+Transcriptase
• EC 2.7.7.49

References

1. ^ Lodish, et al, Molecular Cell Biology (2004), 5th edn, W. H. Freeman and Company, New York, ISBN 0-7167-4366-3
2. ^ Promega kit instruction manual (1999)

Phosphotransferases/kinases (EC 2.7)
2.7.1 - OH acceptor	Hexo- - Gluco- - Fructo- (Hepatic fructo-) - Galacto- - Phosphofructo- (1, 2) - Thymidine - [[NAD+ kinase|NAD+]] - Glycerol - Pantothenate - Mevalonate - Pyruvate - Deoxycytidine - PFP - Diacylglycerol - Bruton's tyrosine - Phosphoinositide 3 (Class I PI 3, Class II PI 3) - Sphingosine
2.7.2 - COOH acceptor	Phosphoglycerate - Aspartate
2.7.3 - N acceptor	Creatine
2.7.4 - PO4 acceptor	Phosphomevalonate - Adenylate - Nucleoside-diphosphate
2.7.6 - P2O7	Ribose-phosphate diphosphokinase - Thiamine pyrophosphokinase
2.7.7 - nucleotidyl-	Integrase - PNPase - Polymerase - RNase PH - UDP-glucose pyrophosphorylase - Galactose-1-phosphate uridylyltransferase -Terminal deoxynucleotidyl transferase - RNA replicase - Reverse transcriptase (Telomerase) - Transposase
2.7.8 - other phos.	N-acetylglucosamine-1-phosphate transferase
2.7.10-11 - protein	Tyrosine - Serine/threonine-specific

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