A reverse transcriptase (RT) is an
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...
used to generate
complementary DNA (cDNA) from an
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
template, a process termed reverse transcription. Reverse transcriptases are used by
virus
A virus is a wikt:submicroscopic, submicroscopic infectious agent that replicates only inside the living Cell (biology), cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and ...
es such as
HIV
The human immunodeficiency viruses (HIV) are two species of '' Lentivirus'' (a subgroup of retrovirus) that infect humans. Over time, they cause acquired immunodeficiency syndrome (AIDS), a condition in which progressive failure of the immu ...
and
hepatitis B to replicate their genomes, by
retrotransposon mobile genetic elements to proliferate within the host genome, and by
eukaryotic cells to extend the
telomeres at the ends of their
linear chromosomes. Contrary to a widely held belief, the process does not violate the flows of genetic information as described by the classical
central dogma
The central dogma of molecular biology is an explanation of the flow of genetic information within a biological system. It is often stated as "DNA makes RNA, and RNA makes protein", although this is not its original meaning. It was first stated by ...
, as transfers of information from RNA to DNA are explicitly held possible.
Retroviral RT has three sequential biochemical activities: RNA-dependent
DNA polymerase activity,
ribonuclease H (RNase H), and DNA-dependent DNA polymerase activity. Collectively, these activities enable the enzyme to convert single-stranded RNA into double-stranded cDNA. In retroviruses and retrotransposons, this cDNA can then integrate into the host genome, from which new RNA copies can be made via host-cell
transcription. The same sequence of reactions is widely used in the laboratory to convert RNA to DNA for use in
molecular cloning,
RNA sequencing,
polymerase chain reaction (PCR), or
genome analysis
Personal genomics or consumer genetics is the branch of genomics concerned with the sequencing, analysis and interpretation of the genome of an individual. The genotyping stage employs different techniques, including single-nucleotide polymorphi ...
.
History
Reverse transcriptases were discovered by
Howard Temin
Howard Martin Temin (December 10, 1934 – February 9, 1994) was an American geneticist and virologist. He discovered reverse transcriptase in the 1970s at the University of Wisconsin–Madison, for which he shared the 1975 Nobel Prize in ...
at the
University of Wisconsin–Madison in ''
Rous sarcoma'' virions
and independently isolated by
David Baltimore in 1970 at
MIT from two RNA tumour viruses:
murine leukemia virus
The murine leukemia viruses (MLVs or MuLVs) are retroviruses named for their ability to cause cancer in murine (mouse) hosts. Some MLVs may infect other vertebrates. MLVs include both exogenous and endogenous viruses. Replicating MLVs have a pos ...
and again
Rous sarcoma virus.
For their achievements, they shared the 1975
Nobel Prize in Physiology or Medicine (with
Renato Dulbecco
Renato Dulbecco ( , ; February 22, 1914 – February 19, 2012) was an Italian–American virologist who won the 1975 Nobel Prize in Physiology or Medicine for his work on oncoviruses, which are viruses that can cause cancer when they infect anima ...
).
Well-studied reverse transcriptases include:
* HIV-1 reverse transcriptase from
human immunodeficiency virus type 1 () has two subunits, which have respective molecular weights of 66 and 51
kDas.
* M-MLV reverse transcriptase from the
Moloney murine leukemia virus Moloney may refer to:
*Moloney (surname), people with the surname ''Moloney''
* ''Moloney'' (TV series), 1996–97 American television police drama
See also
*Maloney
*Moloney's Mimic Bat
Moloney's mimic bat (''Mimetillus moloneyi'') is a species ...
is a single 75 kDa monomer.
* AMV reverse transcriptase from the
avian myeloblastosis virus
Avian may refer to:
*Birds or Aves, winged animals
*Avian (given name) (russian: Авиа́н, link=no), a male forename
Aviation
*Avro Avian, a series of light aircraft made by Avro in the 1920s and 1930s
*Avian Limited, a hang glider manufacture ...
also has two subunits, a 63 kDa subunit and a 95 kDa subunit.
*
Telomerase reverse transcriptase that maintains the
telomeres of
eukaryotic chromosomes.
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
RNA virus
An RNA virus is a virusother than a retrovirusthat has ribonucleic acid (RNA) as its genetic material. The nucleic acid is usually single-stranded RNA ( ssRNA) but it may be double-stranded (dsRNA). Notable human diseases caused by RNA viruses ...
es, such as
retrovirus
A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. Once inside the host cell's cytoplasm, the virus uses its own reverse transcriptase ...
es, 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
hepadnavirus
''Hepadnaviridae'' is a family of viruses. Humans, apes, and birds serve as natural hosts. There are currently 18 species in this family, divided among 5 genera. Its best-known member is hepatitis B virus. Diseases associated with this family i ...
es, 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 failure to replicate.
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
DNA polymerase δ, mistaking the viral DNA-RNA for a primer and synthesizing a double-stranded DNA by a similar mechanism as in
primer removal, where the newly synthesized DNA displaces the original RNA template.
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
drug resistance.
Retroviral reverse transcription
Retroviruses
A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. Once inside the host cell's cytoplasm, the virus uses its own reverse transcriptase e ...
, also referred to as class VI
ssRNA-RT
A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. Once inside the host cell's cytoplasm, the virus uses its own reverse transcriptase e ...
viruses, 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 the human immunodeficiency virus (
HIV
The human immunodeficiency viruses (HIV) are two species of '' Lentivirus'' (a subgroup of retrovirus) that infect humans. Over time, they cause acquired immunodeficiency syndrome (AIDS), a condition in which progressive failure of the immu ...
) and the human T-lymphotropic virus (
HTLV). Creation of double-stranded DNA occurs in the
cytosol as a series of these steps:
#
Lysyl tRNA acts 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 H degrades 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
Retroviral integrase (IN) is an enzyme produced by a retrovirus (such as HIV) that integrates—forms covalent links between—its genetic information into that of the host cell it infects. Retroviral INs are not to be confused with phage int ...
.
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.
Retroviral RNA is arranged in 5’ terminus to 3’ terminus. The site where the
primer
Primer may refer to:
Arts, entertainment, and media Films
* ''Primer'' (film), a 2004 feature film written and directed by Shane Carruth
* ''Primer'' (video), a documentary about the funk band Living Colour
Literature
* Primer (textbook), a t ...
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. The fact that the PBS is located 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 (with respect to the newly synthesized DNA strand). 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.
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
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...
activity that copies the
sense cDNA strand into an ''antisense'' DNA to form a double-stranded viral DNA intermediate (vDNA). The HIV viral RNA structural elements regulate the progression of reverse transcription.
In cellular life
Self-replicating stretches of
eukaryotic genomes known as
retrotransposons utilize reverse transcriptase to move from one position in the genome to another via an 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
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
template; this RNA is used as a template for
DNA replication
In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inherita ...
.
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). 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.
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.
Structure
The reverse transcriptase employs a "right hand" structure similar to that found in other
viral nucleic acid polymerases.
In addition to the transcription function, retroviral reverse transcriptases have a domain belonging to the
RNase H family, which is vital to their replication. By degrading the RNA template, it allows the other strand of DNA to be synthesized. Some fragments from the digestion also serve as the primer for the
DNA polymerase (either the same enzyme or a host protein), responsible for making the other (plus) strand.
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.
Reverse transcriptase has a high error rate when transcribing RNA into DNA since, unlike most other
DNA polymerases, it has no
proofreading
Proofreading is the reading of a galley proof or an electronic copy of a publication to find and correct reproduction errors of text or art. Proofreading is the final step in the editorial cycle before publication.
Professional
Traditional ...
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.
Other than creating
single-nucleotide polymorphisms, reverse transcriptases have also been shown to be involved in processes such as
transcript fusions,
exon shuffling and creating artificial
antisense transcripts.
It has been speculated that this ''template switching'' activity of reverse transcriptase, which can be demonstrated completely ''in vivo'', may have been one of the causes for finding several thousand unannotated transcripts in the genomes of model organisms.
Template switching
Two
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
genomes are packaged into each retrovirus particle, but, after an infection, each virus generates only one
provirus A provirus is a virus genome that is integrated into the DNA of a host cell. In the case of bacterial viruses (bacteriophages), proviruses are often referred to as prophages. However, proviruses are distinctly different from prophages and these ter ...
.
After infection, reverse transcription is accompanied by template switching between the two genome copies (copy choice recombination).
[ 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.] From 5 to 14 recombination events per genome occur at each replication cycle. Template switching (recombination) appears to be necessary for maintaining genome integrity and as a repair mechanism for salvaging damaged genomes.
Applications
Antiviral drugs
As HIV
The human immunodeficiency viruses (HIV) are two species of '' Lentivirus'' (a subgroup of retrovirus) that infect humans. Over time, they cause acquired immunodeficiency syndrome (AIDS), a condition in which progressive failure of the immu ...
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
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
in a technique called reverse transcription polymerase chain reaction
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase cha ...
(RT-PCR). The classical PCR PCR or pcr may refer to:
Science
* Phosphocreatine, a phosphorylated creatine molecule
* Principal component regression, a statistical technique
Medicine
* Polymerase chain reaction
** COVID-19 testing, often performed using the polymerase chain r ...
technique can be applied only to DNA strands, but, with the help of reverse transcriptase, RNA can be transcribed into DNA, thus making PCR PCR or pcr may refer to:
Science
* Phosphocreatine, a phosphorylated creatine molecule
* Principal component regression, a statistical technique
Medicine
* Polymerase chain reaction
** COVID-19 testing, often performed using the polymerase chain r ...
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
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
, it allowed scientists to clone, sequence, and characterise RNA.
See also
* cDNA library
* DNA polymerase
* msDNA
* Reverse transcribing virus
* RNA polymerase
In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that synthesizes RNA from a DNA template.
Using the enzyme helicase, RNAP locally opens the ...
* Telomerase
* Retrotransposon marker
References
External links
*
animation of reverse transcriptase action and three reverse transcriptase inhibitors
(September 2002) at the RCSB PDB
HIV Replication 3D Medical Animation. (Nov 2008). Video by Boehringer Ingelheim.
*
*
TWiV 904: 50 years of reverse transcriptase
Vincent Racaniello travels to Cold Spring Harbor Laboratory to speak with David Baltimore, John Coffin (scientist), and Harold Varmus about the discovery in 1970 of retroviral reverse transcriptase and its impact on life sciences research.
{{Kinases
Genetics
EC 2.7.7
Molecular biology
Viral enzymes
Telomeres
1970 in biology
RNA reverse-transcribing viruses