Reverse Transcriptase

8 Key excerpts on "Reverse Transcriptase"

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  eBook - ePub

  Viruses: More Friends Than Foes

  • Karin Moelling(Author)
  • 2016(Publication Date)
  • WSPC

    (Publisher)

  Interestingly, the RT can perform two reactions: RNA to DNA and DNA to DNA, and between these two the original RNA, after it has been copied, has to be removed by the RNase H. The final result is double-stranded DNA.

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  The discovery of the reverse flow of information came as a great surprise. As a consequence the DNA can then be integrated into the host genomic DNA and be transmitted as long as the cell lives and divides as a cellular gene. Retroviruses normally do not lyse or destroy their host cells but “bud” out of the cells’ membranes. If we believe that RNA came first and that DNA came later during evolution, then “reverse” transcriptase is in fact the wrong name: the step from RNA to DNA is not a reverse one but a straight-ahead one. A strictly correct name would therefore be “real transcriptase”! Indeed, this would allow the same abbreviation to be used — however, nobody is interested in such a change.

  Now that, figuratively speaking, every possible genome has been sequenced, the latest surprise has been that there are numerous Reverse Transcriptases around. They are present in many organisms, in all eukaryotes (animals and plants), and also in archaea, in bacteria, in spliceosomes, in retrotransposons, in a strange chimeric multi-satellite msDNA, and in human and bacterial immune systems. In bacteria alone there are more than 1000 different kinds of Reverse Transcriptases. What are they all there for? In mammalian cells we know about the retrotransposons, which code for Reverse Transcriptases necessary for the “copy-and-paste” mechanism of cellular DNA described below (retrotransposons are reminiscent of simplified retroviruses). How unexpected this was can be described by an anecdote. In 1978 one of the co-discoverers of the Reverse Transcriptase David Baltimore got up in a meeting when someone described the existence of the Reverse Transcriptase in flies: “To my knowledge flies do not have retroviruses.” Only much later now we know the answer — also flies have Reverse Transcriptases not from retroviruses but from their relatives, the retrotransposons, which are precursors or truncated “crippled” retroviruses and widely distributed including flies, they are extremely abundant. The retroviruses as special case happened to be discovered first!

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  Gene and Cell Therapy

  Therapeutic Mechanisms & Applications

  • (Author)
  • 2014(Publication Date)
  • The English Press

    (Publisher)

  The term retro in retrovirus refers to this reversal (making DNA from RNA) of the central dogma of molecular biology. Reverse Transcriptase activity outside of retroviruses has been found in almost all eukaryotes, enabling the generation and insertion of new copies of retrotransposons into the host genome. These inserts are transcribed by enzymes of the host into new RNA molecules that enter the cytosol. Next, some of these RNA molecules are translated into viral proteins. For example, the gag gene is translated into molecules of the capsid protein, the pol gene is transcribed into molecules of Reverse Transcriptase, and the env gene is translated into molecules of the envelope protein. It is important to note that a retrovirus must bring its own Reverse Transcriptase in its capsid, otherwise it is unable to utilize the enzymes of the infected cell to carry out the task, due to the unusual nature of producing DNA from RNA. Industrial drugs that are designed as protease and Reverse Transcriptase inhibitors can quickly be proved ineffective because the gene sequences that code for the protease and the Reverse Transcriptase can undergo many substitutions. These substitutions of nitrogenous bases, which make up the DNA strand, can make either the protease or the Reverse Transcriptase difficult to attack. The amino acid substitution enables the enzymes to evade the drug regiments because mutations in the gene sequences can cause physical or chemical change, which makes them harder to detect by the drug. When the drugs that are supposed to attack enzymes, such as protease, are designed, the manufacturers target specific sites on the enzyme. One way to attack these targets can be through hydrolysis of molecular bonds, which means that the drug will add molecules of H 2 O (water) to specific bonds. By adding molecules of water at a site on the virus, the drug breaks the previous bonds that were linked to each other.

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  DNA Replication and Related Cellular Processes

  • Jelena Kusic-Tisma(Author)
  • 2011(Publication Date)
  • IntechOpen

    (Publisher)

  6 Reverse Transcriptase and Retroviral Replication T. Matamoros, M. Álvarez, V. Barrioluen g o, G. Betancor and L. Menéndez-Arias Centro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Científicas – Universidad Autónoma de Madrid), Campus de Cantoblanco, Madrid Spain 1. Introduction Within each viral particle, retroviruses package two copies of a single-stranded RNA genome of about 10 kb. All of the viral genomes contain three major genes, arranged in the order: 5´-gag–pol–env -3´, and some retroviruses may also have accessory genes ( e.g. vif , vpr , tax , etc…). Structural proteins such as MA (matrix protein), CA (capsid protein) and NC (nucleocapsid protein) are encoded within gag . Envelope proteins that mediate viral entry (surface and transmembrane glycoproteins) derive from expression of the env gene. Virus-encoded enzymes such as the protease, the Reverse Transcriptase (RT) and the integrase, required to complete the viral life cycle, usually derive from the expression of pol . The reverse transcription of the viral single-stranded (+) RNA genome into double-stranded DNA is an essential step in retroviral replication and an important target for therapeutic intervention (for reviews, see Telesnitsky & Goff, 1997; Abbink & Berkhout, 2008; Sarafianos et al., 2009). Reverse transcription is a relatively complex process that requires the intervention of at least three elements: (i) the viral genomic RNA (that serves as template); (ii) a specific primer ( i.e . a transfer RNA); and (iii) the viral RT. Retroviral RTs are enzymes that possess two activities: (i) a DNA polymerase activity that uses either RNA or DNA as template, and (ii) an RNase H activity, which degrades RNA from RNA/DNA hybrids. Unlike eukaryotic DNA polymerases, retroviral RTs are devoid of 3´  5´ exonucleolytic proofreading and show intrinsic error frequencies of around 10 -4 to 10 -5 , well above the values reported for cellular DNA polymerases.

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  Enzymes of Nucleic Acid Synthesis and Modification

  Volume 1: DNA Enzymes

  • Samson T. Jacob(Author)
  • 2018(Publication Date)
  • CRC Press

    (Publisher)

  Table 1 ). Recent discoveries indicate that in addition to the RNase H and DNA polymerase activities of Reverse Transcriptase, the polymerase gene of retroviruses codes for a DNA endonuclease activity which may or may not be found associated with Reverse Transcriptase. This chapter will focus on the enzymatic and nucleic acid binding properties of these proteins and the role they play in the replication of retroviruses, and will attempt to place this analysis in perspective by making comparisons, where possible, with other better understood replicative systems.

  Retroviruses and their resident Reverse Transcriptases have been characterized from many different vertebrates. Most of our knowledge concerning Reverse Transcriptase and the replication of retroviruses is derived from studies with avian leukosis-sarcoma viruses (ALSV) and the enzyme from the avian myeloblastosis virus (AMV) complex of viruses, and to a lesser extent with murine leukemia virus (MuLV) and Moloney MuLV Reverse Transcriptase. This discussion will be confined to these two virus systems, which should portray a general picture applicable to most retroviruses and their enzymes.

  B. Background

  In order to understand the role of Reverse Transcriptase in the retrovirus life cycle a certain amount of background is essential.

  1. Life Cycle of Retroviruses

  The first event in the retrovirus life cycle is adsorption of the virion to the cell surface, followed by penetration of the virion into the cell cytoplasm. Uncoating of the virus particle to expose the internal core20 - 23 probably takes place within the cytoplasm the first 1 to 2 hr after infection.24 Viral RNA in the core is transcribed within the cytoplasm into linear duplex DNA16 , 25 - 27 which is transported to the nucleus where it can be converted to covalently closed circular DNA.28 Mature duplex DNA is integrated into the host genome.29 , 30 The mechanism of integration and the structure of the precursor to integration are not yet established. Subsequently, the integrated viral DNA is transcribed by cellular RNA polymerase31 and transcripts are processed and transported to the cytoplasm. Viral mRNA is translated into a series of precursor polyproteins which encapsidate 35S viral RNA32 and a specific subset population of cell tRNAs33 and the maturing particles move to the cell surface, from which they bud and acquire an outer envelope.32

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  Pratt's Essential Biochemistry

  • Charlotte W. Pratt, Kathleen Cornely(Authors)
  • 2020(Publication Date)
  • Wiley

    (Publisher)

  The result is a double-stranded DNA molecule (see diagram). tRNA viral RNA DNA Viral Reverse Transcriptase has proved to be more than a biological curiosity. It has become a valuable laboratory tool, allowing researchers to purify messenger RNA transcripts from cells, transform them to DNA (called cDNA for complementary DNA), and then quantify them, sequence them, or use them to direct protein synthesis. Reverse Transcriptase activity can be blocked by two different types of drugs. Nucleoside analogs such as AZT and ddC readily enter cells and are phosphorylated. The resulting nucleotides bind in the Reverse Transcriptase active site and are linked, via their 5ʹ phos- phate group, to the growing DNA chain. However, because they lack a 3ʹ OH group, further addition of nucleotides is impossible. Reverse Transcriptase can also be inhibited by non-nucleoside analogs such as nevirapine, a noncompetitive inhibitor that binds to a hydrophobic patch on the surface of Reverse Transcriptase near the base of the thumb domain. This does not interfere with RNA or nucleotide binding, but it does inhibit polymerase activity, probably by restricting thumb movement. HIV infections are typically treated with a “cocktail” of drugs that often includes a Reverse Transcriptase inhibitor along with a protease inhibitor (see Box 7.A). Explain why the drugs described here interfere only mini- mally with nucleic acid metabolism in the human host. 530 DNA Damage and Repair 531 of DNA appears to fold back on itself to form a structure called a T-loop (Fig. 20.16). Multiple copies of six different proteins, collectively known as shelterin, bind to the telomere. These proteins play a critical role in regulating telomere length, which may be an indicator of the cells’ longevity. IS TELOMERASE ACTIVITY LINKED TO CELL IMMORTALITY? Cells that normally undergo a limited number of cell divisions appear to con- tain no active telomerase.

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  A Century of Nature

  Twenty-One Discoveries that Changed Science and the World

  • Laura Garwin, Tim Lincoln, Laura Garwin, Tim Lincoln(Authors)
  • 2010(Publication Date)
  • University of Chicago Press

    (Publisher)

  1970 T W E L V E Viruses reverse the genetic flow Robin A. Weiss In 1970 it emerged that certain viruses that have their genes in the form of RNA can copy the RNA “backward” into DNA in infected cells. This discovery has had an immense impact. The enzyme concerned, Reverse Transcriptase, makes possible the manufacture of specific proteins for use as medicines. It is also the best target for attacking HIV, the cause of AIDS. With the rise of molecular biology in the 1950s and 1960s, it became widely believed that the flow of biological information goes in one direction only— that RNA is made according to information stored in the genetic material DNA, and that protein is then created from the instructions in the RNA. On 27 June 1970, two short papers published in Nature by David Baltimore 1 and by Howard Temin and Satoshi Mizutani 2 showed that the flow can be reversed. As with many key discoveries, the experiments were quite simple. The authors showed that purified particles of two cancer-causing (tumor) viruses—Rous sarcoma virus of chickens and Rauscher leukemia virus of mice—contain a reverse transcription enzyme activity that makes DNA us-ing RNA as the template. Living cells faithfully replicate their genetic material, the DNA, so that an exact copy is reproduced in each daughter cell (see p. 234). Replication is catalyzed by an enzyme, DNA polymerase. To read out the genetic infor-mation, another enzyme, called RNA polymerase or transcriptase, makes RNA that contains the same genetic sequence as one of the DNA strands. This “messenger” RNA is then translated into proteins, the molecules that form the structure of living cells and carry out their functions. But the genetic material of certain viruses—very small parasites inside 168 r o b i n a . w e i s s living cells—is in the form of RNA. This was first shown in 1955 for a virus of plants, tobacco mosaic virus, and it is true of many human viruses too.

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  Essential Biochemistry

  • Charlotte W. Pratt, Kathleen Cornely(Authors)
  • 2017(Publication Date)
  • Wiley

    (Publisher)

  After entering a cell, the HIV particle disassembles. The 9-kb viral RNA is then transcribed into DNA by the action of the viral enzyme Reverse Transcriptase. Another viral enzyme, an integrase, incorporates the resulting DNA into the host genome. Expression of the viral genes produces 15 differ- ent proteins, some of which must be processed by HIV protease to achieve their mature forms. Eventually, new viral particles are as- sembled and bud off from the host cell, which dies. Because HIV preferentially infects cells of the immune system, cell death leads to an almost invariably fatal immunodeficiency. HIV Reverse Transcriptase, which synthesizes DNA from an RNA template (a contradiction of the central dogma outlined in Section 3.3), resembles other polymerases in having fingers, thumb, and palm domains. These parts of the protein (colored red in the model shown here) comprise a polymerase active site that can use either DNA or RNA as a template (no other enzyme has this dual specificity). A separate domain (green) contains an RNase active site that degrades the RNA template. [Structure of HIV Reverse Transcriptase (pdb 1BQN) determined by Y. Hsiou, K. Das, and E. Arnold.] Reverse transcription occurs as follows: The enzyme binds to the RNA template and generates a complementary DNA strand. In a host cell, DNA synthesis is primed by a trans- fer RNA molecule. As polymerization proceeds, the RNase active site degrades the RNA strand of the RNA–DNA hybrid molecule, leaving a single DNA strand that then serves as a template for the Reverse Transcriptase polymerase active site to use in synthesizing a second strand of DNA. The result is a double-stranded DNA molecule (see diagram). tRNA viral RNA DNA Viral Reverse Transcriptase has proved to be more than a biological curiosity.

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  PCR/RT- PCR in situ

  Light and Electron Microscopy

  • Gerard Morel, Mireille Raccurt(Authors)
  • 2002(Publication Date)
  • CRC Press

    (Publisher)

  4.3.2 Deoxynucleotide Triphosphates (dNTPs) In reverse transcription, four triphosphate nucle-otides are used at an equimolar concentration: ➫ In the opposite case, the fidelity of the enzyme and the length of the transcribed cDNA can be affected. • dATP ➫ Deoxyadenosine • dTTP ➫ Deoxythymidine • dCTP ➫ Deoxycytosine • dGTP ➫ Deoxyguanosine TATAA E 2 E 1 E 3 E 4 E 5 ATG I 1 I 2 I 3 I 4 I 4 5 ′ 3 ′ Reverse Transcription (RT) 74 4.3.3 Enzymes The most frequently used enzymes are: • AMV Reverse Transcriptase ➫ Avian myeloblastosis virus • M-MLV Reverse Transcriptase ➫ Moloney murine leukemia virus • Tth DNA polymerase ➫ Thermostable polymerase of Thermus thermophilus Given that the manufacturers of these enzymes give information about their particular charac-teristics, this paragraph will present only their main features and modes of action. ➫ The specifics of the enzyme have to be taken into account in the RT step. 4.3.3.1 AMV Reverse Transcriptase ❶ Origin AMV Reverse Transcriptase is extracted from the avian myeloblastosis virus. It is an αβ -holoenzyme with a molecular weight of 157 kDa. The mature αβ dimer has different enzymatic activities: ➫ The α subunit is derived from the β subunit by proteolysis. • RNA-dependent DNA polymerase • DNA-dependent DNA polymerase • RNase H ❷ Quantity supplied The quantity supplied corresponds to a level of activity varying between 250 and 1000 U. The conservation buffer contains 50% glycerol to avoid freezing. ➫ A unit (U) is defined as the quantity of enzyme which, in 10 min at 37 ° C, incorporates 1 nmol of insoluble dTTP in an acid medium, starting with an RNA [poly (A)] matrix and an oligo (dT) primer ( see Section 4.3.1). ❸ Conservation In order for the enzyme to retain its maximum level of activity, it should in general be stored in aliquots at − 20 ° C. ➫ Conservation time is extended by storage at − 70 ° C, and according to some suppliers an enzyme will remain stable for 24 months in such conditions.

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