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)

Peptidyl transferase, the ribosomal activity responsible for catalysis of peptide bond formation, is resistant to vigorous procedures that are conventionally employed to remove proteins from protein-nucleic acid complexes. When the "fragment reaction" was used as a model assay for peptide bond formation, Escherichia coli ribosomes or 50S subunits retained 20 to 40 percent activity after extensive treatment with proteinase K and SDS, but lost activity after extraction with phenol or exposure to EDTA. Ribosomes from the thermophilic eubacterium Thermus aquaticus remained more than 80 percent active after treatment with proteinase K and SDS, which was followed by vigorous extraction with phenol. This activity is attributable to peptidyl transferase, as judged by specific inhibition by the peptidyl transferase-specific antibiotics chloramphenicol and carbomycin. In contrast, activity is abolished by treatment with ribonuclease T1. These findings support the possibility that 23S ribosomal RNA participates in the peptidyl transferase function.
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PMID:Unusual resistance of peptidyl transferase to protein extraction procedures. 137 96

Destabilizing events required for subsequent cotranslational disassembly of tobacco mosaic virus (TMV) particles in vitro were studied. Brief treatment of U-32P-labelled TMV (strain vulgare or U2) with 1% SDS exposed only 2.5% of the RNA (160 5' nucleotides) in a susceptible subpopulation of virions. Limited uncoating occurred almost immediately and appeared to be synchronous because the amount of 5' oligonucleotide marker (omega) recovered remained constant throughout a 15 min period in SDS. Additional RNase T1-sensitive oligonucleotides were exposed only after 1 to 2 min in SDS. Coat protein (CP) subunits released from virions 'destabilized' by ultracentrifugation at between pH 7.2 and 9.2 were quantified using L-[35S]methionine-labelled particles of TMV strain U2. CP recovery and virus particle translation results were consistent with increasing numbers of virions uncoating for approximately 200 nucleotides. In the presence of sparsomycin (SPN), the TMV strain vulgare 5' leader and the first AUG codon can bind two 80S ribosomes. Electron microscopy of pH 7.5-treated TMV particles incubated in SPN-treated wheatgerm extract or rabbit reticulocyte lysate, showed that approximately 10% of virions complexed with one ribosome and approximately 10% with two bound ribosomes, confirming that omega at least had been uncoated. Nucleocapsids in these complexes were shorter than untreated TMV by 9 to 10 nm (i.e. equivalent to 192 to 217 nucleotides exposed). The template activities of virions pretreated at pH 7.2 to 9.2 were destroyed by RNase H when short cDNAs were hybridized to sequences at, or immediately 3' to, the first AUG codon. We propose that the complete 5' leader of TMV RNA interacts weakly with CP subunits and that this micro-instability is due to the absence of G residues and is essential for initiation of cotranslational virus disassembly.
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PMID:Complete uncoating of the 5' leader sequence of tobacco mosaic virus RNA occurs rapidly and is required to initiate cotranslational virus disassembly in vitro. 184 66

Various agents were tested for their effects on microbial proteases, which activity was monitored by the analysis of cleaved peptide bands in SDS-polyacrylamide gel electrophoresis. Using casein as a substrate, fungal protease (type XIX) was inhibited by the phenyl methyl sulphonyl fluoride, chymostatin, antipain and leupeptin, while bacterial protease (type XXVI) was inhibited by phosphatidyl glycerol, phosphatidyl inositol and sphingosine. MS2 RNA exerted minor inhibition on the bacterial proteolysis of regulatory subunits of cyclic AMP-dependent protein kinase (A-PK). The cleavage of DNA binding protein by both proteases was inhibited, in the presence of MS2 RNA and lambda DNA. In comparison, phosphatidyl serine slightly stimulated the fungal protease on the cleavage of ribonuclease T1. RNA polymerase is a good substrate of the bacterial protease as indicated by the generation of multiple cleaved peptide fragments, whereas alkaline phosphatase is not susceptible to proteolysis.
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PMID:A further study on the regulation of microbial proteases. 222 36

The genome segments of two electrophoretically distinct variants of bluetongue virus (BTV) Serotype 2 (Ona A and Ona B) from the U.S.A. were analyzed by double-dimension gel electrophoresis of RNase T1 produced oligonucleotides. Segments 1, 4, 5, 6, 7 and 10 were examined individually after separation by SDS-PAGE; and Segments 2 and 3, and 8 and 9, which were difficult to resolve, were fingerprinted as pairs. The Ona A and Ona B strains appeared to be closely related since corresponding segments were comparable, sharing 53-89% of the large oligonucleotides counted. Since the strains with the Ona A electropherotype preceded Ona B infection in Florida, U.S.A. and since Ona A was indistinguishable from the early African isolate of Serotype 2, Ona B was thought to be a variant of an Ona A strain. These data tend to support the hypothesis that Ona B could have evolved from Ona A as the result of point mutations or genetic drift.
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PMID:Oligonucleotide fingerprint analysis of the genomes of two variants of bluetongue virus serotype 2 isolated in the U.S.A. 255 47

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

Influenza undergoes extensive antigenic variation in nature. These new antigenic variants invariably supplant the preceding antigenic type that then disappears. In apparent contrast to this, two H3N2 strains that were forwarded to the Laboratory Centre for Disease Control, Ottawa, in 1984 from a Canadian public health laboratory for reference analysis were shown to be most closely related to 1973 and 1974 strains. Laboratory contamination on isolation by the regional public health laboratory was the most likely explanation for the occurrence of these strains, since one of the isolates (RV/76/84) was identical by T1 mapping to an A/Eng-like isolate being grown in the laboratory of isolation. The two isolates, RV/74/84 and RV/76/84, were shown to be distinct from each other and from prototype H3N2 strains by RNase T1 oligonucleotide mapping, SDS-PAGE, peptide mapping of hemagglutinin, and hemagglutination inhibition assay. RV/74 and RV/76 appeared to have been genetically stable for the 10 to 11 years preceding 1984; this is consistent with laboratory frozen storage for this period of time. This paper demonstrates the utility of RNase T1 mapping for the characterization of novel or spurious influenza isolates.
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PMID:The use of T1 oligonucleotide mapping to determine that the emergence of Canadian 1973-like and 1974-like H3N2 strains in 1984 was the result of laboratory contamination. 312 Nov 60

1-ethyl-3-dimethyl aminopropylcarbodiimide (EDC) was used to cross-link 30S ribosomal proteins to 16S rRNA within the E. coli 3OS ribosomal subunit. Covalently linked complexes containing 30S proteins and 16S rRNA, isolated by sedimentation of dissociated crosslinked 30S subunits through SDS containing sucrose gradients, were digested with RNase T1, and the resulting oligonucleotide-protein complexes were fractionated on SDS containing polyacrylamide gels. Eluted complexes containing 30S proteins S9 and S12 linked to oligonucleotides were obtained in pure form. Oligonucleotide 5'terminal labelling was successful in the case of S12 containing but not of the S9 containing complex and led to identification of the S12 bound oligonucleotide as CAACUCG which is located at positions 1316-1322 in the 16S rRNA sequence. Protein S12 is crosslinked to the terminal G of this heptanucleotide.
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PMID:Protein-RNA crosslinking in Escherichia coli 30S ribosomal subunits. Identification of a 16S rRNA fragment crosslinked to protein S12 by the use of the chemical crosslinking reagent 1-ethyl-3-dimethyl-aminopropylcarbodiimide. 676 Jan 29

Two strains of recently isolated Venezuelan equine encephalitis (VEE) complex virus from southern Brazil, avirulent for 6- to 8-week-old mice and short-haired guinea pigs, were characterized by biologic, serologic, and biochemical means. They were shown serologically to represent a single, newly recognized variant of subtype I. Two-dimensional polyacrylamide gel electrophoresis (PAGE) of ribonuclease T1 digests of viral ribonucleic acid showed considerable homology between the genomes of the new variant prototype and variant IA. Three structural proteins were visualized by discontinuous sodium dodecyl sulfate-PAGE (SDS-PAGE). Although the smallest protein of both recent isolates migrates with the capsid proteins of other subtype I viruses, the larger structural proteins of the new variants differ in molecular weight from the E1 and E2 envelope glycoproteins of the other subtype I variants. The new isolates produced peptide fragment patterns that were identical to each other, but different from the patterns of other subtype I viruses, following SDS-PaGE of dissociated virions digested with Staphylococcus aureus V8 protease. Since these two isolates were from Culex (Melanoconion) species mosquitoes and from a bat (Carollia perspicillata), were postulated that this is an enzootic VEE virus variant for which the classification IF is suggested.
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PMID:Identification of a new Venezuelan equine encephalitis virus from Brazil. 714 12

Protein folding, associated with isomerization of disulfide bonds, was studied using the mixed disulfide between glutathione and reduced ribonuclease T1 (GS-RNase T1) as a stable soluble and homogeneous starting material; conditions were selected to model those within the lumen of the endoplasmic reticulum where native disulfide bonds are formed in protein biosynthesis. Folding was initiated by addition of free glutathione (GSH +/- GSSG) to promote thiol-disulfide interchange and was monitored by intrinsic protein fluorescence, appearance of native ribonuclease activity, HPLC, and nonreducing SDS-PAGE. All the analyses indicated that native RNase T1 was recovered in high yield in a variety of redox conditions. Appearance of native activity followed first-order kinetics; kinetic analysis of the intrinsic fluorescence changes indicated an additional rapid process in some conditions, interpreted as the formation of a nonnative intermediate state. Analysis by HPLC and SDS-PAGE also indicated the formation of transient intermediates. In 1.5 M NaCl, GS-RNase T1 adopts a compact native-like conformation; refolding by thiol-disulfide interchange in these conditions was accelerated approximately 2-fold. Refolding of GS-RNase T1 was catalyzed by protein disulfide isomerase (PDI); substoichiometric quantities of PDI accelerated refolding several-fold. GS-RNase T1 refolding was inhibited by BiP; refolding was completely blocked in presence of a 5-fold molar excess of BiP, and the yield of refolding was substantially reduced by equimolar concentrations of BiP; the refolding was then restored by the addition of ATP. GS-RNase T1 is a convenient model substrate for studying protein folding linked to native disulfide formation in conditions comparable to those within the lumen of the endoplasmic reticulum.
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PMID:Refolding by disulfide isomerization: the mixed disulfide between ribonuclease T1 and glutathione as a model refolding substrate. 762 8

Reduced oxygen tension (hypoxia) induces a 3-fold increase in stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. To investigate the possibility that RNA-protein interactions are involved in mediating this increase in stability, RNA gel shift assays were performed using different fragments of labeled TH mRNA and the S-100 fraction of PC12 cytoplasmic protein extracts. We identified a sequence within the 3'-untranslated region of TH mRNA that binds cytoplasmic protein. RNase T1 mapping revealed that the protein was bound to a 28 nucleotide long sequence that is located between bases 1551-1579 of TH mRNA. Moreover, protein binding to this fragment was prevented with an antisense oligonucleotide directed against bases 1551-1579 and subsequent RNase H digestion. This fragment of the 3'-untranslated region of TH mRNA is rich in pyrimidine nucleotides, and the binding of cytoplasmic protein to this fragment was reduced by competition with other polypyrimidine sequences including poly(C) but not poly(U) polymers. The binding of the protein to TH mRNA was increased when cytoplasmic proteins were extracted from PC12 cells exposed to hypoxia (5% O2) for 24 h. Electrophoresis of the UV cross-linked RNA-protein complex on SDS-polyacrylamide gel electrophoresis revealed a complex of 74 kDa. The potential role of this protein-TH mRNA interaction in regulation of TH mRNA stability during hypoxia is discussed.
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PMID:Hypoxia stimulates binding of a cytoplasmic protein to a pyrimidine-rich sequence in the 3'-untranslated region of rat tyrosine hydroxylase mRNA. 790 89


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