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:3.1.27.3 (RNase T1)
1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glu 58 is one of the amino acids which participates in its catalytic action of ribonuclease T1. We mutated this residue to Gln 58 or Asp 58 by genetic engineering using chemically synthesized genes. The mutant enzymes were expressed in E. coli as fused proteins and purified to homogeniety on SDS-PAGE after cleavage with cyanogen bromide. Their activities in hydrolyzing pGpC were reduced to 10% in the Asp 58 mutant and about 1% in the Gln 58 mutant compared to that of the wild-type enzyme. These results suggest that Glu 58 is important but not essential for catalysis of ribonuclease T1.
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PMID:Modification of Glu 58, an amino acid of the active center of ribonuclease T1, to Gln and Asp. 287 6

The complete amino acid sequence of ribonuclease N1 (RNase N1), a guanine-specific ribonuclease from a fungus, Neurospora crassa, was determined by conventional protein sequencing, using peptide fragments obtained by tryptic digestion of cyanogen bromide-treated RNase N1 and by Staphylococcus aureus V8 protease digestion of heat-denatured RNase N1. The results showed that the protein is composed of a single polypeptide chain of 104 amino acid residues cross-linked by two disulfide bonds and has a molecular weight of 11,174: (sequence; see text) (Disulfide bonds: C2-C10, C6-C103) The amino acid sequence was homologous with those of RNase T1 (65% identity) and related microbial RNases.
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PMID:The amino acid sequence of ribonuclease N1, a guanine-specific ribonuclease from the fungus Neurospora crassa. 297 30

The genes for ribonuclease T1 and its site-specific mutants were chemically synthesized and introduced to Escherichia coli. All enzymes were fusion products produced by joining the synthetic gene at specific restriction sites to the synthetic gene for human growth hormone in a plasmid containing the E. coli trp promoter. The fusion protein from this plasmid contained 66% of the amino-terminal sequences of the human growth hormone, which were recognizable immunologically. RNase T1 or its mutants were cleaved from the fusion protein with cyanogen bromide. The synthetic RNase T1 endowed with the revised wild-type triad Gly-Ser-Pro, residues 71-73, was fully functional, readily hydrolyzing pGpC bonds, whereas a mutant enzyme having the originally reported, erroneous triad Pro-Gly-Ser was totally inactive. Various amino acid substitutions were also introduced to the guanosine recognition region comprised of residues 42-45, Tyr-Asn-Asn-Tyr. Substitution of either of the tyrosine residues noted above with phenylalanine had no dramatic effect on the enzyme's function. Replacement of asparagine-43 with arginine or alanine also caused only a small change in the hydrolyzing activity--a mutant enzyme maintained greater than 50% of the wild-type activity. In sharp contrast, when aspartic acid or alanine was substituted for asparagine-44, the activity was dramatically reduced to a few percent of the wild-type activity.
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PMID:Inquiries into the structure-function relationship of ribonuclease T1 using chemically synthesized coding sequences. 301 4

Examination of the intestinal contents of free-living Oryzomys nigripes rats by PAGE revealed two sharply defined bands that could be stained by ethidium bromide or by silver nitrate with comparable intensities. The molecules forming these bands were susceptible to digestion by pancreatic RNase A but not by RNase T1 or by DNase I. Their lengths were estimated to be about 2.6 and 1.5 kbp, respectively, by comparison with rotavirus SA11 genome segments. They cosedimented in CsCl gradients at a density of 1.39 to 1.40 g/ml, together with uniform particles approximately 35 nm in diameter with indistinct surface structure. It is suggested that these particles represent an as yet undescribed virus with a bisegmented double-stranded RNA genome, for which the name 'picobirnavirus' is proposed.
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PMID:A virus with a bisegmented double-stranded RNA genome in rat (Oryzomys nigripes) intestines. 305 86

The preparation and analysis of a mutant ribonuclease (RNase) T1 which possesses higher nucleolytic activity than the wild-type enzyme are described. The gene for the mutant RNase T1 (Tyr45----Trp45), in which a single amino acid at the binding site of the guanine base has been changed, was constructed by the cassette mutangenesis method using a chemically synthesized gene [Ikehara, M. et al. (1986) Proc. Natl Acad. Sci. USA 83, 4695-4699]. In order to reduce the nucleolytic activity of the enzyme in vivo, this gene was expressed in Escherichia coli as a fused protein connected through methionine residues to other proteins at both the N- and C-termini. After liberation from the fused protein by cleavage with cyanogen bromide at the methionine junctions, the mutant RNase T1 was purified by column chromatography. The nucleolytic activity toward pGpC increased to 120% of that of wild-type RNase T1. The kinetic parameters of the mutant enzyme demonstrate that this higher nucleolytic activity is due to a higher affinity for the substrate, probably because of an increased stacking effect in the binding pocket for the guanine base. This mutant enzyme also possessed a higher nucleolytic activity against pApC than wild-type RNase T1.
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PMID:Increase in nucleolytic activity of ribonuclease T1 by substitution of tryptophan 45 for tyrosine 45. 312 93

The complete amino acid sequence of an extracellular guanyl-specific RNase from Aspergillus pallidus fungi has been established. The RNase contains 104 amino acid residues (Mr 11,029). Its primary structure was analyzed basing on the automated Edman degradation of the carboxymethylated RNase followed by tryptic digestion and sequencing of the resultant hydrolysate. An additional structural information was obtained by means of the automatic sequencing of the cyanogen bromide peptide mixture and by studying the kinetics of the RNase's digestion with carboxypeptidase Y.
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PMID:[Amino acid sequence of ribonuclease Ap1 from Aspergillus pallidus]. 313 2

Conformational states of the ribosomal 5 S RNA molecule and its associated protein L1a in the yeast RNA-protein (RNP) complex were determined using controlled RNase T1 digestion in conjunction with fluorescence probes, ethidium bromide and bisanilinonaphthalenesulfonic acid. Fluorescence measurements indicated that the RNA molecule in the RNP complex appeared to exhibit a slightly lower degree of secondary structure than that in the free form. Controlled digestion of the intact RNP complex with RNase T1 resulted in an initial increase in ethidium fluorescence followed by a gradual decrease. In free RNA, a similar profile, except that a larger increase in ethidium fluorescence at the initial stage of digestion, was observed. During digestion of the RNP complex, increases in bisanilinonaphthalenesulfonic acid fluorescence and in light scattering were observed. These findings implied that as regions of the 5 S RNA molecule were perturbed, hydrophobic regions in the protein became exposed. Polyacrylamide gel analysis of the digestion products revealed a temporal appearance of discrete RNA fragments. Sequence analysis of these fragments generated information about the structural arrangement of the RNA molecule within the RNP complex. Results from the present investigation indicate that interactions between the 5 S RNA and protein L1a can stabilize functionally relevant conformations of the components that are individually labile. Properties of the separated components also suggest that special conditions, such as those suggested by Steitz et al. (Steitz, J. A., Berg, C., Hendrick, J. P., LaBranche-Chabot, H., Metspalu, A., Rinke, J., and Yario, T. (1988) J. Cell Biol. 106, 545-556) may be involved for these components to associate during ribosomal assembly.
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PMID:Probing the RNA structure within the yeast 5 S RNA.L1a protein complex by fluorescence and enzymatic digestion. 314 71

The amino acid sequence of ribonuclease T2 (RNase T2) from Aspergillus oryzae has been determined. This has been achieved by analyzing peptides obtained by digestions with Achromobacter lyticus protease I, Staphylococcus aureus V8 protease, and alpha-chymotrypsin of two large cyanogen bromide peptides derived from the reduced and S-carboxymethylated or S-aminoethylated protein. Digestion with A. lyticus protease I was successfully used to degrade the N-terminal half of the S-aminoethylated protein at cysteine residues. RNase T2 is a glycoprotein consisting of 239 amino acid residues with a relative molecular mass of 29,155. The sugar content is 7.9% (by mass). Three glycosylation sites were determined at Asns 15, 76 and 239. Apparently RNase T2 has a very low degree of sequence similarity with RNase T1, but a considerable similarity is observed around the amino acid residues involved in substrate recognition and binding in RNase T1. These similar residues may be important for the catalytic activity of RNase T2.
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PMID:Amino-acid sequence of ribonuclease T2 from Aspergillus oryzae. 316 20

RNase T1 gene and several mutant genes were constructed by joining of chemically synthesized deoxyoligonucleotides. These genes were inserted into an expression vector and expressed as fused protein in E. coli. RNase T1 and its mutant enzymes were liberated by cyanogen bromide treatment and their activities were measured.
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PMID:Synthesis and expression of the native RNase T1 gene and several mutant genes. 393 39

Previously we have isolated the specific RNA methyltransferase from the nucleoli of Ehrlich ascites tumor cells. The purified enzyme was found to be specific for methylation of C5 position of cytosine residue in ribosomal RNA in vitro (Obara, 1982b). In the present study, we have investigated the recognition mechanisms of RNA structure by this enzyme from the points of view of both primary and secondary structures. Analysis of in vitro methylation product by ribonuclease T1 digestion indicated the methylation-site(s) was limited to a certain number of nonanucleotide. The next experiments with either Sl nuclease or actinomycin D and ethidium bromide suggested that the enzyme modified only cytidine residue in or located close to the double stranded part of RNA. On the other hand, the characterization of analogues of cytidine residue in the RNA at molecular level showed that the methylation of rRNA was inhibited by either cytidine, CDP or CTP, but little inhibition was observed in the presence of cytosine, 5-methylcytidine and CMP.
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PMID:Recognition of the ribosomal RNA structures by purified nucleolar RNA methyltransferase. 667 41


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