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)

The energetics of thermal denaturation of two isoforms of ribonuclease T1 (Gln25 and Lys25) in various solvents have been studied by differential scanning calorimetry. It has been shown that the thermal transition of both forms of RNase T1 is strongly affected by slow kinetics, which cause an apparent deviation of the transition from a simple two-state model. By decreasing the heating rate or increasing the transition temperature, the denaturation of RNase approaches an equilibrium two-state transition. This permits determination of the thermodynamic parameters characterizing unfolding of the native structure. These thermodynamic parameters were correlated with the structural features of protein. Analysis of different contributions to the stability of RNase T1 shows that van der Waals interactions and hydrogen bonding are the major contributors to the conformational stability of the protein.
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PMID:Energetics of ribonuclease T1 structure. 813 67

A ribonuclease T1 homologue, ribonuclease Ms (RNase Ms) from Aspergillus saitoi, has been crystallized as a complex with a substrate analogue GfpC where the 2'-hydroxyl (2'-OH) group of guanosine in guanylyl-3',5'-cytidine (GpC) is replaced by the 2'-fluorine (2'-F) atom to prevent transesterification. The crystal structure of the complex was solved at 1.8-A resolution to a final R-factor of 0.204. The role of His92 (RNase T1 numbering) as the general acid catalyst was confirmed. Of the two alternative candidates for a general base to abstract a proton from the 2'-OH group, His40 and Glu58 were found close to the 2'-F atom, making the decision between the two groups difficult. We then superposed the active site of the RNase Ms/GfpC complex with that of pancreatic ribonuclease S (RNase S) complexed with a substrate analogue UpcA, a phosphonate analogue of uridylyl-3',5'-adenosine (UpA), and found that His12 and His119 of RNase A almost exactly coincided with Glu58 and His92, respectively, of RNase Ms. Similar superposition with a prokaryotic microbial ribonuclease, RNase St [Nakamura, K. T., Iwahashi, K., Yamamoto, Y., Iitaka, Y., Yoshida, N., & Mitsui, Y. (1982) Nature 299, 564-566], also indicated Glu58 as a general base. Thus the present comparative geometrical studies consistently favor, albeit indirectly, the traditional as well as the most recent notion [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Biochemistry 29, 9064-9072] that Glu58, rather than His40, must be the general base catalyst in the intact enzymes of the RNase T1 family.
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PMID:Crystal structure of ribonuclease Ms (as a ribonuclease T1 homologue) complexed with a guanylyl-3',5'-cytidine analogue. 821 54

A method is described for the detection, chemical characterization and sequence placement of post-transcriptionally modified nucleotides in RNA. Molecular masses of oligonucleotides produced by RNase T1 hydrolysis can be measured by electrospray mass spectrometry with errors of less than 1 Da, which provides exact base composition, and recognition of modifications resulting from incremental increases in mass. Used in conjunction with combined liquid chromatography-mass spectrometry and gene sequence data, modified residues can be completely characterized at the nucleoside level, and assigned to sequence sites within oligonucleotides defined by selective RNase cleavage. The procedures are demonstrated using E.coli 5S rRNA, in which all RNase T1 fragments predicted from the rDNA sequence are identified solely on the basis of their molecular masses, and using E.coli 16S rRNA for analysis of post-transcriptional modification, including placement of 3-methyluridine at position 1498. The principles described are generally applicable to other covalent structural modifications of RNA which produce a change in mass, such as those resulting from editing, photochemical cross-linking, or xenobiotic modification.
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PMID:A novel method for the determination of post-transcriptional modification in RNA by mass spectrometry. 823 93

We describe the construction and testing of a structural model at the nucleotide level for conformation CH of the central hairpin of genomic RNA from coliphage Q beta. The model was developed with the computer program MFOLD using both optimal and suboptimal predictions. Structural information obtained by electron microscopic analysis of Kleinschmidt spreadings of Q beta RNA was used to guide the modeling. The model was tested in solution with three enzymatic probes: RNase T1, RNase T2, and RNase V1, as well as four chemical probes: dimethylsulfate, diethylpyrocarbonate, kethoxal and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMCT). The structural analyses in solution are consistent with the predicted structural model. The model is also supported by comparative structural analysis with the related coliphage SP. The model provides a structural basis for published biochemical and genetic studies implicating large, long-range structural features in the co-regulation of viral coat and replicase expression. In addition, we show that the read-through region of the viral protein A1 forms a separate structural domain, and we suggest that it functions as a nucleation site that participates in the folding and refolding of the molecule during replication and translation. In addition to the central hairpin, we have analyzed the structure of the viral coat initiation region. Our studies show that the entire region consists of small local hairpins and that 26 nucleotides immediately surrounding the coat initiation codon are single-stranded.
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PMID:A two-dimensional model at the nucleotide level for the central hairpin of coliphage Q beta RNA. 837 1

We have chosen two members of the microbial RNase family, barnase and binase, which have 85% identity (17 substitutions and 1 deletion) and almost identical three-dimensional structure, to study the evolution of protein stability. The 17 residues that differ are scattered throughout the molecule. Each of the 17 differing residues has been mutated independently and the effect on protein stability analysed. Each point mutation has an effect on protein stability that ranges from +1.1 to -1.1 kcal mol-1. These changes in energy are additive. There is no clear correlation between the type of mutation and the effect on protein stability. A multiple mutant having six of the single mutations that increase the stability of barnase is 3.3 kcal mol-1 more stable than wild type and has the same activity. There could be selective pressure to maintain proteins at a certain stability and, consequently, mutations that decrease stability tend to be counterbalanced by stabilizing mutations. Alternatively, there could simply be pressure to maintain stability above a certain level, and any further increases in stability need not be maintained during evolution. These results suggest a simple way to improve the stability of proteins: choose two homologous proteins that have high similarity, mutate individually all of the residues that differ between the two, and combine the mutations that increase the stability in a multiple mutant.
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PMID:Step-wise mutation of barnase to binase. A procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability. 837 5

Regulation of the biosynthesis of extracellular ribonucleases of Bacillus amyloliquefaciens H2 (barnase), Bacillus intermedius 7P (binase), and Bacillus pumilus KMM 62 (RNase Bp) was studied in their native strains and recombinant Escherichia coli strains. Recombinant plasmids were obtained that contained genes encoding barnase, binase, and RNase Bp under the control of their own regulatory sequences (plasmids pMT415, pML5, and pML61), genes encoding barnase and binase under the control of the tac promoter and the phoA leader sequence (plasmids pMT416 and pML163), and the binase-encoding gene under the control of the regulatory sequences of the barnase and RNase Bp genes (plasmids pML53 and pML67, respectively). Inorganic phosphate (Pi) inhibited the biosynthesis of binase and RNase Bp in natural strains B. intermedius 7P and B. pumilus KMM 62 and recombinant E. coli strains when genes encoding these RNases were under the control of their own regulatory sequences. In contrast the biosynthesis of barnase-either in the natural B. amylolique-faciens strain or in recombinant E. coli strains-was not affected by Pi. Neither did inorganic phosphate produce any effect on the biosynthesis of binase when the binase-encoding gene was under the control of the barnase gene promoter (pML53). The leader sequences and promoters were found to be similar in the binase and RNase Bp genes and differed considerably from the leader sequence and promoter of the barnase gene. The promoter regions of the binase and RNase Bp genes, but not of the barnase gene, contained sequences that resembled the Pho box of the phosphate regulon from E. coli.
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PMID:[Biosynthetic regulation of extracellular ribonucleases in native strains of bacilli and in recombinant strains of Escherichia coli]. 853 11

The three-dimensional structure of ribonuclease Rh (RNase Rh), a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.0 A resolution. The overall structure of RNase Rh is completely different from those of other previously studied RNases, such as RNase A from bovine pancreas and RNase T1 from Aspergillus oryzae. In the structure of RNase Rh, two histidine residues (His46 and His109) and one glutamic acid residue (Glu105), which were predicted to be critical to the activity from the chemical modification and mutagenesis experiments, are found to be located close together, constructing the active site. The indole ring of Trp49 plays an important role in preserving the active site structure by its stacking interactions with the imidazole ring of His 109, and by hydrogen bonding with the carboxyl group of Glu105. There exists a hydrophobic pocket around the active site, which contains the aromatic side-chain of Trp49 and Tyr57. The results of mutagenesis studies suggest that this pocket is the base binding site of the substrate.
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PMID:The crystal structure of ribonuclease Rh from Rhizopus niveus at 2.0 A resolution. 855 22

The results of genotoxicity testing of microbial ribonucleases from Bacillus species with different catalytic activity obtained by site-directed mutagenesis in SOS chromotest are reported. At the concentrations 0.1-1 mg/ml, the induction factor for wild-type bacillar binase, barnase and mutant Arg58Lys binase with 100% activity was found to be significantly higher than 1.5 (1.8-2.8). Mutant RNases having decreased catalytic activity (binases with replacements Lys26Ala, Arg61Gln, His101Glu) or through natural inhibitor barstar inactivated wild-type RNase exhibited no SOS-inducing potency. The ability of native bacillar RNases and mutant enzymes possessing high catalytic activity comparable with the activity of wild-type RNase to cause the SOS response indicates that genotoxicity is mediated through the probable cleavage of cellular RNA.
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PMID:SOS-inducing ability of native and mutant microbial ribonucleases. 876 49

Poly[2'-O-(2,4-dinitrophenyl)]poly(A)[DNP-poly(A)] has been found to be a potent inhibitor in solution for RNases A, B, S, T1, T2 and H as well as phosphodiesterases I and II. Kinetic measurements with RNase B and RNase T1 showed DNP-poly(A) to be a reversible competitive inhibitor with K1 equal to 1.03 and 1.05 microM, respectively. Data on the quenching of fluorescence of RNase T1 by DNP-poly(A) indicate the existence of more than one RNase-binding site in each DNP-poly(A) molecule. By attaching each DNP-poly(A) molecule at one end covalently to oxirane acrylic beads, an affinity column was prepared for selective removal of RNases from aqueous solutions by simple filtration. It was found that a 1000-fold reduction in RNase concentration can be obtained by passing either 7.0 microM or 7.0 nM RNase A solution through a 5-cm-long column. The column can be saturated by passing through a concentrated RNase solution and subsequently regenerated by washing with salt solution. The regenerated column can be used repeatedly with no significant decrease in RNase-binding affinity and capacity. By titration of the derivatized beads with RNase, the first dissociation constant (Kd) and binding capacity for the bound enzyme can be determined. The (Kd) was found to be 0.66 microM for RNase B and 0.48 microM for RNase T1; the corresponding binding capacities were found to be 21.0 x (10)-8 and 9.6 x (10)-8 mol/g, respectively.
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PMID:Selective removal of ribonucleases from solution with covalently anchored macromolecular inhibitor. 877 29

The effect of the concentrations of peptone, yeast extract, and inorganic phosphate on the expression of genes of extracellular ribonucleases from Bacillus intermedius 7P (binase) and Bacillus amyloliquefaciens H2 (barnase) was studied in Escherichia coli cells transformed with plasmids containing the structural genes of binase or barnase under the control of their own or synthetic regulatory region or the structural binase gene under the control of the regulatory regions of the genes of barnase or Bacillus pumilus RNase. Inorganic phosphate inhibited the expression of the binase gene under the control of its own regulatory region or the regulatory region of the RNase Bp gene. In all other cases, inorganic phosphate produced no effect on the synthesis of RNases by E. coli cells. This difference in the effects of phosphate may be due to the presence of a nucleotide sequence similar to the E. coli Pho box in the promoters of the binase and RNase Bp genes and the absence of this sequence in the barnase gene promoter. It was shown that high peptone and yeast extract concentrations in the cultivation medium are required for good growth of the recombinant E. coli strains and the biosynthesis of RNases.
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PMID:[Effect of culture medium components on the accumulation of extracellular bacillary ribonucleases in culture fluid of recombinant strains of Escherichia coli]. 910 46


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