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)

1. RNase Ms, a base non-specific RNase from Aspergillus saitoi was reduced and carboxymethylated (RCM-RNase Ms). RCM-RNase Ms was hydrolyzed with trypsin, and the trypsin digests were then treated with chymotrypsin. Trypsin digests were also treated with Staphylococcus protease and with chymotrypsin, separately. 2. By the analyses of the amino acid sequences of the peptides formed, the alignment of these peptides in RCM-RNase Ms was determined. 3. From the digest of heat-denatured RNase Ms with Bacillus subtilis protease, two peptides containing disulfide bridges were isolated. From the analysis of these two peptides, the locations of the bridges were determined. 4. The amino acid sequence of RNase Ms was compared with those of RNase T1 (Asp. oryzae, guanine specific), RNase U1 (Ustilago sphaerogena, guanine specific) and RNase U2 (Ustilago sphaerogena, purine specific). There are very similar sequences between these for RNases irrespective of their differences in base specificity. These were, in RNase Ms, tripeptide sequence containing His39 (Tyr-Pro-His), the tetrapeptide containing Glu57 (Glu-Tyr-Pro-Ile), the hexapeptide containing Arg76 (Asp-Arg-Val-Ile-Phe-Asp) and the hexapeptide containing His 91 (Ile-Thr-His-Thr-Gly-Ala). The other sequences common for all four RNases are Tyr67, Phe100, and Cys103 in RNase Ms. Since among these peptides His39, Glu57, His91, and Arg76 in RNase Ms corresponded to His40, Glu58, His92, and Arg77 in RNase T1 which are known to be involved in the active site of RNase T1, the possible role of these amino acids in the active site of RNase Ms is discussed. 5. The sequence similarity of RNase Ms to that of RNase T1 was about 60% and to those of RNase U1 and RNase U2 was about 30%. 6. The details of the experimental evidence used to elucidate the amino acid sequence of RNase Ms are described in the supplemental miniprint.
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PMID:Primary structure of a minor ribonuclease from Aspergillus saitoi. 709 2

The higher order structure of the functionally important 530 loop in Escherichia coli 16S rRNA was studied in mutants with single base changes at position 517, which significantly impair translational fidelity. The 530 loop has been proposed to interact with the EF-Tu-GTP-aatRNA ternary complex during decoding. The reactivity at G530, U531 and A532 to the chemical probes kethoxal, CMCT and DMS respectively was increased in the mutant 16S rRNA compared with the wild-type, suggesting a more open 530 loop structure in the mutant ribosomes. This was supported by oligonucleotide binding experiments in which probes complementary to positions 520-526 and 527-533, but not control probes, showed increased binding to the 517C mutant 70S ribosomes compared with the non-mutant control. Furthermore, enzymatic digestion of 70S ribosomes with RNase T1, specific for single-stranded RNA, substantially cleaved both wild-type and mutant rRNAs between G524 and C525, two of the nucleotides involved in the 530 loop pseudoknot. This site was also cleaved in the 517C mutant, but not wild-type rRNA, by RNase V1. Such a result is still consistent with a more open 530 loop structure in the mutant ribosomes, since RNase V1 can cut at appropriately stacked single-stranded regions of RNA. Together these data indicate that the 517C mutant rRNA has a rather extensively unfolded 530 loop structure. Less extensive structural changes were found in mutants 517A and 517U, which caused less misreading. A correlation between the structural changes in the 530 loop and impaired translational accuracy is proposed.
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PMID:Structural changes in the 530 loop of Escherichia coli 16S rRNA in mutants with impaired translational fidelity. 756 70

Binase, the extracellular ribonuclease of Bacillus intermedius, is inhibited by barstar, the natural protein inhibitor of the homologous RNase, barnase, of B. intermedius. The dissociation constants of the binase complexes with barstar and its double Cys40,82Ala mutant are about 10(-12) M, only 5 to 43 times higher than those of the barnase-barstar complex. As with barnase, the denaturation temperature of binase is raised dramatically in the complex. Calorimetric studies of the formation and stability of the binase-barstar complex show that the binase reaction with barstar is qualitatively similar to that of barnase but some significant quantitative differences are reported.
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PMID:Dissociation constants and thermal stability of complexes of Bacillus intermedius RNase and the protein inhibitor of Bacillus amyloliquefaciens RNase. 778 35

A guanine nucleotide-specific RNase (RNase Po1) was isolated from caps of the fruit bodies of Pleurotus ostreatus. RNase Po1 is most active towards RNA at pH 8.0. The effect of heating on the molar ellipticity at 210 nm of RNase Po1 showed that RNase Po1 is more stable than RNase T1. The primary structure of RNase Po1 was determined to be < ETGVRSCNCAGRSFTGTDVTNAIRSARAGGSGNYPHVYNNFEGFSFSCTPTFFEFPVFRGSVYSGGSPG ADRVIYD- QSGRFCACLTHTGAPSTNGFVECRF. It consisted of 101 amino acid residues, with a molecular weight of 10,760. RNase Po1 has relatively higher sequence homology with RNase T1 family RNase. It contains 6 half cystine residues. The locations of four of them are superimposable on those of RNase U1 and RNase U2. The amino acid residues forming the active site of RNase T1 were well conserved in this RNase. Therefore, RNase Po1 is a unique member of the RNase T1 family in respect of the location of one disulfide bridge, and its stability.
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PMID:Purification and primary structure of a new guanylic acid specific ribonuclease from Pleurotus ostreatus. 779 82

The initial step in mouse hepatitis virus (MHV) RNA replication is the synthesis of negative-strand RNA from a positive-strand genomic RNA template. Our approach to begin studying MHV RNA replication is to identify the cis-acting signals for RNA synthesis and the proteins which recognize these signals at the 3' end of genomic RNA of MHV. To determine whether host cellular and/or viral proteins interact with the 3' end of the coronavirus genome, an RNase T1 protection/gel mobility shift electrophoresis assay was used to examine cytoplasmic extracts from mock- and MHV-JHM-infected 17Cl-1 murine cells for the ability to form complexes with defined regions of the genomic RNA. We demonstrated the specific binding of host cell proteins to multiple sites within the 3' end of MHV-JHM genomic RNA. By using a set of RNA probes with deletions at either the 5' or 3' end or both ends, two distinct binding sites were located. The first protein-binding element was mapped in the 3'-most 42 nucleotides of the genomic RNA [3' (+42) RNA], and the second element was mapped within an 86-nucleotide sequence encompassing nucleotides 171 to 85 from the 3' end of the genome (171-85 RNA). A single potential stem-loop structure is predicted for the 3' (+)42 RNA, and two stem-loop structures are predicted for the 171-85 RNA. Proteins interacting with these two elements were identified by UV-induced covalent cross-linking to labeled RNAs followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The RNA-protein complex formed with the 3'-most 42 nucleotides contains approximately five host polypeptides, a highly labeled protein of 120 kDa and four minor species with sizes of 103, 81, 70, and 55 kDa. The second protein-binding element, contained within a probe representing nucleotides 487 to 85 from the 3' end of the genome, also appears to bind five host polypeptides, 142, 120, 100, 55, and 33 kDa in size, with the 120-kDa protein being the most abundant. The RNA-protein complexes observed with MHV-infected cells in both RNase protection/gel mobility shift and UV cross-linking assays were identical to those observed with uninfected cells. The possible involvement of the interaction of host proteins with the viral genome during MHV replication is discussed.
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PMID:Specific binding of host cellular proteins to multiple sites within the 3' end of mouse hepatitis virus genomic RNA. 788 46

An in vitro transcription system from Candida utilis is described. The template used is a hybrid plasmid containing Saccharomyces cerevisiae CYC1 promoter linked to a synthetic 377-bp G-minus casette (1). In vitro transcriptions are carried out in the presence of RNase. T1. Under these conditions only the transcripts that are resistant to RNase T1 accumulate. Using this protocol, it has been shown that in the absence of cytosolic factors RNA polymerase II (pol II) from C. utilis initiated RNA synthesis randomly. But both C. utilis and S. cerevisiae cell-free extracts could direct pol II from C. utilis to initiate transcription accurately. Results also indicated that the general transcription factors are functionally interchangeable between S. cerevisiae and C. utilis.
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PMID:Accurate transcription initiation by RNA polymerase II from Candida utilis. 798 59

The gene for the extracellular ribonuclease of B. pumilus KMM62 (RNase Bp) has been cloned and sequenced. The structural gene for this enzyme is similar to those of the extracellular ribonucleases of B. intermedius 7P (binase) and B. amyloliquefaciens H2 (barnase), as are the regulatory regions of binase and RNase Bp. The regulatory region of the barnase gene, however, is quite different from the other two. In the promoter of the genes for binase and RNase Bp, but not in that for barnase, is a region similar to the Pho box of E. coli. We have established that inorganic phosphate suppresses the synthesis of the binase and RNase Bp, but does not effect the synthesis of barnase.
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PMID:Phosphate regulation of biosynthesis of extracellular RNases of endospore-forming bacteria. 800 70

The synthesis and enzymatic characterization of DUPAAA, a novel fluorogenic substrate for RNases of the pancreatic type is described. It consists of the dinucleotide uridylyl-3',5'-deoxyadenosine to which a fluorophore, o-aminobenzoic acid, and a quencher, 2,4-dinitroaniline, have been attached by means of phosphodiester linkages. Due to intramolecular quenching the intact substrate displayed very little fluorescence. Cleavage of the phosphodiester bond at the 3'-side of the uridylyl residue by RNase caused a 60-fold increase in fluorescence. This allowed the continuous and highly sensitive monitoring of enzyme activity. The substrate was turned over efficiently by RNases of the pancreatic type, but no cleavage was observed with the microbial RNase T1. Compared to the dinucleotide substrate UpA, the specificity constant with RNase A, RNase PL3 and RNase U(s) increased 6-, 18-, and 29-fold, respectively. These differences in increased catalytic efficiency most likely reflect differences in the importance of subsites on the enzyme in the binding of elongated substrates. Studies on the interactions of RNase inhibitor with RNase A using DUPAAA as a reporter substrate showed that it was well suited for monitoring this very tight protein-protein interaction using pre-steady-state kinetic methods.
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PMID:A novel fluorogenic substrate for ribonucleases. Synthesis and enzymatic characterization. 805 28

The chemical-shift dependences of the proton signals of the guanosine and uridine moieties were measured as a function of the relative amount of GpcU complexed with RNase Pb1 (EC 3.1.27.3). The equal values of the chemical-shift changes of the guanosine C8-protons on complex formation between GpcU and RNase Pb1 and that of the 3'-GMP and RNase Pb1 allow to conclude that the guanosine base is bound in the same manner in these protein-ligand complexes. The guanosine moiety of GpcU is also most probably bound in the syn-conformation. The absence of changes in both the linewidths and the chemical shifts of the C1', C5 and C6-proton signals of the uridine on complex formation indicates that the uridine moiety of the dinucleoside phosphonate is not immobilized in the complex. The pH dependences of the chemical shifts of the C2-protons of the histidine-imidazole ring of RNase Pb1 and that of the 31P of GpcU in the RNase complex were studied. The results suggest that there is a direct interaction between the phosphonate group of the ligand and the protonated imidazole ring of His-90. The side groups of His-38 and Glu-56 are hydrogen bonded to each other at neutral pH and they are located in the vicinity of the phosphonate group of GpcU. When the carboxyl group of Glu-56 is protonated the His-38 imidazole ring forms a new hydrogen bond with one of the phosphoryl oxygens of the phosphonate group. On the basis of these results we propose the mechanism of action of RNase Pb1 which is probably also true for RNase T1.
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PMID:NMR studies of a complex of RNAse from Penicillium brevicompactum with dinucleoside phosphonate and the implications for the mechanism of enzyme action. 810 37

The action of the guanylyl-preferring RNase from Bacillus intermedius (binase) on a mixture of oligoadenylates with randomly distributed 2'-5' and 3'-5' internucleotide bonds [(A2'/3'p)n] under conditions sufficient for complete hydrolysis of poly(A) results in a mixture containing a single circular oligoadenylate and two series of linear oligoadenylates ending in cyclic 2',3'-phosphate. Individual compounds formed upon digestion of (A2'/3'p)n with binase have been isolated. Their structure was determined on the basis of their chemical and enzymatic conversions and confirmed by 1H-, 13C- and 31P-NMR spectra. According to these data, the circular triadenylate contains one 2'-5' and two 3'-5' internucleotide bonds, linear oligoadenylates of one series contain exclusively 2'-5' internucleotide bonds [(A2'p)nA > p], while each compound of the other series contains a single 3'-5' internucleotide bond connecting the 5'-ultimate nucleotide residue with the penultimate one [A3'p(A2'p)n-1A > p]. The incubation of compounds of the former series A3' p(A2'p)n > p at pH 1.0 and the subsequent action of phosphatase results in successive formation of compounds of two other new series: A3'p(A2'p)nA2'(3')p and A3'p(A2'p)nA.
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PMID:Preparation of cyclic 2',3'-monophosphates of oligoadenylates (A2'p)nA > p and A3'p(A2'p)n-1A > p. 811 3


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