reverse transcriptase
| Dictionary: reverse transcriptase |
n.
A polymerase that catalyzes the formation of DNA on an RNA template, found in oncogenic viruses containing RNA, especially the retroviruses.
| 5min Related Video: reverse transcriptase |
| Britannica Concise Encyclopedia: reverse transcriptase |
For more information on reverse transcriptase, visit Britannica.com.
| Sci-Tech Encyclopedia: Reverse transcriptase |
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.
| Dental Dictionary: reverse transcriptase |
n
An enzyme within a retrovirus that converts its ribonucleic acid into deoxyribonucleic acid (DNA), which then penetrates the cell nucleus and joins the host’s DNA.
| Genetics Encyclopedia: 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 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
| Wikipedia: Reverse transcriptase |
| RNA-directed DNA polymerase | |||||||
|---|---|---|---|---|---|---|---|
 |
|||||||
| Crystallographic structure of HIV reverse transcriptase where the P51 subunit is colored green and the P66 subunit is colored cyan.[1] | |||||||
| Identifiers | |||||||
| EC number | 2.7.7.49 | ||||||
| CAS number | 9068-38-6 | ||||||
| IntEnz | IntEnz view | ||||||
| BRENDA | BRENDA entry | ||||||
| ExPASy | NiceZyme view | ||||||
| KEGG | KEGG entry | ||||||
| MetaCyc | metabolic pathway | ||||||
| PRIAM | profile | ||||||
| PDB | structures | ||||||
| Gene Ontology | AmiGO / EGO | ||||||
|
|||||||
In molecular biology and biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into double-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse transcribed into a single strand cDNA. 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 at MIT. The two shared the 1975 Nobel Prize in Physiology or Medicine with Renato Dulbecco for their discovery.
Well studied reverse transcriptases include:
- HIV-1 reverse transcriptase from 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
Contents |
Function in 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, can allow RNA to serve as a template in assembling, and making DNA strands. HIV infects humans with the use of this enzyme. Without reverse transcriptase, the viral genome would not be able to incorporate into the host cell, resulting in the failure of the ability to replicate. Unlike bacteria, retroviruses use preexisting host-encoded transfer RNAs as primers.
Process of reverse transcription
Reverse transcriptase creates single stranded DNA from a RNA template.
In virus species with reverse transcriptase lacking DNA-dependent DNA polymerase activity, creation of double-stranded DNA can possibly be done by host-encoded DNA polymerase δ, mistaking the viral DNA-RNA for a primer and synthesizing a double-stranded DNA by similar mechanism as in primer removal, where the newly synthesized DNA displaces the original RNA template.
The process of reverse transcription is extremely error-prone and it is during this step that mutations may occur. Such mutations may cause drug resistance.
Process 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 Human Immunodeficiency Virus (HIV) and Human T-Lymphotropic virus (HTLV). It takes place in the cytosol.[2] Creation of double stranded DNA 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
Creation of double-stranded DNA also involves strand transfer, in which there is a translocation of short DNA product from initial RNA dependent DNA synthesis to acceptor template regions at the other end of the genome, which are later reached and processed by the reverse transcriptase for its DNA-dependent DNA activity.[3]
Retroviral RNA is arranged in 5’ terminus to 3’ terminus. The site where the primer (molecular biology) is annealed to viral RNA is called the primer-binding site (PBS). The RNA 5’end to the PBS site is called U5, and the RNA 3’ end to the PBS is called the leader. The tRNA primer is unwound between 14 and 22 nucleotides and forms a base-paired duplex with the viral RNA at PBS. That PBS locates near the 5’ terminus of viral RNA is unusual because reverse transcriptase synthesize DNA from 3’ end of the primer in the 5’ to 3’ direction. Therefore, the primer and reverse transcriptase must be relocated to 3’ end of viral RNA. In order to accomplish this reposition, multiple steps and various enzymes including DNA polymerase, ribonuclease H(RNase H) and polynucleotide unwinding are needed.[4] }}</ref>
The HIV reverse transcriptase also has ribonuclease activity that degrades the viral RNA during the synthesis of cDNA, as well as DNA-dependent DNA polymerase activity that copies the sense cDNA strand into an antisense DNA to form a double-stranded viral DNA intermediate (vDNA).[5]
In eukaryotes
Self-replicating stretches of eukaryotic genomes known as retrotransposons utilize 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.[6]
In prokaryotes
Reverse transcriptases are also found in bacterial Retron msr RNAs, distinct sequences which code for reverse transcriptase, and are used in the synthesis of msDNA. In order to initiate synthesis of DNA, a primer is needed. In bacteria, the primer is synthesized during replication.[7]
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
There are three different replication systems during the life cycle of a retrovirus. First of all, the reverse transcriptase synthesize viral DNA from viral RNA, and then from newly made complementary DNA strand. The second replication process occurs when host cellular DNA polymerase replicates the integrated viral DNA. Lastly, RNA polymerase II transcribes the proviral DNA into RNA which will be packed into virions. Therefore, mutation can occur during one or all of these replication steps.[8]
Reverse transcriptase has a high error rate when transcribing RNA into DNA since, 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[9]
Applications
Antiviral drugs
For more details on this topic, see Reverse transcriptase inhibitor.
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 (trade name Retrovir), lamivudine (Epivir) and tenofovir (Viread), as well as non-nucleoside inhibitors, such as nevirapine (Viramune).
Molecular biology
For more details on this topic, see Reverse transcription polymerase chain reaction.
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 be applied only 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 used also 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.
History
The idea of reverse transcription was very unpopular at first as it contradicted the central dogma of molecular biology which states that DNA is transcribed into RNA which is then 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.
See also
- cDNA library
- DNA polymerase
- msDNA
- Reverse transcribing virus
- RNA polymerase
- Telomerase
- Retrotransposon marker
References
- ^ PDB 1HMV; Rodgers DW, Gamblin SJ, Harris BA, Ray S, Culp JS, Hellmig B, Woolf DJ, Debouck C, Harrison SC (February 1995). "The structure of unliganded reverse transcriptase from the human immunodeficiency virus type 1". Proc. Natl. Acad. Sci. U.S.A. 92 (4): 1222–6. PMID 7532306.
- ^ Bio-Medicine.org - Retrovirus Retrieved on 17 Feb, 2009
- ^ Telesnitsky, A., Goff, S.P. (1993). "Strong-stop strand transfer during reverse transcription". in Skalka, M. A., Goff, S.P. Reverse transcriptase (1st ed.). New York: Cold Spring Harbor. pp. 49. ISBN 0-87969-382-7.
- ^ Bernstein, A.; Weiss, Robin; Tooze, John (1985). "RNA tumor viruses". Molecular Biology of Tumor Viruses (2nd ed.). Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory.
- ^ Doc Kaiser's Microbiology Home Page > IV. VIRUSES > F. ANIMAL VIRUS LIFE CYCLES > 3. The Life Cycle of HIV Community College of Baltimore County. Updated: Jan., 2008
- ^ Monty Krieger; Matthew P Scott; Matsudaira, Paul T.; Lodish, Harvey F.; Darnell, James E.; Lawrence Zipursky; Kaiser, Chris; Arnold Berk (2004). Molecular cell biology (5th ed.). New York: W.H. Freeman and CO. ISBN 0-7167-4366-3.
- ^ Hurwitz J, Leis JP (January 1972). "RNA-dependent DNA polymerase activity of RNA tumor viruses. I. Directing influence of DNA in the reaction". J. Virol. 9 (1): 116–29. PMID 4333538.
- ^ Bbenek, K., Kunkel, A. T (1993). "The fidelity of retroviral reverse transcriptases". in Skalka, M. A., Goff, P. S.. Reverse transcriptase. New York: Cold Spring Harbor Laboratory Press. pp. p. 85. ISBN 0-87969-382-7.
- ^ Promega kit instruction manual (1999)
External links
- MeSH RNA+Transcriptase
- animation of reverse transcriptase action and three reverse transcriptase inhibitors
- Molecule of the month (September 2002) at the Protein Data Bank
- HIV Replication 3D Medical Animation. (Nov 2008). Video by Boehringer Ingelheim.
|
||||||||||||||||||||||||||||||||||||||||||
|
||||||||||||||||||||||||||||||||||||||||||||
This entry is from Wikipedia, the leading user-contributed encyclopedia. It may not have been reviewed by professional editors (see full disclaimer)
Learn More
 | Dna Microarrays |
| Dna Polymerases |
| Evolutionof Genes |
Help us answer these
 | Does RNA virus have Reverse transcriptases in them? |
| What enzyme reverse transcriptase uses? |
| What is the defference between Real-time PCR and reverse transcriptase PCR? |
Post a question - any question - to the WikiAnswers community:
Copyrights:
 |
 | Dictionary. The American Heritage® Dictionary of the English Language, Fourth Edition Copyright © 2007, 2000 by Houghton Mifflin Company. Updated in 2009. Published by Houghton Mifflin Company. All rights reserved. Read more |
 |
| Britannica Concise Encyclopedia. Britannica Concise Encyclopedia. © 2006 Encyclopædia Britannica, Inc. All rights reserved. Read more |
 |
| Sci-Tech Encyclopedia. McGraw-Hill Encyclopedia of Science and Technology. Copyright © 2005 by The McGraw-Hill Companies, Inc. All rights reserved. Read more |
 |
| Dental Dictionary. Mosby's Dental Dictionary. Copyright © 2004 by Elsevier, Inc. All rights reserved. Read more |
 |
| Genetics Encyclopedia. Genetics. Copyright © 2003 by The Gale Group, Inc. All rights reserved. Read more |
 |
| Wikipedia. This article is licensed under the Creative Commons Attribution/Share-Alike License. It uses material from the Wikipedia article "Reverse transcriptase". Read more |
Related answers
- What is the purpose of reverse transcriptase?
- Why does the HIV virus carry copies of reverse transcriptase enzyme?
- What is reverse?
ADVERTISEMENT
Answer these
- What does reverse transcriptase and hiv have in common?
- Can reverse transcriptase be denatured?
- Reverse transcriptase enzyme was invented by?
Mentioned in
- transcriptase
- reverse transcription
- Non-nucleoside reverse transcriptase inhibitors (in medicine)
- Retrovirus (in medicine)
- complementary DNA (Single-stranded DNA)
- nucleoside analogue
- complementary deoxyribonucleic acid (cell and molecular biology)
- AZT (nucleoside analogue antiviral drug)
- DDC (nucleoside analogue antiviral drug)
- DDI (nucleoside analogue antiviral drug)
