Reverse Transcriptase

G. Maga , in Brenner's Encyclopedia of Genetics (Second Edition), 2013

Opposite Transcriptase and Retrotransposons

Retrotransposons are genetic mobile elements that apply reverse transcription to generate a DNA copy of themselves to exist inserted into eukaryotic genomes. Similarly to retroviruses, their genomes possess the gene for opposite transcriptase, RNase H, and protease, merely they lack the genes for the proteins of the viral capsid, and so they practise not generate virions and therefore do not spread among individuals, merely are genetically inherited by the descendants if present in the chromosomes of germline cells. Some of these elements encode reverse transcriptases with special backdrop.

The Schizosaccharomyces pombe Tf1 transposon opposite transcriptase initiates DNA synthesis from an eleven-nt RNA primer generated by cocky-annealing of the 11 terminal bases of the RNA genome to a complementary sequence located within the 5′ end and subsequently cleaved by its RNAse H action to generate the three′-hydroxyl end of the primer. Another unique feature of this enzyme is its power to add one or ii nucleotides to the 3′-concluding cease of the Dna re-create in a template-contained fashion. This machinery protects the 3′ end of the transposon Deoxyribonucleic acid from degradation by cellular exonucleases.

The Bombyx mori retrotransposon R2 also encodes a reverse transcriptase with unusual enzymatic backdrop. It can utilise a 3′-hydroxyl end from any RNA or Dna molecule to prime its reaction without the demand of whatsoever complementarity betwixt the primer and the template.

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Dna Polymerases

Hyone-Myong Eun , in Enzymology Primer for Recombinant Dna Engineering science, 1996

ii. Nucleotide sequencing.

Reverse transcriptases (both AMV and MoLV) complement DNA-dependent DNA polymerases (e.g., Politico Ik) in the dideoxynucleotide sequencing of Dna, especially at the regions of loftier GC content and/or secondary structures ( 108). Opposite transcriptase has been shown to be peculiarly useful in sequencing with fluorescence tag-modified ddNTPs that are not substrates for Politician Ik (109).

Reverse transcriptases are besides valuable reagents in the direct dideoxynucleotide sequencing of RNA (110). In fact, RTases have been instrumental in obtaining nucleotide sequence information directly from viral RNA genomes, 16S rRNA (111), and mRNAs (112). The higher thermostability of AMV RTase compared with that of MoLV RTase makes the AMV RTase more useful for nucleotide sequencing at elevated temperatures.

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HIV and Acquired Immunodeficiency Syndrome

Tak W. Mak , Mary Eastward. Saunders , in The Immune Response, 2006

i) Reverse Transcriptase

Contrary Transcriptase (RT) is essential for HIV replication because the viral RNA genome on its own is highly susceptible to degradation by intracellular RNases. RT rapidly makes a much more nuclease-resistant double-stranded DNA copy of the RNA template that later integrates to form the proviral Dna. HIV RT is a heterodimer equanimous of a 66 kDa subunit (p66) and a 51 kDa subunit (p51) created by cleavage of a separate molecule of p66. All the catalytic activity of HIV RT is attributable to p66, while p51 supports the functions of p66. As well every bit its Deoxyribonucleic acid polymerase office, RT has an RNase H function that degrades the RNA template used to brand the viral Deoxyribonucleic acid. The complex method by which the RT synthesizes the viral DNA from the genomic ssRNA is described in Figure 25-four.

Figure 25-4. Synthesis of HIV Deoxyribonucleic acid from Genomic RNA

(ane) A cellular tRNAlys3 molecule hybridizes with the primer bounden site (PBS) on the genomic +RNA strand and HIV RT commences synthesis of the negative strand DNA (—Deoxyribonucleic acid) in the three′ management. (2) Equally the — DNA elongates, the 5′ department of genomic + RNA used as the template is degraded by the RNase H activity of RT. (3) The newly synthesized fragment of —DNA "jumps" to the 3′ finish of the genomic RNA where the R' sequence in the − DNA hybridizes to the R sequence in the + RNA. (four) Synthesis of the —DNA strand and then continues in the 5′ to 3′ management until it is completed. (five) The genomic +RNA is degraded by RT RNase H activity with the exception of two poly-purine tracts (cPPT and PPT) that remain hybridized to the — Dna. Using the —Dna as the template and both PPTs every bit primers, the positive strand DNA (+DNA) is elongated toward its 3′ end until a PBS site is created (6). (7) The tRNAlys3 at the 5′ end of the –DNA strand and the PPTs are degraded. (8) Hybridization of the PBS' with the PBS site results in circularization of the — and + DNA strands and elongation of the +DNA (9) The U3′-R-U5′ sequence of the − Deoxyribonucleic acid is used every bit a template to complete the iii′ end of the + Dna strand and form the 3′ LTR of the proviral DNA. Elongation of the —Deoxyribonucleic acid strand through to its three′ end copies the v′+Dna to produce the five′ LTR of the provirus. Elongation of the +Dna strand ends at a termination site 3′ of the cPPT'. The resulting overlap in the +DNA strand is non shown in the viral DNA. (ten) Notation that R-U5 and U3-R from the genomic RNA accept now both go U3-R-U5 in the viral Deoxyribonucleic acid, giving it the required LTR at each end. The R site in the 3′ LTR contains a cleavage site and a polyadenylation site used for the transcription of viral mRNA.

 Adapted from Gotte M. et al. (1999) Archives of Biochemistry and Biophysics 365(2), 199–210. Copyright © 1999

HIV RT is responsible for much of the antigenic variation of HIV that confounds both the natural immune response and vaccine development. HIV RT lacks the proof-reading capabilities inherent in cellular polymerases, meaning that its duplication of the HIV genome is highly mistake-prone. Mutations due to uncorrected RT action appear in the HIV genome at a rate of well-nigh 1 in every 1500–4000 nucleotides per replication bicycle. Equally a point of comparing, the boilerplate rate of mutation of the human cellular genome is 1 in 10seven–ten8 base pairs.

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Viral Tools for In Vitro Manipulations of Nucleic Acids

Boriana Marintcheva , in Harnessing the Power of Viruses, 2018

two.3.two.2 Reverse Transcriptases

Reverse transcriptases (RTs) are RNA dependent DNA pols initially isolated from retroviruses. In improver, RTs are coded past dsRNA viruses that utilize reverse transcription such as hepatitis B virus (replication of hepatitis is discussed in Chapter 1); and various retroelements in eukaryotes and prokaryotes. The enzyme telomerase maintaining the ends of the eukaryotic chromosomes is technically also a reverse transcriptase, although its machinery is very distinct from conventional RTs. Historically, the discovery of RT revolutionized molecular biology leading to the revision of the primal dogma and enabling scientists to develop new research tools that heavily influenced cloning, assay of gene expression and RNA biology. HIV RT is one of the almost extensively studied polymerases in the context of understanding the biology of this devastating virus and designing RT inhibitors as drugs to manage HIV infections. RTs exhibit three cardinal enzyme activities (Fig. 2.13): (1) RNA-dependent Dna pol that uses ssRNA template and a primer (tRNALys for HIV RT) to synthesize ssDNA/cDNA, which remains hybridized to its RNA template; (ii) RNAse H endonuclease, which selectively degrades the RNA strand of Dna/RNA hybrids and (3) DNA dependent Dna polymerase action, converting the unmarried-stranded cDNA into dsDNA. Conventional RT enzymes have ii active sites, one executing the polymerase activities and another executing the endonuclease action. RT are monomeric or dimeric proteins and some lack intrinsic RNAse H activeness. The RTs of Moloney murine leukemia virus (M-MLV) and Avian myeloblastosis virus (AMV) are most often used as molecular tools in RT-PCR, RT-qPCR, cDNA cloning, RNA sequencing and whatever other experimental technique/approach that requires conversion of RNA to DNA. Site-directed mutagenesis and protein evolution take been utilized to optimize those enzymes improving thermostability and modulating RNAseH activity. Using thermostable version of RT is benign for lowering the nonspecific nucleic acrid amplification and minimizing affect of circuitous secondary structures. Robust RNAseH action is an reward in RT-PCR, whereas lower RNAseH activity is beneficial in cDNA cloning protocols, particularly when very long mRNA transcripts are reverse transcribed. In some cases RT is used only to produce the RNA/Dna hybrid and a conventional Dna politico carries out the cDNA to dsDNA polymerization step.

Figure ii.13. Opposite transcriptase activities and mechanism of activeness.

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Viral Replication Enzymes and their Inhibitors Function B

Nicolas Sluis-Cremer , in The Enzymes, 2021

Abstract

Contrary transcriptase (RT) is a multifunctional enzyme that has RNA- and Deoxyribonucleic acid-dependent Dna polymerase activity and ribonuclease H (RNase H) activity, and is responsible for the reverse transcription of retroviral unmarried-stranded RNA into double-stranded DNA. The essential role that RT plays in the man immunodeficiency virus (HIV) life cycle is highlighted by the fact that multiple antiviral drugs—which can be classified into ii distinct therapeutic classes—are routinely used to treat and/or prevent HIV infection. This book chapter provides detailed insights into the three-dimensional structure of HIV RT, the biochemical mechanisms of DNA polymerization and RNase H activity, and the mechanisms past which nucleoside/nucleotide and nonnucleoside RT inhibitors block reverse transcription.

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DNA Polymerases: Opposite Transcriptase Integrase, and Retrovirus Replication

Yard.-L. Andréola , ... S. Litvak , in Encyclopedia of Biological Chemistry (Second Edition), 2013

Abstruse

Reverse transcriptase (RT), also known equally RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into DNA. This enzyme is able to synthesize a double helix Deoxyribonucleic acid once the RNA has been reverse transcribed in a beginning pace into a unmarried-strand Dna. RNA viruses, such as retroviruses, utilize the enzyme to reverse-transcribe their RNA genomes into Dna, which is then integrated into the host genome and replicated along with information technology. During the replication of some DNA viruses, such as the hepadnaviruses or pararetroviruses, besides conveying a RT, the Dna genome is transcribed to RNA that serves as a template to make new viral DNA strands.

Although RT was discovered in retroviruses and thought to exist a epitome of these infectious agents, it is currently known that RT is plant in many other eukaryotic and prokaryotic systems like telomerase, retrotransposons, retrons, and are found abundantly in the genomes of plants and animals. Retroviral RT has a domain carrying a ribonuclease H (RNase H) activeness that is crucial to their replication. RNase H is an endonuclease able to degrade the RNA moiety of Deoxyribonucleic acid–RNA hybrids family. Another viral-encoded enzyme, integrase, is constitute either in the mature dimeric form of RT or as a free enzyme. Integrase is vital for the insertion of the double-stranded Deoxyribonucleic acid synthesized past RT in the genome of the host-infected prison cell. Inhibitors of the DNA polymerase activeness of retroviral RT are widely used in the handling of pathologies produced by retroviruses like in the case of acquired immune deficiency syndrome.

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Deoxyribonucleic acid Replication, Repair, and Mutagenesis

Due north.V. Bhagavan , Chung-Eun Ha , in Essentials of Medical Biochemistry (Second Edition), 2015

Contrary Transcriptase

Contrary transcriptase is an RNA-dependent Dna polymerase that was discovered in many retroviruses such as human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) in 1970. The contrary transcriptase catalyzes the conversion of RNA template molecules into a Deoxyribonucleic acid double helix and provides a very useful tool for molecular biological science research. Reverse transcriptases are commonly used to produce complementary Dna (cDNA) libraries from various expressed mRNAs and are also used to quantify the level of mRNA synthesis when combined with the polymerase chain reaction technique, chosen RT-PCR. Reverse transcriptase contains three enzymatic activities: (1) RNA-dependent DNA polymerase, (2) RNase H, and (three) DNA-dependent DNA polymerase. Outset, RNA-dependent DNA polymerase synthesizes a Dna strand complementary to the RNA template. Then RNase H removes the RNA strand from the RNA–Dna hybrid double helix. Then the Deoxyribonucleic acid-dependent DNA polymerase completes double-stranded Deoxyribonucleic acid synthesis. Dissimilar other Deoxyribonucleic acid polymerases, opposite transcriptase lacks a proofreading capability and therefore has loftier fault rates during Dna synthesis, up to one error in 2000 base incorporations. The loftier error rates of viral reverse transcriptases provide selective advantage for their survival in the host system.

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Antiviral Therapy

Wang-Shick Ryu , in Molecular Virology of Human Pathogenic Viruses, 2017

26.3.2 HIV RT Inhibitors

HIV RT is involved in viral opposite transcription, which converts the viral RNA into the Deoxyribonucleic acid (come across Fig. 26.4). HIV RT has been the most exploited antiviral drug target ever, resulting in the evolution of 12 drugs. HIV RT inhibitors can be divided into 2 classes depending on the mode of action: nucleoside RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). In fact, 7 NRTIs and five NNRTIs are currently available. Zidovudine (AZT) and lamivudine (3TC), lacking the three′OH group, deed every bit a concatenation terminator of the viral reverse transcription (Fig. 26.7). These nucleoside analogs are prodrugs in that they act as an inhibitor only after conversion into a triphosphate course. On the other hand, NNRTIs, such every bit nevirapine (NVP) and efavirenz (ETR), bind to a region that is distinct from the dNTP-bounden site on the viral RT protein. In other words, these NNRTIs act as allosteric inhibitors.

Figure 26.vii. HIV RT inhibitors.

Zidovudine (AZT) and lamivudine (3TC) are NRTIs that act as chain terminators of HIV RT post-obit conversion into a triphosphate form in cells. Nevirapine (NVP) and efavirenz (ETR) are NNRTIs. Iii-letter acronyms are shown in parenthesis.

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Polymerase Chain Reaction

David P. Clark , Nanette J. Pazdernik , in Molecular Biology (Second Edition), 2013

four Contrary Transcriptase PCR

The coding sequence of virtually eukaryotic genes is interrupted by intervening sequences, or introns (see Ch. 12 for introns and RNA processing). Consequently, the original version of a eukaryotic gene is very large, difficult to dispense, and virtually impossible to express in whatsoever other type of organism. Since mRNA has had the introns removed naturally, it may exist used as the source of an uninterrupted coding sequence that is much more convenient for engineering and expression. This involves converting the RNA back into a DNA copy, known every bit complementary DNA (cDNA) past reverse transcriptase . Thus, when amplifying eukaryotic genes by PCR the cDNA version is oftentimes used (rather than the truthful chromosomal cistron sequence) since this lacks the introns.

Reverse transcriptase is an enzyme establish in retroviruses that converts the RNA genome carried in the retrovirus particle into double-stranded Deoxyribonucleic acid. Opposite transcriptase kickoff transcribes a complementary strand of DNA to make an RNA:DNA hybrid. Next, opposite transcriptase or RNase H degrades the RNA strand of the hybrid. The single-stranded Dna is then used every bit a template for synthesizing double-stranded DNA (cDNA). Once the cDNA has been fabricated, PCR can be used to dilate the cDNA and generate multiple copies (Fig. 6.13). This combined procedure is referred to as reverse transcriptase PCR (RT-PCR) and allows genes to be amplified and cloned equally intron-costless DNA copies starting from mRNA.

Figure half dozen.13. Reverse Transcriptase PCR

RT-PCR is a two-stride procedure that involves making a cDNA copy of the mRNA, and so using PCR to amplify the cDNA. First, a sample of mRNA (which lacks introns) is isolated. Contrary transcriptase is used to make a cDNA copy of the mRNA. The cDNA sample is and then amplified by PCR. This yields multiple copies of cDNA without introns.

Contrary transcription followed past PCR allows cloning of genes starting from the messenger RNA, and thus, identifying the expressed exons of the eukaryotic gene.

Performing RT-PCR on an organism under unlike growth conditions reveals when a factor of interest is expressed (i.e., when the corresponding mRNA is present) and what surround induces factor expression. To compare two unlike conditions, mRNA is extracted from cells growing in both conditions. RT-PCR is performed on the ii samples of mRNA using PCR primers that match the particular cistron of interest. If the gene is expressed, a PCR product will exist produced, whereas if the gene is switched off, its mRNA will exist missing (Fig. 6.14).

Figure half dozen.14. RT-PCR for Gene Expression

RT-PCR tin determine the amount of mRNA for a particular gene in two different growth conditions. In this instance, the gene of interest is expressed in condition ane but non in condition ii. Therefore, in condition 1   mRNA from the factor of interest is present, contrary transcriptase generates a cDNA, and PCR amplifies this cDNA into many copies. In condition 2 the mRNA is absent-minded and then the RT-PCR procedure does not generate the respective Deoxyribonucleic acid.

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Chemical and Constructed Biology Approaches To Understand Cellular Functions - Part A

Justin Grand. Thomas , ... Hashemite kingdom of jordan L. Meier , in Methods in Enzymology, 2019

ii.3 Nucleotide resolution sequencing of individual ac4C sites

ii.3.1 Materials

TGIRT-III opposite transcription enzyme (INGEX)

5   × TGIRT reaction buffer (user prepared with nuclease-costless reagents, 2.25   Thousand NaCl, 100   mM Tris-HCL pH   7.5)

RNasin plus, 40   units/μL (Promega, N2611)

Optimized dNTP mix [ten   mM dTTP, 10   mM dCTP, 10   mM dATP, v   mM dGTP] (prepared from 100   mM dNTPs set, Nib N0446S)

Phusion High-Allegiance PCR Kit (New England Biolabs, E0553S)

Agarose LE, Molecular Biology Course (Thomas Scientific, C996H59)

100   bp Deoxyribonucleic acid Ladder (New England Biolabs, N3231S)

SYBR Safe DNA Gel Stain (Invitrogen, S33102)

UltraPure TBE Buffer, ten   × (Invitrogen, 15581044)

UV transilluminator (UVP MultiDoc-It Imager Benchtop UV Transilluminator)

6   × DNA gel loading dye (Nib, B7021S)

QIAquick Gel extraction kit (Qiagen, 28704)

Thermocycler

Agarose gel electrophoresis equipment.

ii.iii.2 Opposite transcription of borohydride-treated RNA

Contrary transcription of sodium borohydride-treated ac4c results in partial incorporation of adenosine in the cDNA strand opposite the reduced ac4C. Information technology should besides exist noted that reduced ac4C results in partial termination of reverse transcription at or next to its location, often referred to equally RT-stops (Fig. iv A). Of the reverse transcriptase enzymes screened by our lab TGIRT-Iii RT (Ingex) produces the highest proportion of full-length to stop product, besides equally the most robust C-to-T signature of the RT enzymes screened by our lab ( Fig. 4B). The following protocol describes the use of TGIRT RT for sequence-divers cDNA generation, which we have used to profile ac4C in model substrates and human 18S rRNA (Thomas et al., 2018).

1.

Dilute RNA from Section ii.2.2 to 100   pg/μL in water for utilize as template.

2.

Perform TGIRT RT reactions. Nosotros typically carry out RT in twenty   μL volumes in PCR tubes. Exemplary protocol: combine 4   μL 5   × TGIRT reaction buffer with 200   pg (2   μL) of template, 4 pM DNA primer, 2   μL 50   mM MgCl2 and sufficient ultra-pure water to bring the mixture to 17   μL. Note 1: TGIRT reactions are incubated at 57   °C, higher than many other commercially available opposite transcription enzymes. Therefore, it is essential to design the reverse transcription primer to form a stable duplex with the target sequence at this temperature. Note 2: Fluorophore or radiolabeled primers can be substituted at this step to assess RT finish via primer extension assay (Fig. 4)

iii.

Rut to 75   °C for 3   min and placed on ice for i   min to anneal the primer and RNA.

4.

After cooling, add 0.five   μL TGIRT-III, 0.5   μL Rnasin Plus and 100   μL 100   mM DTT. Incubate 20   min at room temperature. Initiate the contrary transcription reaction by adding 1   μL of optimized dNTP mix. In optimization studies we have establish that decreasing the dGTP concentration from 500 to 250   μM increases the sensitivity of C-to-T misincorporation. Incubate the reaction for one hour at 57   °C and proceed to PCR amplification step (Section 2.3.3).

5.

Note on boosted controls: Nosotros recommend performing a reverse transcription reaction in the absence of template to ensure the reagents being used are free from contaminating RNA/Dna. It is also essential to include control reactions that lack RT enzyme for each biological replicate, which allows confirmation that the PCR reaction (Section 2.3.3) is amplifying but cDNA generated through reverse transcription and non contaminating gDNA from other sources.

2.3.iii PCR distension and purification of cDNAs

i.

In order to PCR amplify cDNA, first set 50   μL PCR reactions using Phusion Hot Start Flex DNA polymerase according to the manufacturers recommended protocol using i   μL TGIRT reaction production as template. Terminal reaction conditions used are one   × HF buffer, 200   μM dNTPs, 0.five   μM forward primer, 0.5   μM contrary primers, i unit of measurement polymerase, and 1   μL TGIRT reaction production. Note: A PCR reaction lacking template is recommended to check that PCR reagents are non contaminated with amplifiable genomic Dna. Annealing temperature and number of amplification cycles should be determined empirically for each cDNA to be analyzed. To amplify cDNAs derived from the ac4C-continaing helix 45 of human 18S rRNA, we use an annealing temperature of 67.4   °C and amplify for 33   cycles (Thomas et al., 2018).

2.

Purifying the PCR product by agarose gel purification/extraction is recommended prior to submission of PCR product for sequencing for best results. Mix PCR product with 6   × loading buffer and run on ii% agarose gel containing 1   × SYBRsafe Deoxyribonucleic acid gel stain until bromophenol bluish mark dye runs approximately two-thirds of the way across the gel. 1   μg of 100   bp dsDNA ladder should be run aslope PCR reaction products.

3.

Visualize PCR product on UV transilluminator and excise bands of expected size with a clean razor blade. Control reactions (minus RT and minus template) should exist run alongside and visualized to ensure no PCR product is nowadays in these samples.

four.

Process agarose gel slices using commercially available gel extraction kit, elute Dna and submit for Sanger sequencing with an advisable primer.

ii.3.iv Sanger sequencing-based analysis of cytidine acetylation

ane.

To calculate ac4C-dependent misincorporation, open processed Sanger sequencing traces using sequence trace viewing software. Free software which allow height heights to be calculated include 4peaks (Mac) and Applied Biosystems Sequence Scanner ii.0 (Windows). Simply high-quality sequence traces with low background noise and lilliputian or no acme ghosting should be analyzed.

two.

Place sites of ac4C by locating bases that announced as a mixture of C and T in borohydride treated samples (+   borohydride). Sites of high ac4C stoichiometry may produce more intense bespeak for T than C in sequencing chromatograms and may be assigned as T in base called sequences. Sites of ac4C exhibit substantially decreased C-to-T mutation in alkali pretreated controls (+   borohydride +   alkali) and just background levels of misincorporation (<   x%) in untreated controls (−   borohydride) (Fig. 5).

iii.

Quantify misincorporation at ac4C sites by measuring the meridian of C and T peaks in the sequencing traces and calculate misincorporation using the equation: pct misincorporation   =   100*(T-superlative)/((C-peak)   +   (T-peak)).

iv.

To estimate ac4C stoichiometry at a given site, fit calculated misincorporation to a standard curve. A standard curve for ac4C dependent misincorporation tin exist generated by plotting the average fraction of C-to-T conversion at the site of interest across a series of RNA mixtures prepared from divers ratios of wild-type and Nat10 knockout cells (100:0, fourscore:twenty, 60:40, etc.; Fig. 5).

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