Reverse transcriptase

Reverse transcriptase (RNA-dependent DNA polymerase) Crystallographic structure of HIV reverse transcriptase where the P51 subunit is colored green and the P66 subunit is colored cyan.[1] Identifiers Symbol RVT_1 Pfam PF00078 Pfam clan CL0027 InterPro IPR000477 PROSITE PS50878 SCOP 1hmv Available PDB structures: 1bqm, 1bqn, 1c0t, 1c0u, 1c1b, 1c1c, 1d0e, 1d1u, 1dlo, 1dtq, 1dtt, 1eet, 1ep4, 1fk9, 1fko, 1fkp, 1har, 1hmv, 1hni, 1hnv, 1hpz, 1hqe, 1hqu, 1hvu, 1hys, 1i6j, 1ikv, 1ikw, 1ikx, 1iky, 1j5o, 1jkh, 1jla, 1jlb, 1jlc, 1jle, 1jlf, 1jlg, 1jlq, 1klm, 1lw0, 1lw2, 1lwc, 1lwe, 1lwf, 1mml, 1mu2, 1n4l, 1n5y, 1n6q, 1nnd, 1qai, 1qaj, 1qe1, 1r0a, 1rev, 1rt1, 1rt2, 1rt3, 1rt4, 1rt5, 1rt6, 1rt7, 1rtd, 1rth, 1rti, 1rtj, 1rw3, 1s1t, 1s1u, 1s1v, 1s1w, 1s1x, 1s6p, 1s6q, 1s9e, 1s9g, 1suq, 1sv5, 1t03, 1t05, 1tkt, 1tkx, 1tkz, 1tl1, 1tl3, 1tv6, 1tvr, 1uwb, 1vrt, 1vru, 1ztt, 1ztw, 2b5j, 2b6a, 2ban, 2be2, 2fjv, 2fjw, 2fjx, 2fvp, 2fvq, 2fvr, 2fvs, 2hmi, 2hnd, 2hny, 2hnz, 2i5j, 2iaj, 2ic3, 2opp, 2opq, 2opr, 2ops, 2r2r, 2r2s, 2r2t, 2r2u, 2rf2, 2rki, 2vg5, 2vg6, 2vg7, 2zd1, 2ze2, 3bgr, 3c6t, 3c6u, 3di6, 3dle, 3dlg, 3dlk, 3dm2, 3dok, 3dol, 3drp, 3drr, 3drs, 3hvt

RNA-directed DNA polymerase Identifiers EC number 2.7.7.49 CAS number 9068-38-6 Databases IntEnz IntEnz view BRENDA BRENDA entry ExPASy NiceZyme view KEGG KEGG entry MetaCyc metabolic pathway PRIAM profile PDB structures Gene Ontology AmiGO / EGO Search PMC articles PubMed articles

In the fields of 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

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.

Process of reverse transcription

Reverse transcriptase creates single 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 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). Creation of double-stranded DNA occurs in the cytosol^[2] as a series of steps:

1. 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
2. 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
3. A domain on the reverse transcriptase enzyme called RNAse H degrades the 5’ end of the RNA which removes the U5 and R region
4. 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
5. The first strand of complementary DNA (cDNA) is extended and the majority of viral RNA is degraded by RNAse H
6. Once the strand is completed, second strand synthesis is initiated from the viral RNA
7. There is then another ‘jump’ where the PBS from the second strand hybridizes with the complementary PBS on the first strand
8. 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 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]

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][7]

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.^[8]

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.^[9]

Reverse transcriptase has a high error rate when transcribing RNA into DNA since, unlike any 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^[10]

Applications

The molecular structure of zidovudine (AZT), 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 (trade name 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 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

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.