Reverse Transcriptase

G. Maga , in Brenner's Encyclopedia of Genetics (2d Edition), 2013

Reverse Transcriptase and Retrotransposons

Retrotransposons are genetic mobile elements that use reverse transcription to generate a Deoxyribonucleic acid copy of themselves to be inserted into eukaryotic genomes. Similarly to retroviruses, their genomes possess the gene for reverse transcriptase, RNase H, and protease, but they lack the genes for the proteins of the viral capsid, so they do not generate virions and therefore practice not spread among individuals, only 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 contrary transcriptase initiates DNA synthesis from an 11-nt RNA primer generated by self-annealing of the eleven terminal bases of the RNA genome to a complementary sequence located inside the five′ end and subsequently cleaved by its RNAse H activity to generate the 3′-hydroxyl end of the primer. Some other unique feature of this enzyme is its ability to add together i or two nucleotides to the 3′-concluding end of the DNA copy in a template-independent style. This mechanism protects the 3′ finish of the transposon Dna from deposition past cellular exonucleases.

The Bombyx mori retrotransposon R2 also encodes a reverse transcriptase with unusual enzymatic properties. It tin utilize a 3′-hydroxyl end from any RNA or Dna molecule to prime its reaction without the need of any complementarity between the primer and the template.

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Dna Polymerases

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

ii. Nucleotide sequencing.

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

Reverse transcriptases are likewise valuable reagents in the direct dideoxynucleotide sequencing of RNA (110). In fact, RTases accept been instrumental in obtaining nucleotide sequence information directly from viral RNA genomes, 16S rRNA (111), and mRNAs (112). The college 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 Caused Immunodeficiency Syndrome

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

i) Reverse Transcriptase

Reverse Transcriptase (RT) is essential for HIV replication because the viral RNA genome on its own is highly susceptible to degradation by intracellular RNases. RT speedily makes a much more nuclease-resistant double-stranded DNA re-create of the RNA template that subsequently integrates to form the proviral Deoxyribonucleic acid. HIV RT is a heterodimer composed of a 66 kDa subunit (p66) and a 51 kDa subunit (p51) created by cleavage of a separate molecule of p66. All the catalytic action of HIV RT is attributable to p66, while p51 supports the functions of p66. Too as its Dna polymerase function, RT has an RNase H office that degrades the RNA template used to make the viral Deoxyribonucleic acid. The complex method by which the RT synthesizes the viral Deoxyribonucleic acid from the genomic ssRNA is described in Figure 25-4.

Figure 25-iv. Synthesis of HIV DNA from Genomic RNA

(one) A cellular tRNAlys3 molecule hybridizes with the primer binding site (PBS) on the genomic +RNA strand and HIV RT commences synthesis of the negative strand Dna (—DNA) in the 3′ direction. (2) As the — Deoxyribonucleic acid elongates, the v′ 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 iii′ end of the genomic RNA where the R' sequence in the − Dna hybridizes to the R sequence in the + RNA. (4) Synthesis of the —Deoxyribonucleic acid strand then continues in the 5′ to 3′ direction until it is completed. (v) The genomic +RNA is degraded by RT RNase H action with the exception of 2 poly-purine tracts (cPPT and PPT) that remain hybridized to the — Deoxyribonucleic acid. Using the —Deoxyribonucleic acid every bit the template and both PPTs as primers, the positive strand DNA (+DNA) is elongated toward its three′ end until a PBS site is created (half-dozen). (7) The tRNAlys3 at the v′ terminate of the –DNA strand and the PPTs are degraded. (viii) Hybridization of the PBS' with the PBS site results in circularization of the — and + DNA strands and elongation of the +Deoxyribonucleic acid (9) The U3′-R-U5′ sequence of the − Deoxyribonucleic acid is used equally a template to complete the 3′ end of the + DNA strand and form the 3′ LTR of the proviral Deoxyribonucleic acid. Elongation of the —Deoxyribonucleic acid strand through to its 3′ finish copies the 5′+Dna to produce the 5′ LTR of the provirus. Elongation of the +DNA strand ends at a termination site 3′ of the cPPT'. The resulting overlap in the +Deoxyribonucleic acid strand is not shown in the viral DNA. (10) Note that R-U5 and U3-R from the genomic RNA have now both become U3-R-U5 in the viral Dna, giving it the required LTR at each end. The R site in the iii′ LTR contains a cleavage site and a polyadenylation site used for the transcription of viral mRNA.

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

HIV RT is responsible for much of the antigenic variation of HIV that confounds both the natural allowed 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 error-decumbent. Mutations due to uncorrected RT activity appear in the HIV genome at a charge per unit of nearly 1 in every 1500–4000 nucleotides per replication cycle. As a point of comparison, the boilerplate rate of mutation of the human cellular genome is 1 in xvii–108 base of operations pairs.

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Viral Tools for In Vitro Manipulations of Nucleic Acids

Boriana Marintcheva , in Harnessing the Power of Viruses, 2018

2.three.2.2 Reverse Transcriptases

Reverse transcriptases (RTs) are RNA dependent DNA pols initially isolated from retroviruses. In addition, RTs are coded by dsRNA viruses that apply reverse transcription such as hepatitis B virus (replication of hepatitis is discussed in Chapter 1); and diverse 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 singled-out from conventional RTs. Historically, the discovery of RT revolutionized molecular biology leading to the revision of the central dogma and enabling scientists to develop new research tools that heavily influenced cloning, analysis of factor expression and RNA biology. HIV RT is one of the well-nigh extensively studied polymerases in the context of agreement the biology of this devastating virus and designing RT inhibitors as drugs to manage HIV infections. RTs exhibit 3 primal enzyme activities (Fig. two.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; (2) RNAse H endonuclease, which selectively degrades the RNA strand of Dna/RNA hybrids and (3) DNA dependent Deoxyribonucleic acid polymerase activity, converting the single-stranded cDNA into dsDNA. Conventional RT enzymes have two active sites, one executing the polymerase activities and another executing the endonuclease activity. RT are monomeric or dimeric proteins and some lack intrinsic RNAse H activeness. The RTs of Moloney murine leukemia virus (One thousand-MLV) and Avian myeloblastosis virus (AMV) are most oft used as molecular tools in RT-PCR, RT-qPCR, cDNA cloning, RNA sequencing and any other experimental technique/arroyo that requires conversion of RNA to Deoxyribonucleic acid. Site-directed mutagenesis and protein evolution take been utilized to optimize those enzymes improving thermostability and modulating RNAseH action. Using thermostable version of RT is benign for lowering the nonspecific nucleic acid amplification and minimizing impact of circuitous secondary structures. Robust RNAseH activity is an advantage in RT-PCR, whereas lower RNAseH activity is beneficial in cDNA cloning protocols, especially 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 pol carries out the cDNA to dsDNA polymerization step.

Effigy ii.xiii. Opposite transcriptase activities and mechanism of activeness.

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Viral Replication Enzymes and their Inhibitors Part B

Nicolas Sluis-Cremer , in The Enzymes, 2021

Abstract

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

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DNA Polymerases: Reverse Transcriptase Integrase, and Retrovirus Replication

Chiliad.-L. Andréola , ... Due south. Litvak , in Encyclopedia of Biological Chemistry (Second Edition), 2013

Abstract

Contrary 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 Dna once the RNA has been contrary transcribed in a first step into a single-strand Dna. RNA viruses, such equally retroviruses, use the enzyme to opposite-transcribe their RNA genomes into Dna, which is and then integrated into the host genome and replicated forth with it. During the replication of some Deoxyribonucleic acid viruses, such equally the hepadnaviruses or pararetroviruses, as well conveying a RT, the Deoxyribonucleic acid genome is transcribed to RNA that serves every bit a template to make new viral Deoxyribonucleic acid strands.

Although RT was discovered in retroviruses and idea to be a paradigm of these infectious agents, information technology is currently known that RT is institute in many other eukaryotic and prokaryotic systems similar telomerase, retrotransposons, retrons, and are plant abundantly in the genomes of plants and animals. Retroviral RT has a domain conveying a ribonuclease H (RNase H) activity 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 class of RT or as a free enzyme. Integrase is vital for the insertion of the double-stranded Dna synthesized by RT in the genome of the host-infected cell. Inhibitors of the DNA polymerase activity of retroviral RT are widely used in the handling of pathologies produced by retroviruses similar in the example of acquired immune deficiency syndrome.

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DNA Replication, Repair, and Mutagenesis

North.5. Bhagavan , Chung-Eun Ha , in Essentials of Medical Biochemistry (2d Edition), 2015

Opposite Transcriptase

Opposite transcriptase is an RNA-dependent Dna polymerase that was discovered in many retroviruses such as man immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) in 1970. The opposite transcriptase catalyzes the conversion of RNA template molecules into a Deoxyribonucleic acid double helix and provides a very useful tool for molecular biology research. Reverse transcriptases are commonly used to produce complementary DNA (cDNA) libraries from various expressed mRNAs and are too used to quantify the level of mRNA synthesis when combined with the polymerase chain reaction technique, called RT-PCR. Contrary transcriptase contains 3 enzymatic activities: (1) RNA-dependent DNA polymerase, (2) RNase H, and (3) DNA-dependent DNA polymerase. First, RNA-dependent DNA polymerase synthesizes a Dna strand complementary to the RNA template. And then RNase H removes the RNA strand from the RNA–Dna hybrid double helix. Then the Deoxyribonucleic acid-dependent DNA polymerase completes double-stranded Dna synthesis. Dissimilar other Dna polymerases, reverse transcriptase lacks a proofreading capability and therefore has high fault rates during DNA synthesis, up to i mistake in 2000 base incorporations. The high mistake 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.two HIV RT Inhibitors

HIV RT is involved in viral opposite transcription, which converts the viral RNA into the DNA (run into Fig. 26.4). HIV RT has been the nigh exploited antiviral drug target ever, resulting in the development 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, seven NRTIs and five NNRTIs are currently available. Zidovudine (AZT) and lamivudine (3TC), lacking the 3′OH grouping, human activity as a chain terminator of the viral reverse transcription (Fig. 26.seven). These nucleoside analogs are prodrugs in that they act as an inhibitor only after conversion into a triphosphate form. On the other hand, NNRTIs, such as nevirapine (NVP) and efavirenz (ETR), demark to a region that is distinct from the dNTP-binding site on the viral RT poly peptide. In other words, these NNRTIs act as allosteric inhibitors.

Effigy 26.7. HIV RT inhibitors.

Zidovudine (AZT) and lamivudine (3TC) are NRTIs that act as chain terminators of HIV RT following conversion into a triphosphate form in cells. Nevirapine (NVP) and efavirenz (ETR) are NNRTIs. Three-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

4 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 factor is very large, hard to manipulate, and almost incommunicable to express in whatsoever other type of organism. Since mRNA has had the introns removed naturally, information technology may be used as the source of an uninterrupted coding sequence that is much more convenient for technology and expression. This involves converting the RNA dorsum into a Dna copy, known as complementary Dna (cDNA) by reverse transcriptase . Thus, when amplifying eukaryotic genes by PCR the cDNA version is oft used (rather than the true chromosomal gene sequence) since this lacks the introns.

Opposite transcriptase is an enzyme found in retroviruses that converts the RNA genome carried in the retrovirus particle into double-stranded DNA. Reverse transcriptase first transcribes a complementary strand of Deoxyribonucleic acid to make an RNA:Deoxyribonucleic acid hybrid. Next, opposite transcriptase or RNase H degrades the RNA strand of the hybrid. The single-stranded DNA is then used as a template for synthesizing double-stranded Deoxyribonucleic acid (cDNA). Once the cDNA has been fabricated, PCR can exist used to dilate the cDNA and generate multiple copies (Fig. vi.13). This combined process is referred to every bit reverse transcriptase PCR (RT-PCR) and allows genes to be amplified and cloned as intron-free DNA copies starting from mRNA.

Figure 6.xiii. Contrary Transcriptase PCR

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

Reverse transcription followed by 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 dissimilar growth conditions reveals when a gene of interest is expressed (i.e., when the corresponding mRNA is present) and what environment induces gene expression. To compare two different weather, mRNA is extracted from cells growing in both weather. RT-PCR is performed on the 2 samples of mRNA using PCR primers that match the particular gene of interest. If the cistron is expressed, a PCR product volition be produced, whereas if the gene is switched off, its mRNA volition exist missing (Fig. 6.14).

Figure 6.14. RT-PCR for Factor Expression

RT-PCR can determine the corporeality of mRNA for a particular gene in two dissimilar growth conditions. In this case, the factor of interest is expressed in condition 1 merely not in condition 2. Therefore, in status 1   mRNA from the gene of involvement is present, reverse transcriptase generates a cDNA, and PCR amplifies this cDNA into many copies. In status 2 the mRNA is absent and and so the RT-PCR procedure does not generate the respective Dna.

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

Justin One thousand. Thomas , ... Hashemite kingdom of jordan Fifty. Meier , in Methods in Enzymology, 2019

2.3 Nucleotide resolution sequencing of individual ac4C sites

ii.3.ane Materials

TGIRT-III reverse transcription enzyme (INGEX)

v   × TGIRT reaction buffer (user prepared with nuclease-complimentary reagents, ii.25   Grand NaCl, 100   mM Tris-HCL pH   7.5)

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

Optimized dNTP mix [10   mM dTTP, 10   mM dCTP, x   mM dATP, 5   mM dGTP] (prepared from 100   mM dNTPs prepare, Nib N0446S)

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

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

100   bp DNA Ladder (New England Biolabs, N3231S)

SYBR Safe Dna Gel Stain (Invitrogen, S33102)

UltraPure TBE Buffer, x   × (Invitrogen, 15581044)

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

half-dozen   × DNA gel loading dye (Neb, B7021S)

QIAquick Gel extraction kit (Qiagen, 28704)

Thermocycler

Agarose gel electrophoresis equipment.

two.iii.2 Reverse transcription of borohydride-treated RNA

Reverse transcription of sodium borohydride-treated ac4c results in partial incorporation of adenosine in the cDNA strand opposite the reduced ac4C. It should also be noted that reduced ac4C results in partial termination of opposite transcription at or next to its location, often referred to as RT-stops (Fig. 4 A). Of the reverse transcriptase enzymes screened by our lab TGIRT-3 RT (Ingex) produces the highest proportion of full-length to stop product, too as the almost robust C-to-T signature of the RT enzymes screened by our lab ( Fig. 4B). The following protocol describes the utilise of TGIRT RT for sequence-defined cDNA generation, which we accept used to profile ac4C in model substrates and human 18S rRNA (Thomas et al., 2018).

1.

Dilute RNA from Department two.two.2 to 100   pg/μL in water for use as template.

2.

Perform TGIRT RT reactions. Nosotros typically carry out RT in twenty   μL volumes in PCR tubes. Exemplary protocol: combine four   μL five   × TGIRT reaction buffer with 200   pg (ii   μL) of template, 4 pM Deoxyribonucleic acid 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 bachelor reverse transcription enzymes. Therefore, it is essential to blueprint 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)

3.

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

4.

After cooling, add 0.5   μL TGIRT-III, 0.5   μL Rnasin Plus and 100   μL 100   mM DTT. Incubate twenty   min at room temperature. Initiate the reverse transcription reaction past calculation i   μL of optimized dNTP mix. In optimization studies nosotros accept constitute 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 go along to PCR amplification footstep (Section 2.3.three).

5.

Note on additional controls: We recommend performing a reverse transcription reaction in the absence of template to ensure the reagents being used are complimentary 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 only cDNA generated through contrary transcription and not contaminating gDNA from other sources.

2.3.iii PCR distension and purification of cDNAs

i.

In order to PCR amplify cDNA, first prepare fifty   μL PCR reactions using Phusion Hot Starting time Flex Deoxyribonucleic acid polymerase according to the manufacturers recommended protocol using 1   μL TGIRT reaction product as template. Concluding reaction conditions used are ane   × HF buffer, 200   μM dNTPs, 0.v   μM frontward primer, 0.5   μM reverse primers, 1 unit of measurement polymerase, and 1   μL TGIRT reaction product. Note: A PCR reaction lacking template is recommended to check that PCR reagents are not contaminated with amplifiable genomic DNA. Annealing temperature and number of distension cycles should exist determined empirically for each cDNA to exist analyzed. To amplify cDNAs derived from the ac4C-continaing helix 45 of man 18S rRNA, we utilize an annealing temperature of 67.four   °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 six   × loading buffer and run on 2% agarose gel containing one   × SYBRsafe Dna gel stain until bromophenol blueish marker dye runs approximately 2-thirds of the way beyond the gel. 1   μg of 100   bp dsDNA ladder should be run alongside 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 be 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 appropriate primer.

2.iii.4 Sanger sequencing-based analysis of cytidine acetylation

i.

To calculate ac4C-dependent misincorporation, open processed Sanger sequencing traces using sequence trace viewing software. Free software which allow peak heights to be calculated include 4peaks (Mac) and Applied Biosystems Sequence Scanner 2.0 (Windows). Simply high-quality sequence traces with low background noise and little or no acme ghosting should be analyzed.

2.

Identify sites of ac4C by locating bases that appear as a mixture of C and T in borohydride treated samples (+   borohydride). Sites of loftier ac4C stoichiometry may produce more intense signal for T than C in sequencing chromatograms and may exist assigned as T in base called sequences. Sites of ac4C exhibit substantially decreased C-to-T mutation in alkali pretreated controls (+   borohydride +   brine) and but background levels of misincorporation (<   10%) in untreated controls (−   borohydride) (Fig. v).

iii.

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

four.

To estimate ac4C stoichiometry at a given site, fit calculated misincorporation to a standard bend. A standard curve for ac4C dependent misincorporation can be generated past plotting the average fraction of C-to-T conversion at the site of involvement beyond a serial of RNA mixtures prepared from divers ratios of wild-blazon and Nat10 knockout cells (100:0, eighty:xx, lx:40, etc.; Fig. 5).

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