A reverse transcriptase (RT) is an enzyme used to produce complementary DNA (cDNA) from an RNA template, a process called reverse transcription . It is primarily associated with retroviruses. However, non-retroviruses also use RT (for example, hepatitis B virus, a member of Hepadnaviridae, which is a dsDNA-RT virus, whereas retroviruses are ssRNA viruses). RT inhibitors are widely used as antiretroviral drugs. RT activities are also associated with replication of the ends of chromosomes (telomerase) and some elements of cellular genetics (retrotransposons).
Retroviral RT has three consecutive biochemical activities:
- (a) RNA-dependent DNA polymerase activity,
- (b) ribonuclease H, and
- (c) DNA-dependent DNA polymerase activity.
This activity is used by retroviruses to convert single stranded genomic RNAs into double-stranded cDNAs that can integrate into the host genome, potentially resulting in long-term infections that are very difficult to eradicate. The same reaction sequences are widely used in laboratories to convert RNA into DNA for use in molecular cloning, RNA sequencing, polymerase chain reaction (PCR), or genome analysis.
Well-studied reverse transcriptases include:
- The HIV-1 reverse transcriptase of human immunodeficiency virus type 1 ( GDP: 1HMV ) has two subunits, which have molecular weights of 66 and 51 kDa respectively. M-MLV reverse transcriptase from Moloney murine leukemia virus is a 75 kDa single monomer.
- AMV reverse transcriptase from the avian myeloblastosis virus also has two subunits, 63 kDa subunits and 95 kDa subunits.
- Telomerase reverse transcriptase that maintains a telechere of eukaryotic chromosomes.
Video Reverse transcriptase
Histori
Inverted transcripts were discovered by Howard Temin at the University of Wisconsin-Madison in RSV virions and independently isolated by David Baltimore in 1970 at MIT from two tumor RNA tumors: R-MLV and RSV again. For their achievement, they shared the 1975 Nobel Prize in Physiology or Medicine (with Renato Dulbecco).
The idea of ​​inverted transcription is very unpopular at first, as opposed to the core dogma of molecular biology, which states that DNA is transcribed into RNA, which is then translated into proteins. However, in 1970, when scientists Howard Temin and David Baltimore both independently found the enzyme responsible for reverse transcription, named reverse transcriptase, the possibility that genetic information could be transmitted in this way was eventually accepted.
Maps Reverse transcriptase
Works in virus
Enzymes are encoded and used by viruses that use reverse transcription as a step in the replication process. Reverse-transcribing RNA viruses, such as retroviruses, use enzymes to reverse their RNA genome into DNA, which is then integrated into the host genome and replicated at the same time. Reverse-transcribed DNA viruses, such as hepadnaviruses, can allow RNA to function as templates in assembling and making DNA strands. HIV infects humans with the use of this enzyme. Without reverse transcriptase, the viral genome will not be inserted into the host cell, resulting in failure to replicate.
Inverted transcription process
Inverted transcripts create double-stranded DNA from RNA templates.
In virus species with reverse transcriptase that does not have DNA-dependent DNA polymerases, the creation of double-stranded DNA can be performed by hosted DNA polymerases, mistaking DNA DNA-RNA as primary and synthesizing double-stranded DNA by the like. mechanisms such as primary removal, where newly synthesized DNA replaces the original RNA template.
The reverse transcription process is very error-prone, and during this step mutations can occur. Such mutations can cause drug resistance.
Retroviruses, also referred to as class VI virus ssRNA-RT, are reverse-transcriptional RNA viruses with DNA intermediates. Their genome consists of two positive-stranded RNA molecules with 5 'cap and 3' polyadenylated tail. Examples of retroviruses include human immunodeficiency virus (HIV) and human T-lymphotropic virus (HTLV). The creation of double-stranded DNA occurs in the cytosol as a series of these steps:
- Lysyl tRNA acts as a primer and hybridizes into a complementary part of the viral RNA genome called the primary binding site or PBS.
- Inverted transcriptase then adds DNA nucleotides to the 3 'primer end, synthesizes DNA that complements the U5 region (non-coding region) and R (direct repetition found on both ends of RNA molecules) from viral RNA.
- The domain of a reverse transcriptase enzyme called RNAse H decreases the U5 and R regions at the 5 'end of the RNA.
- The primer then "jumps" to the 3 'end of the viral genome, and a new DNA strand is synthesized hybridization into the complementary R region of the RNA.
- The complementary DNA (cDNA) added in (2) is extended, and then most of the viral RNA is degraded by RNAse H, leaving only the PP sequence.
- The synthesis of a second DNA strand begins, using the remaining viral RNA fragments as primers.
- Then there is another "jump" in which the PBS of the second strand hybridizes with the complementary PBS on the first strand.
- The two 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 genome by the integrase enzyme.
The creation of double-stranded DNA also involves strand transfers , in which there is a short DNA product translocation from RNA DNA synthesis depending on the beginning to the acceptor templates at the other end of the genome, which are later achieved and processed by reverse transcripts for DNA activity DNA dependent.
Retroviral RNAs are set in terminus 5 'terminus to 3'. The site where the primer is an annealing viral RNA is called the primary-binding site (PBS). RNA is 5'end to the PBS website called U5, and the 3 'RNA end to the PBS is called the leader. The tRNA primer is released between 14 and 22 nucleotides and forms a base pair duplex with viral RNA in PBS. The fact that PBS is located near the end of the RNA 5 terminal is unusual because reverse transcriptase synthesizes DNA from the 3 'primary end in the 5' to 3 'direction (with respect to newly synthesized DNA strands). Therefore, the primary and reverse transcriptase must be moved to the 3 'end of the viral RNA. To achieve this repositioning, several steps and enzymes including DNA polymerase, H (RNase H) ribonuclease and polynucleotide binding are required.
Reverse transcriptase HIV also has a ribonuclease activity that decreases viral RNA during cDNA synthesis, as well as DNA-dependent DNA polymerase activity that copies the cDNA senses into antisense DNA to form stranded middle-virus DNA (vDNA).
In eukaryotes
Self replication of the eukaryotic genome known as retrotransposons uses reverse transcriptase to move from one position in the genome to another via intermediate RNA. They are found abundantly in the genomes of plants and animals. Telomerase is another reverse transcript found in many eukaryotes, including humans, carrying its own RNA template; This RNA is used as a template for DNA replication.
In prokaryote
Initial reverse transcriptase reports in prokaryotes come as far back as 1971 (Beljanski et al., 1971a, 1972). It has since been broadly described as part of the bacterial retro, a distinct sequence that encodes reverse transcriptase, and is used in msDNA synthesis. To begin DNA synthesis, a primer is required. In bacteria, primers are synthesized during replication.
Evolutionary role
Valerian Dolja of Oregon State argues that the virus, because of its diversity, has played an evolutionary role in the development of cellular life, with reverse transcriptase playing a central role.
Structure
Reverse transcriptase enzymes include RNA-dependent DNA polymerase and DNA-dependent DNA polymerase, which work together to reverse transcription. In addition to the transcription function, reverse transcriptases retroviral has the domain belonging to the RNase H family, which is very important for their replication.
Replicating allegiance
There are three different replication systems during the retrovirus lifecycle. First of all, reverse transcriptase synthesizes viral DNA from viral RNA, and then from a new complementary strand of DNA is created. The second process of replication occurs when host cellular polymerase DNA replicates the DNA of an integrated virus. Finally, RNA polymerase II transcribes proviral DNA to RNA, which will be packed into virions. Therefore, mutations may occur during one or all of these replication steps.
Inverted transcripts have a high error rate when transcribing RNA into DNA because, unlike most other DNA polymerases, it lacks proofreading ability. This high error rate allows the mutation to accumulate at the rate of acceleration relative to the improved form of replication. The commercially available reverse transcripts produced by Promega are cited by their manual as having an error rate in the range of 1 in 17,000 bases for AMV and 1 in 30,000 bases for M-MLV.
In addition to creating a single nucleotide polymorphism, reverse transcriptases have also been shown to be involved in processes such as transcript fusion, exon shaking and creating artificial antisense transcripts. It has been speculated that the switching template activity of this reverse transcriptase, which can be fully proven in vivo , may have been one of the causes of finding several thousand transcripts not denoted in the genomes of model organisms.
Apps
Because HIV uses reverse transcriptase to copy its genetic material and produces a new virus (part of the retroviral proliferation cycle), a special drug has been designed to disrupt the process and thereby suppress its growth. Collectively, the drug is known as a reverse-transcriptase inhibitor and includes nucleotides and nucleotides analog 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 polymerase chain reaction techniques to RNA in a technique called reverse transcription polymerase chain reaction (RT-PCR). Classical PCR techniques can only be applied to DNA strands, but, with the help of reverse transcriptase, RNA can be transcribed into DNA, allowing PCR analysis of RNA molecules. Reverse transcriptase is also used to create a cDNA library of mRNAs. The commercial availability of reverse transcriptase greatly increases knowledge in the field of molecular biology, because, along with other enzymes, it allows scientists to clone, sort, and characterize RNA.
Reverse transcriptase has also been used in the production of insulin. By introducing eukaryotic mRNAs for insulin production along with reverse transcriptase to bacteria, mRNAs can be incorporated into the prokaryotic genome. A substantial amount of insulin can then be made, setting aside the need to harvest pancreas and other traditional sources. Incorporating eukaryotic DNA into the bacteria directly will not work because it carries the intron, so it will not successfully translate using a bacterial ribosome. Processing in eukaryotic cells during mRNA production removes these introns to provide an appropriate template. Reverse transcriptase converts this edited RNA back into DNA so it can be incorporated into the genome.
See also
- cDNA library
- DNA polymerase
- msDNA
- Reversed virus transcription
- RNA polymerase
- Telomerase
- Retrotransposon marker
References
External links
- RNA Transcriptase in the US National Library of Medicine Subject Medical Headings (MeSH) Animation
- from reverse transcriptase action and three reverse transcriptase inhibitors
- This month's molecule (September 2002) in RCSB PDB
- 3D Medical Replication of HIV Animation. (Nov 2008). Video by Boehringer Ingelheim.
- DS Goodsell. "Molecule of the Month: Reverse Transcriptase (Sep 2002)". Collaborative Research for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) . Retrieved 2013-01-13 .
Source of the article : Wikipedia
0 Comments