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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)

Various plant viral RNAs possess a 3' terminus with tRNA-like properties. These viral RNAs are charged with an amino acid upon incubation with the cognate aminoacyl-tRNA synthetase and ATP. We have studied the structure of end-labelled 3'-terminal fragments of turnip yellow mosaic virus RNA and brome mosaic virus RNA 2 with chemical modifications of the adenosine and cytidine residues and with enzymatic digestions using RNase T1, nuclease S1 and the double-strand-specific ribonuclease from cobra venom. The data indicate that the 3' termini of these plant viral RNAs lack a cloverleaf structure as found in classical tRNA. The three-dimensional folding, however, reveals a striking resemblance with classical tRNA. The models proposed are supported by phylogenetic data. Apparently distinct three-dimensional solutions have evolved to meet the requirements for faithful recognition by tRNA-specific enzymes. The way in which the aminoacyl acceptor arms of these tRNA-like structures are constructed reveal novel features in RNA folding which may have a bearing on the secondary and tertiary structures of RNA in general. The dynamic behaviour of brome mosaic virus RNA 2 in solution presumably is illustrative of conformational transitions, which RNAs generally undergo on changing the ionic conditions.
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PMID:Three-dimensional models of the tRNA-like 3' termini of some plant viral RNAs. 662 63

The specificity of transcription of Euglena gracilis Z chloroplast DNA by chloroplast DNA-dependent RNA polymerase in a transcriptionally active chromosome (Hallick, R.B., Lipper, C., Richards, O.C., and Rutter, W.J. (1976) Biochemistry 15, 3039-3045) has been studied. RNA molecules are both initiated and elongated in vitro. The RNA transcripts have been characterized as to their size, nuclease sensitivity, 5'-terminal oligonucleotides, and coding locus on the chloroplast genome. RNA labeled in vitro at the 5' end with [gamma-32P]ATP was digested with RNase T1, RNase A, and S1 nuclease. The resulting 5'-gamma-32P-oligonucleotides were fractionated by gel electrophoresis. In each case, one or two discrete products were obtained, consistent with initiation in vitro only at defined loci. RNA labeled in vitro with [alpha-32P]ATP or CTP has been hybridized to Southern (Southern, E.M. (1975) J. Mol. Biol. 98, 503-517) transfers of restriction endonuclease fragments of chloroplast DNA. The most abundant in vitro transcripts hybridize to chloroplast DNA fragments coding for 23 S, 16 S, and 5 S rRNAs. Only the coding strands of the rRNA genes are transcribed. Non-rDNA sequences of chloroplast DNA are also selectively transcribed but at much lower levels. The transcriptionally active chromosome has proved to be an ideal biochemical preparation for the study of selective transcription of cell organelle DNA.
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PMID:Selective in vitro transcription of Euglena chloroplast ribosomal RNA genes by a transcriptionally active chromosome. 676 27

Escherichia coli tRNAArg was digested with ribonuclease T1 under restrictive conditions in order to dissect a minimum number of diester bonds. The number of diester bonds cleaved and their locations were determined by phosphorylation of the newly formed 5' hydroxyl groups with [32P] ATP and polynucleotide kinase. There was complete loss of aminoacylation of tRNAARg when two diester bonds were cleaved at the anticodon. However, this material retained the specific properties of synthetase recognition. Two fragments were derived by further digestion of this tRNA. One 19 nucleotide-long fragment derived from the 3' end of tRNAArg and another 18 nucleotide-long fragment derived from the 5' end of the molecule were required to maintain the properties of the specific recognition by the arginyl tRNA synthetase in the absence of the rest of the structure including the anticodon.
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PMID:Recognition of E coli tRNAArg by arginyl tRNA synthetase. 677 88

Vaccinia virus mRNAs synthesized in vitro and in vivo, polyadenylated leader sequences synthesized in vitro in the absence of added GTP, CTP, or UTP or in the presence of 20 micrograms of actinomycin D per ml, and high-molecular-weight RNA synthesized in vitro under limiting ATP concentrations were labeled specifically in the cap structure using [alpha-32P]GTP and vaccinia-soluble enzyme extracts. The complexity of RNase T1-resistant 5'-terminal oligonucleotides was analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 190 unique T1-resistant 5'-terminal oligonucleotides were observed from vaccinia virus 8 to 12S RNA synthesized in vitro. A somewhat greater complexity was observed with polyadenylated leader sequences and actinomycin D RNAs where unique T1-resistant oligonucleotides ranged from approximately 210 to 280 5'-terminal fragments. On a composite fingerprint of the above RNAs, more than 300 identifiable unique T1-resistant 5'-terminal oligonucleotides were observed. Significantly, close to 300 T1-resistant fragments were derived from RNA sedimenting faster than 18S on denaturing sucrose gradients. Analysis of vaccinia RNAs synthesized in vivo in the absence of either de novo protein synthesis or DNA replication or in the presence of actinomycin D gave essentially similar profiles of 5'-terminal T1-resistant oligonucleotide fingerprints consisting of approximately 200 fragments. Analysis of the 5'-terminal T1-resistant oligonucleotides of vaccinia RNAs present after DNA replication showed essentially the same pattern of early T1-fragments albeit in reduced amounts but in addition revealed a complex pattern of T1-resistant oligonucleotides unique to this class of vaccinia RNA.
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PMID:Analysis of vaccinia virus transcriptional complexity in vitro and in vivo: characterization of RNase T1-resistant 5'-terminal oligonucleotides. 680 83

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

To elucidate the metabolic function of mRNA polyadenylation in Escherichia coli. we searched for a polyadenylate-binding protein as a potential mediator of the function of the poly(A) moiety. Using a nitrocellulose filter-binding assay and a Northwestern blot technique, a protein in the ribosomal supernatant fraction of E coli was identified and purified to homogeneity. N-terminal sequence analysis yielded a 25-residue sequence which corresponded to the 25 N-terminal amino acids of protein S1, one of the proteins of the E coli 30S ribosomal subunit. Poly(A) binding to S1 protein was inhibited by Mg2+ and Mn2+ and by ATP and stimulated 8-fold by 100 mM KCl. The binding of S1 to poly(A) occurred with an association constant of 3 x 10(6) M-1 and seemed to be only mildly cooperative. Competition studies of the binding of poly(A) and poly(C) to purified S1 protein were consistent with the presence of two polynucleotide binding sites, of which one binds poly(A) five times more strongly than poly(C), whereas the other binds poly(C) 50 times more strongly than poly(A). Poly(A) bound to 30S ribosomal subunits but not to 50S ribosomes. To study possible association of S1 with the poly(A) tracts of E coli mRNA in the process of translation, poly(A) RNA was isolated from polysomes by oligo(dT) cellulose chromatography and the poly(A) RNA with bound protein was eluted either directly or after digestion with RNase T1 and A. When subjected to Western blot analysis with antibody to S1, both poly(A) RNA and isolated poly(A) tracts revealed bound S1 protein. The implications of these results for the possible interaction of poly(A) tracts of mRNA and the translational machinery of E coli are discussed.
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PMID:Identification of ribosomal protein S1 as a poly(A) binding protein in Escherichia coli. 945 50

Peptidyl prolyl cis-trans isomerases can enzymatically assist protein folding, but these enzymes exclusively target the peptide bond preceding proline residues. Here we report the identification of the Hsp70 chaperone DnaK as the first member of a novel enzyme class of secondary amide peptide bond cis-trans isomerases (APIases). APIases selectively accelerate the cis-trans isomerization of nonprolyl peptide bonds. Results from independent experiments support the APIase activity of DnaK: (i) exchange crosspeaks between the cis-trans conformers appear in 2D (1)H NMR exchange spectra of oligopeptides (ii) the rate constants for the cis-trans isomerization of various dipeptides increase and (iii) refolding of the RNase T1 P39A variant is catalyzed. The APIase activity shows both regio and stereo selectivity and is stimulated two-fold in the presence of the complete DnaK/GrpE/DnaJ/ATP refolding system. Moreover, known DnaK-binding oligopeptides simultaneously affect the APIase activity of DnaK and the refolding yield of denatured firefly luciferase in the presence of DnaK/GrpE/DnaJ/ATP. These results suggest a new role for the chaperone as a regioselective catalyst for bond rotation in polypeptides.
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PMID:The hsp70 chaperone DnaK is a secondary amide peptide bond cis-trans isomerase. 1203 50

Sso7d is a small basic protein consisting of 62 amino acids isolated from the thermoacidophilic archeobacterium Sulfolobus solfataricus. The protein is endowed with DNA binding properties, RNase activity, and the capability of rescuing aggregated proteins in the presence of ATP. In this study, the electrostatic properties of Sso7d are investigated by using the Poisson-Boltzmann calculation of the surface potential distribution and following by NMR spectroscopy the proton chemical shift pH titration of acidic residues. Although the details of the catalytic mechanism still have to be defined, the results from NMR experiments confirm the possible involvement of Glu35 as the proton acceptor in the catalytic reaction, as seen by its abnormally high pK(a) value. Poisson-Boltzmann calculations and NMR titration shifts suggest the presence of a possible hydrogen bond between Glu35 and Tyr33, with a consequent rather rigid arrangement at these positions. Comparison with RNase T1 suggests that Tyr7 may be a good candidate for acting as a proton donor in the active site of Sso7d as shown by its low phenolic pK(a) of approximately 9.3. Titration experiments performed with the UpA, a RNA dinucleotide model, showed that the protein residues affected by the interaction are mainly located in a different region with respect to the surface affected by DNA recognition, in good agreement with the surface potential distribution found with electrostatic calculations.
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PMID:Investigations of Sso7d catalytic residues by NMR titration shifts and electrostatic calculations. 1257 54

DEx(D)/(H) proteins catalyze structural rearrangements in RNA by coupling ATP hydrolysis to the destabilization of RNA helices or RNP complexes. The Escherichia coli DEx(D)/(H) protein DbpA specifically recognizes a region within the catalytic core of 23S rRNA. To better characterize the interaction of DbpA with this region and to identify changes in the complex between different nucleotide-bound states of the enzyme, RNase T1, RNase T2, kethoxal and DMS footprinting of DbpA on a 172 nt fragment of 23S rRNA were performed. A number of protections identified in helices 90 and 92 were consistent with biochemical experiments measuring the RNA binding and ATPase activity of DbpA with truncated RNAs. When DbpA was bound with AMPPNP, but not ADP, several additional footprints were detected in helix 93 and the single-stranded region 5' of helix 90, suggesting binding of the helicase domains of DbpA at these sites. These results propose that DbpA can act at multiple sites and hint at the targets of its biological activity on rRNA.
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PMID:Interaction of Escherichia coli DbpA with 23S rRNA in different functional states of the enzyme. 1517 85


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