Reverse transcriptase ribonuclease

HIV contains a cylindrical core within its capsid. In addition, to two copies of its (+)-ssRNA genome, the core contains several enzymes reverse transcriptase, ribonuclease, integrase, and protease. The RNA molecules are coated with multiple copies of two low-molecular-weight proteins, p7 and p9. (The numbers in these and other protein names indicate their kilodalton mass for example, p7 is a protein with a 7-kD mass.) The bullet-shaped core itself is composed of hundreds of copies of p6, and copies of pl7 form an inner lining of the viral envelope. The envelope of HIV contains two major viral proteins, gpl20 and gp41, in addition to host proteins. 

Retrovirus RNA-Dependant DNA polymerase (reverse transcriptase) Ribonuclease H Endoribonuclease Protein-cleaving enzyme Protein kinase... 

After the virus has attached to CD4 and chemokine receptors, another viral glycoprotein (gp41) assists with viral fusion to the cell and internalization of the viral contents. The viral contents include single-stranded RNA, an RNA-dependent DNA polymerase (also known as reverse transcriptase), and other enzymes. Using the single-stranded viral RNA as a template, reverse transcriptase synthesizes a complementary strand of DNA. The single-stranded viral RNA is removed from the newly formed DNA strand by ribonuclease H, and reverse transcriptase completes the synthesis of double-stranded DNA. The... 

Divalent metal ions are essential for ribonuclease H activity. Two Mn(II) ions have been located in the catalytic site of ribonuclease H domain of HIV-1 reverse transcriptase in close proximity to the four acidic residues Asp443, Glu478, Asp498, and Asp549 after soaking crystals in 45 mM MnCl2 (406). 

List of Abbreviations PCR, polymerase chain reaction RT-PCR, reverse transcription polymerase chain reaction DNA, deoxyribonucleic acid RNA, ribonucleic acid RNase, ribonuclease mRNA, messenger RNA GABAa, y-aminobutyric acid type A cRNA, copy RNA dNTPs, deoxy nucleoside triphosphates MMLV, Mouse Moloney murine leukemia vims RT, reverse transcriptase bp, base pair Tm, melting temperature DEPC, diethylpyrocarbonate OD, optical density mL, milliliter SA-PMPs, streptavidin paramagnetic particles dT, deoxy thymidine DTT, dithiothreitol DNase, deoxyribonuclease RNasin, ribonuclease inhibitor UV, ultraviolet TBE, Tris-borate, 1 mM EDTA EDTA, ethylenediaminetetraacetic acid Buffer RET, guanidium thiocyanate lysis buffer PBS, phosphate buffered saline NT2, Ntera 2 neural progenitor cells... 

List of Abbreviations cDNA, complementary DNA ddH20, double-distilled H2O dNTP, deoxyribonu-cleotide triphosphate EDTA, ethylenediaminetetraacetic acid MgCl2, magnesium chloride mRNA, messenger ribonucleic acid NaOH, sodium hydroxide PCR, polymerase chain reaction qRT PCR, quantitative reverse transcriptase polymerase chain reaction RNase, ribonuclease RT PCR, reverse transcriptase polymerase chain reaction UTR, untranslated region... 

Palaniappan C, Fay PJ, Bambara RA. Nevirapine alters the cleavage specificity of ribonuclease H of human immunodeficiency virus 1 reverse transcriptase. J Biol Chem 1995 270 4861-4869. 

Champoux JJ. Roles of ribonuclease H in reverse transcription. In Skalka AM, Goff SP, eds. Reverse Transcriptase. Plainview, New York Cold Spring Harbor Laboratory Press, 1993 103-117. 

Davies JF, Hostomska Z, Hostomsky Z, Jordan SR, Matthews DA. Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase. Science 1991 252 88-95. 

Hizi A, Hughes SH, Shaharabany M. Mutational analysis of the ribonuclease H activity of human immunodeficiency virus 1 reverse transcriptase. Virology 1990 175 575-580. 

Cirino NM, Cameron CE, Smith JS, Rausch JW, Roth MJ, Benkovic SJ, et al. Divalent cation modulation of the ribonuclease functions of human immunodeficiency vims reverse transcriptase. Biochemistry 1995 34 9936-9943. 

Mizrahi V, Brooksbank RL, Nkabinde NC. Mutagenesis of the conserved aspartic acid 443, glutamic acid 478, asparagine 494, and aspartic acid 498 residues in the ribonuclease H domain of p66/p51 human immunodeficiency vims type 1 reverse transcriptase. JBiol Chem 1994 269 19245-19249. 

Synthesis of cDNA, usually in radiolabeled form is accomplished with reverse transcriptase, the enzyme from retroviruses that synthesize a DNA-RNA hybrid from ssRNA.570 572 A short oligo (dT) primer is usually hybridized to the 3 poly (A) tail to initiate synthesis. Reverse transcriptase also has ribonuclease (RNase H) activity and will digest away the RNA. If desired, synthesis of the second strand can be carried out by a DNA polymerase to give a complete DNA duplex. Many gene sequences have been deduced from cDNA copies. 

Other DNA polymerases. Reverse transcriptases synthesize DNA using an RNA template strand. They are best known for their function in retroviruses (Chapter 28). The HIV reverse transcriptase is a heterodimer of 51- and 66-kDa subunits. The larger subunit contains a ribonuclease H domain.288-2893 The enzyme is a prime target for drugs such as AZT and others.290 291 A different reverse transcriptase is found in all eukaryotic cells in telom-erase, an enzyme essential for replication of chromosome ends. Reverse transcriptases have also been found in rare LI sequences that are functioning ret-rotransposons (Section D).292... 

Rennin—see chymosin Repertoire selection 415-418 Reporter group 276 Resonance Raman spectra 476 Restriction endonucleases 406-408 Restriction fragment 408 Retention of stereochemical configuration 253, 254 Reverse genetic s 438 -442 Reverse transcriptase 3 8 Rhizopus-pepsin 486, 490 Ribonuclease A 9... 

Essential degradation. Reverse transcriptase has ribonuclease activity as well as polymerase activity. What is the role of its ribonuclease activity ... 

Katayamgi, K., Miyagawa, M., Matsushima, M., Ishikawa, M., Kanaya, S., Nakamura, H., Ikehara, M., Matsuzaki, T., and Morikawa, K. (1992). Structural details of ribonuclease H itom Escherichia coli as refined to an atomic resolution./. Mol. Biol. 223, 1029-1052. Unge, T., Knight, S., Bhikhabhai, R., Lovgren, S., Dauter, Z., Wilson, K., and Strandberg, B. (1994). 2.2 A resolution structure of the amino-terminal half of HIV-1 reverse transcriptase (fingers and palm subdomains). Structure 2,953-961. 

After internalization, the virus is uncoated in preparation for replication. The genetic material of HIV is positive-sense singlestrand RNA (ssRNA) the virus must transcribe this RNA into DNA to optimally replicate in human cells (transcription normally occurs from DNA to RNA—HIV works backward, hence the name retrovirus). To do so, HIV is equipped with a unique enzyme, RNA-dependent DNA polymerase (reverse transcriptase). Reverse transcriptase first synthesizes a complementary strand of DNA using the viral RNA as a template. The RNA portion of this DNA-RNA hybrid is then partially removed by ribonuclease H (RNase H), allowing reverse transcriptase to complete the synthesis of a double-stranded DNA (dsDNA) molecule. Unfortunately, the fidelity of reverse transcriptase is poor. 

There are two different methods to identify modified residues and ribonuclease scissions in RNA molecules the reverse transcriptase method or the end-labelling method. The choice of method depends both on the length of the studied RNA and the method of probing (as discussed in Sections 4.4.1 and 4.4.2). The reverse transcription method uses extension of a primer and therefore is independent of the length of the RNA, while the end-labelling method is restricted to analysis of small RNA molecules (n < 300). The latter method requires scission of the RNA. 

Modified bases in the RNA will stop or pause the reverse transcriptase 3 to the modified base. Breaks in the RNA induced by ribonucleases or chemicals will also terminate the polymerase. The electrophoretic analysis of the reverse transcript reveals the stops as bands on an autoradiograph. The position of the modification or scission can be determined by co-electrophoresis of a dideoxy sequence reaction, and the intensity of the band will reflect the reactivity of the base (Figs 4.7 and 4.10). 

The use of isolated enzymes to form or cleave P-O bonds is an important application of biocatalysts. Restriction endonucleases, (deoxy)ribonucleases, DNA/ RNA-ligases, DNA-RNA-polymerases, reverse transcriptases etc. are central to modern molecular biology(1). Enzyme catalyzed phosphoryl transfer reactions have also found important applications in synthetic organic chemistry. In particular, the development of convenient cofactor regeneration systems has made possible the practical scale synthesis of carbohydrates, nucleoside phosphates, nucleoside phosphate sugars and other natural products and their analogs. This chapter gives an overview of this field of research. 

Manganese(II) ions may also be employed in the hydrolases that compose the ribonuclease H domain of reverse transcriptases (3, 4). Many of these enzymes, which may employ either Mn" or Mg11, are found in a variety of organisms where they may or may not be essential (e.g., Escherichia coli) (3, 4). This class of enzymes catalyzes the hydrolysis of DNA-RNA hybrids. However, retroviral reverse transcriptases are critical for the replication of retroviruses, and Mn11 may be the required cofactor for these ribonuclease hydrolases to function (3, 4). One example of this family of hydrolase enzymes is the RNase H enzyme from HIV-I. This enzyme has been crystallographically characterized (11). In the crystal structure at 2.4-A resolution, the two Mn 1 ions are separated by a distance of about 4 A. They are bound to carboxylate residues that are located near the surface of the enzyme. One of these carboxylates bridges the two manganese... 

The biological applications of manganese are numerous and quite varied. Although most of these biological roles utilize manganese in structural or hydrolytic systems, some very striking redox systems are also known. The hydrolytic roles are, of course, not to be overlooked with respect to the widespread and critical arginase enzyme, which exhibits a strict requirement for two Mn" ions. The ribo-nuclease hydrolases of retroviral reverse transcriptase are another key nonredox application—for example, the ribonuclease hydrolase from HIV-1. 

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