Reverse transcriptase
Reverse transcriptase (RNA-dependent DNA polymerase) | |||||||||
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CDD | cd00304 | ||||||||
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RNA-directed DNA polymerase | |||||||||
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Identifiers | |||||||||
ExPASy | NiceZyme view | ||||||||
KEGG | KEGG entry | ||||||||
MetaCyc | metabolic pathway | ||||||||
PRIAM | profile | ||||||||
PDB structures | RCSB PDB PDBe PDBsum | ||||||||
Gene Ontology | AmiGO / QuickGO | ||||||||
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A reverse transcriptase (RT) is an
Retroviral RT has three sequential biochemical activities: RNA-dependent
History
Reverse transcriptases were discovered by
Well-studied reverse transcriptases include:
- HIV-1 reverse transcriptase from kDas.[7]
- M-MLV reverse transcriptase from the Moloney murine leukemia virus is a single 75 kDa monomer.[8]
- AMV reverse transcriptase from the avian myeloblastosis virus also has two subunits, a 63 kDa subunit and a 95 kDa subunit.[8]
- chromosomes.[9]
Function in viruses
The enzymes are encoded and used by viruses that use reverse transcription as a step in the process of replication. Reverse-transcribing
Process of reverse transcription or retrotranscription
Reverse transcriptase creates double-stranded DNA from an 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
The process of reverse transcription, also called retrotranscription or retrotras, is extremely error-prone, and it is during this step that mutations may occur. Such mutations may cause
Retroviral reverse transcription
as a series of these steps:- tRNAacts as a primer and hybridizes to a complementary part of the virus RNA genome called the primer binding site or PBS.
- Reverse transcriptase then adds DNA nucleotides onto the 3' end of the primer, synthesizing DNA complementary 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 Hdegrades the U5 and R regions on the 5’ end of the RNA.
- The tRNA 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 complementary DNA (cDNA) added in (2) is further extended.
- The majority of viral RNA is degraded by RNAse H, leaving only the PP sequence.
- Synthesis of the second DNA strand begins, using the remaining PP fragment of viral RNA as a primer.
- The tRNA primer leaves and a "jump" happens. The PBS from the second strand hybridizes with the complementary PBS on the first strand.
- Both strands are extended to form a complete double-stranded DNA copy of the original viral RNA genome, which can then be incorporated into the host's 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.[11]
Retroviral RNA is arranged in 5’ terminus to 3’ terminus. The site where the
The HIV reverse transcriptase also has
In cellular life
Self-replicating stretches of
Initial reports of reverse transcriptase in prokaryotes came as far back as 1971 in France (Beljanski et al., 1971a, 1972) and a few years later in the USSR (Romashchenko 1977[17]). These have since been broadly described as part of bacterial Retrons, distinct sequences that 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.[18]
Valerian Dolja of Oregon State argues that viruses, due to their diversity, have played an evolutionary role in the development of cellular life, with reverse transcriptase playing a central role.[19]
Structure
The reverse transcriptase employs a "right hand" structure similar to that found in other
Replication fidelity
There are three different replication systems during the life cycle of a retrovirus. The first process is the reverse transcriptase synthesis of viral DNA from viral RNA, which then forms newly made complementary DNA strands. 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. Mutation can occur during one or all of these replication steps.[23]
Reverse transcriptase has a high error rate when transcribing RNA into DNA since, unlike most other 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.[24]
Other than creating
Template switching
Two RNA genomes are packaged into each retrovirus particle, but, after an infection, each virus generates only one provirus.[28] After infection, reverse transcription is accompanied by template switching between the two genome copies (copy choice recombination).[28] There are two models that suggest why RNA transcriptase switches templates. The first, the forced copy-choice model, proposes that reverse transcriptase changes the RNA template when it encounters a nick, implying that recombination is obligatory to maintaining virus genome integrity. The second, the dynamic choice model, suggests that reverse transcriptase changes templates when the RNAse function and the polymerase function are not in sync rate-wise, implying that recombination occurs at random and is not in response to genomic damage. A study by Rawson et al. supported both models of recombination.[28] From 5 to 14 recombination events per genome occur at each replication cycle.[29] Template switching (recombination) appears to be necessary for maintaining genome integrity and as a repair mechanism for salvaging damaged genomes.[30][28]
Applications
Antiviral drugs
As
Molecular biology
Reverse transcriptase is commonly used in research to apply the
See also
- cDNA library
- DNA polymerase
- msDNA
- Reverse transcribing virus
- RNA polymerase
- Telomerase
- Retrotransposon marker
References
- PMID 20852643.
- S2CID 4164029.
- ^ Sarkar S (1996). The Philosophy and History of Molecular Biology: New Perspectives. Dordrecht: Kluwer Academic Publishers. pp. 187–232.
- S2CID 67861162.
- S2CID 4187764.
- S2CID 4222378.
- PMID 1691562.
- ^ S2CID 207096569.
- PMID 16756500.
- ^ Bio-Medicine.org - Retrovirus Retrieved on 17 Feb, 2009
- ISBN 978-0-87969-382-4.
- ^ Bernstein A, Weiss R, Tooze J (1985). "RNA tumor viruses". Molecular Biology of Tumor Viruses (2nd ed.). Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory.
- S2CID 42378727.
- ^ Kaiser GE (January 2008). "The Life Cycle of HIV". Doc Kaiser's Microbiology Home Page. Community College of Baltimore Count. Archived from the original on 2010-07-26.
- S2CID 221636459.
- ISBN 978-0-7167-4366-8.
- ^ Romashchenko AG, et al. (1977). "Otdelenie ot preparatov DNK-polimeraz I RNK-zavisimoy DNK-polimeraz; oshistka i svoystva fermenta". Proceedings of the USSR Academy of Sciences. 233: 734–737.
- PMID 4333538.
- ^ Arnold C (17 July 2014). "Could Giant Viruses Be the Origin of Life on Earth?". National Geographic. Archived from the original on July 18, 2014. Retrieved 29 May 2016.
- ^ PMID 19022262.
- PMID 9309225.
- PMID 18261820.
- ISBN 978-0-87969-382-4.
- ^ "Promega kit instruction manual" (PDF). 1999. Archived from the original (PDF) on 2006-11-21.
- PMID 20805885.
- S2CID 24619868.
- PMC 3134445.
- ^ PMID 30307534.
- S2CID 20086739.
- PMID 1700865.
External links
- RNA+Transcriptase at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
- animation of reverse transcriptase action and three reverse transcriptase inhibitors
- Molecule of the month (September 2002) at the RCSB PDB
- HIV Replication 3D Medical Animation. (Nov 2008). Video by Boehringer Ingelheim.
- Goodsell DS. "Molecule of the Month: Reverse Transcriptase (Sep 2002)". Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB). Retrieved 2013-01-13.
- Overview of all the structural information available in the PDB for UniProt: P03366 (Human immunodeficiency virus Reverse transcriptase) at the PDBe-KB.
- TWiV 904: 50 years of reverse transcriptase Harold Varmusabout the discovery in 1970 of retroviral reverse transcriptase and its impact on life sciences research.