Ribonuclease H RNase

However, there are a number of other miscellaneous biological roles played by this complex. The [Co(NH3)6]3+ ion has been shown to inhibit the hammerhead ribozyme by displacing a Mn2+ ion from the active site.576 However, [Co(NH3)6]3+ does not inhibit ribonuclease H (RNase),577 topoisomerase I,578 or hairpin ribozyme,579 which require activation by Mg2+ ions. The conclusions from these studies were that an outer sphere complex formation between the enzyme and Mgaq2+ is occuring rather than specific coordination of the divalent ion to the protein. These results are in contrast to DNase I inhibition by the same hexaammine complex. Inhibition of glucose-induced insulin secretion from pancreatic cells by [Co(NH3)6]3+ has been found.580 Intracellular injection of [Co(NH3)6]3+ into a neurone has been found to cause characteristic changes to the structure of its mitochondria, and this offers a simple technique to label neuronal profiles for examination of their ultrastructures.581... 

The ribonuclease H (RNase H) family of enzymes are ubiquitous nucleases that catalyze the hydrolysis of RNA in RNA DNA hybrids (for review, see 1). In contrast to the well-studied ribonucleases A and Tl, RNase H does not employ the 2 -OH in RNA as a nucleophile but instead activates water as the nucleophile for hydrolysis in a metal-dependent reaction. The number and role(s) of divalent metal in the RNase H reaction mechanism are still unclear. 

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. 

HIV-l reverse transcriptase (RT) possesses DNA and RNA template-dependent polymerase activity, as well as an endonucleolytic ribonuclease H (RNase H) activity responsible for degradation of the RNA in an RNA/ DNA duplex. Because RT plays a critical role in the HIV life cycle, it remains a primary therapeutic target for anti-HIV drug development. ... 

Ribonuclease H (RNase H), also termed hybridase in some earlier literature, is an endoribonuclease that specifically degrades the RNA strand of DNA RNA heteroduplexes (Scheme 3.3). The oligoribonucleotide products have 5 -P and 3 -OH termini. 

Ribonuclease H is a small, single domain protein that eleaves RNA from RNA-DNA hybrids. RNase H from E. coli T = 66 °C) has been structurally studied by NMR spectroscopy.Hydrogen exchange NMR experiments have been used to examine the structural distribution of stability in RNase H from T. thermophiliis = 86 °C) and to compare its stability with that of the homologous RNase H from the mesophilic E. The general distribution... 

Emulsion activity (EA) of ribonuclease A (RNase A, 10 mg mL 1) has been shown to be enhanced by glycation with glucose-6-phosphate (G6P), using capillary electrophoresis to follow the changes in RNase A.506 A cluster of about 20 peaks formed and migrated more slowly than RNase A monomer. The area of the cluster increased with temperature and with G6P concentration. EA decreased with the time of incubation in the absence of G6P. In its presence, EA reached a maximum with time of incubation, being highest for 60 mM G6P after 96 h at 30 °C and 18-24 h at 40 °C. 

The reaction of H atoms with bovine pancreatic ribonuclease A (RNAse A) has been studied by steady-state /-radiolysis of lipid vesicle suspensions containing RNAse A. The inactivation of RNAse A caused by interaction of H atoms with protein involved selective attack on methionine residues and was connected with release of diffusible thiyl radicals [reaction (36)] ... 

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... 

Fig. 1.4. Ribonucleases of the RNase H family. The residue numbering above the sequences is that for E. coli RNase H. H-Eco, RNase H from E. coli [83K2] H-Tth, RNase H from Thermus thermophilus [92K5] HIV-H, RNase H domain from HIV-1 reverse transcriptase [86J1].

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. 

RNase H, wild type E. coli ribonuclease HI with all three free cysteines replaced by alanine, was used in all experiments. RNase H labeled with 35 expressed in E. coli cells grown in M9... 

Prepn. 3, 9 (1953). Bovine pancreatic RNase A is a single-chain peptide of 124 amino add residues. Primary structure C. H. W. Hits et al J. Biol. Chem. 235, 633 (I960). Amino acid sequence D. H. Spackman et al.. Ibid. 648. Pictorial representation of entire structure Stein, Moore, Sci. Am. 204, no. 2, 81-92 (Feb. 1961). Ribonuclease from plant leaves has slightly different characteristics Markham, Strominger, Biochem. J. 64, 46p (1956). Can be obtained as a by-product of microbial erythromydn production Japan, pat. 263>38( 63) (to Shionogi). Chemical synthesis of materials possessing partial RNase enzyme activity Gutte,... 

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