Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The three-dimensional structure of RNase H from Escherichia coli was determined at 1.8 A resolution by X-ray crystallography. The enzyme was found to belong to the alpha + beta class of structures, consisting of two distinct domains. The structure implies a possible region interacting with a DNA-RNA hybrid. The Mg2(+)-binding site essential for activity is located near a cluster of four acidic amino acids--one glutamic and three aspartic acid residues. These residues are completely conserved in the homology alignment of sequences of RNase H and reverse transcriptases from retroviruses and retrovirus-like entities. The structural motif of beta strands around the Mg2(+)-binding site has similarities to that in DNase I.
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PMID:Three-dimensional structure of ribonuclease H from E. coli. 169 62

We have purified and determined functional parameters of reconstituted, recombinant HIV-1 reverse transcriptase (RT) heterodimers within which either the p66 or p51 polypeptide was selectively mutated in one or both aspartic acid residues constituting the proposed polymerase active site (-Y-M-D-D-). Heterodimers containing a mutated p51 polypeptide retain almost wild type levels of both RNA-dependent DNA polymerase and ribonuclease H (RNaseH) activity. In contrast, heterodimers whose p66 polypeptide was likewise mutated exhibit wild type RNaseH activity but are deficient in RNA-dependent DNA polymerase activity. These results indicate that in heterodimer RT, the p51 component cannot compensate for active site mutations eliminating the activity of p66, indirectly implying that solely the p66 aspartic acid residues of heterodimer are crucial for catalysis.
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PMID:Subunit-selective mutagenesis indicates minimal polymerase activity in heterodimer-associated p51 HIV-1 reverse transcriptase. 171 45

Selected conserved amino acids in the putative RNase H domain of reverse transcriptase (RT) were modified in a molecularly cloned infectious provirus and in a Moloney murine leukemia virus RT expression vector by site-directed mutagenesis. Substitution of either of two conserved aspartic acid residues in proviral DNA prevented production of infectious particles in transfected NIH 3T3 cells, and the same modifications depressed RT-associated RNase H activity by more than 25-fold with little or no effect on polymerase activity.
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PMID:Inhibition of RNase H activity and viral replication by single mutations in the 3' region of Moloney murine leukemia virus reverse transcriptase. 246 6

The conserved aspartic acid residue 488 in the RNase H domain of HIV-1 reverse transcriptase (RT) was mutated to alanine. RT was expressed in Escherichia coli alone or with the entire pol-gene polyprotein consisting of proteinase, RT, and integrase and processed by the HIV-1 proteinase in the bacterial cell. Expression of mutant RT together with the proteinase resulted in an overproduction of RT p51 vs p66. The mutation also altered the conformation of the RT p66/p51 heterodimer as shown by the loss of binding of monoclonal antibodies to mutant RT in ELISA. Crystallographic data shows that a salt bridge exists between Asp 488 and Lys 465 of RNase H which stabilizes the uncleavable form of RT p66, and that substitution of Asp for Ala would prevent the formation of this salt bridge. Our results indicate that disruption of this salt bridge through mutation of Asp 488 interferes with the conformational changes that regulate the limited processing of p66 to 51 by the virus proteinase. Homology data suggest that such a bridge may be present in other lentiviruses. The mutation introduced caused a moderate decrease in both the RNase H activity and the polymerase activity of RT, indicating that the proper folding of the RNase H domain of RT is necessary to achieve full polymerase activity.
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PMID:Disruption of a salt bridge between Asp 488 and Lys 465 in HIV-1 reverse transcriptase alters its proteolytic processing and polymerase activity. 769 May 4

We have analyzed a total of 12 different global and local multiple protein-sequence alignment methods. The purpose of this study is to evaluate each method's ability to correctly identify the ordered series of motifs found among all members of a given protein family. Four phylogenetically distributed sets of sequences from the hemoglobin, kinase, aspartic acid protease, and ribonuclease H protein families were used to test the methods. The performance of all 12 methods was affected by (1) the number of sequences in the test sets, (2) the degree of similarity among the sequences, and (3) the number of indels required to produce a multiple alignment. Global methods generally performed better than local methods in the detection of motif patterns.
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PMID:Comparative analysis of multiple protein-sequence alignment methods. 807 98

Multiple sequence alignment of distantly related viral proteins remains a challenge to all currently available alignment methods. The hidden Markov model approach offers a new, flexible method for the generation of multiple sequence alignments. The results of studies attempting to infer appropriate parameter constraints for the generation of de novo HMMs for globin, kinase, aspartic acid protease, and ribonuclease H sequences by both the SAM and HMMER methods are described.
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PMID:Parameterization studies for the SAM and HMMER methods of hidden Markov model generation. 887 15

The genes encoding the alpha (63-kDa) and beta (95-kDa) subunits of Rous sarcoma virus (RSV) reverse transcriptase (RT) or the entire Pol polypeptide (99 kDa) were mutated in the conserved aspartic acid residue Asp 181 of the polymerase active site (YMDD) or in the conserved Asp 505 residue of the RNase H active site. We have analyzed heterodimeric recombinant RSV alphabeta and alphaPol RTs within which one subunit was selectively mutated. When alphabeta heterodimers contained the Asp 181-->Asn mutation in their beta subunits, about 42% of the wild-type polymerase activity was detected, whereas when the heterodimers contained the same mutation in their alpha subunits, only 7.5% of the wild-type polymerase activity was detected. Similar results were obtained when the conserved Asp 505 residue of the RNase H active site was mutated to Asn. RNase H activity was clearly detectable in alphabeta heterodimers mutated in the beta subunit but was lost when the mutation was present in the alpha subunit. In summary, our data imply that the polymerase and RNase H active sites are located in the alpha subunit of the heterodimeric RSV RT alphabeta.
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PMID:Asymmetric subunit organization of heterodimeric Rous sarcoma virus reverse transcriptase alphabeta: localization of the polymerase and RNase H active sites in the alpha subunit. 1070 41

Heterodimeric reverse transcriptase (RT) alphabeta from Rous sarcoma virus (RSV) possesses an asymmetric subunit organization with the polymerase and RNase H active sites localized in the alpha subunit. To determine whether homodimeric RSV RTs alpha (63 kDa) or beta (95 kDa) assume alpha subunit organization similar to that of the heterodimer, an essential aspartic acid residue was mutated in the active site of either the polymerase (Asp181 > Asn) or the RNase H (Asp505 > Asn). Homodimeric alpha or beta RT consisting of one wild-type and one mutated subunit exhibit polymerase or RNase H activity, respectively, whereas the corresponding doubly mutated enzymes are inactive, indicating that the catalytic sites of the polymerase and RNase H domains are formed by only one subunit of the homodimer.
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PMID:Homodimeric reverse transcriptases from rous sarcoma virus mutated within the polymerase or RNase H active site of one subunit are active. 1090 7

The deduced amino acid sequence of the region downstream of the reverse transcriptase (RT) motif of the Trypanosoma cruzi L1Tc non-LTR retrotransposon shows a significant homology with the sequence coding for proteins with RNase H activity from different organisms and retroelements. The 25-kDa His(6)-tagged recombinant protein bearing only the L1Tc RNase H domain, named RHL1Tc, exhibits RNase H activity as measured on the [(3)H]poly(rA)/poly(dT) hybrid used as substrate as well as on specific homologous and heterologous [(32)P]RNA/DNA hybrids. The mutation of the conserved aspartic acid at position 39 of the enzyme catalytic site, but not of the serine at position 56 (non-conservative amino acid), abolishes protein RNase H activity. The RNase H activity of the RHL1Tc protein is Mg(2+)-dependent, and it is also active in the presence of the Mn(2+) ion. The optimal condition of RNase H activity is found at pH 8 and 37 degrees C, although it also has significant enzymatic activity at 19 degrees C and pH 6. However, it cannot be excluded that the RNase H activity level and its optimal conditions may be different from that of a protein containing both RT and RNase H domains.
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PMID:The non-LTR (long terminal repeat) retrotransposon L1Tc from Trypanosoma cruzi codes for a protein with RNase H activity. 1203 56

We present the crystal structure of the catalytic domain of Mos1 transposase, a member of the Tc1/mariner family of transposases. The structure comprises an RNase H-like core, bringing together an aspartic acid triad to form the active site, capped by N- and C-terminal alpha-helices. We have solved structures with either one Mg2+ or two Mn2+ ions in the active site, consistent with a two-metal mechanism for catalysis. The lack of hairpin-stabilizing structural motifs is consistent with the absence of a hairpin intermediate in Mos1 excision. We have built a model for the DNA-binding domain of Mos1 transposase, based on the structure of the bipartite DNA-binding domain of Tc3 transposase. Combining this with the crystal structure of the catalytic domain provides a model for the paired-end complex formed between a dimer of Mos1 transposase and inverted repeat DNA. The implications for the mechanisms of first and second strand cleavage are discussed.
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PMID:Mechanism of Mos1 transposition: insights from structural analysis. 1651 70


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