Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.27.3 (RNase T1)
 1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied the distribution of repetitive sequence elements capable of forming double-stranded regions in nuclear RNA of HeLa, KB, and L cells. In human RNA populations, we called these regions duplex Alu family RNA (dAfRNA) because they represent transcripts of the highly reiterated family of DNA regions known as "Alu family DNA" (Rubin et al., Nature (London) 284:372-374, 1980). Although the dAfRNA populations of both human cell lines (HeLa and KB) have low sequence complexity, they represent 5% of the total heterogeneous nuclear RNA and have identical fingerprints; mouse L-cell dAf-like RNA (which has a similar complexity) represents only 2% of the total heterogeneous nuclear RNA and has an entirely different fingerprint. We utilized Escherichia coli RNase III as a highly specific reagent for the recognition of RNA:RNA duplex structure. This enzyme cleaves within the six characteristic RNase T1-resistant oligonucleotides of HeLa- and KB-cell dAfRNA (Robertson et al., J. Mol. Biol. 115:571-589, 1977). In addition, the size of heterogeneous nuclear RNA from all three cell types is reduced from greater than 32S to about 15S after RNase III treatment. We conclude that this size shift is a result of cleavage within dAfRNA regions and that such regions are present in most or all of the large RNA transcripts of these cells.
Mol Cell Biol 1984 Feb
PMID:Structure and distribution of Alu family sequences or their analogs within heterogeneous nuclear RNA of HeLa, KB, and L cells. 670 May 93

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.
 ...
PMID:Selective in vitro transcription of Euglena chloroplast ribosomal RNA genes by a transcriptionally active chromosome. 676 27

A rapid and simple method of oligonucleotide cataloging for phylogenetic studies is presented. It involves in vitro 5'-32P-labelling of RNase T1 - resistant oligonucleotides of ribosomal 16S RNA and finger-printing by high voltage electrophoresis and gradient thinlayer chromatography. Oligonucleotide sequences are established by the mobility shift method, using controlled alkali cleavage, high voltage electrophoresis and homochromatography. These procedures facilitate in particular the analysis of long RNase T1 - resistant oligonucleotides. Oligonucleotide catalogs are established fo three Actinomycetes, namely Oerskovia turbata, Actinoplanes philippinensis and Ampullariella regularis. These catalogs are equivalent to those obtained by methods which were described by Sanger and Woese.
J Mol Evol 1981
PMID:Rapid cataloging of ribonuclease T1 resistant oligonucleotides from ribosomal RNAs for phylogenetic studies. 679 Jul 18

In oxidative protein folding the interdependence between the acquisition of an ordered native-like conformation and disulfide bond formation was investigated by using the C2S/C10N variant of ribonuclease T1 as a model. This protein of 104 residues has a single disulfide bond between Cys6 and Cys103. In the reduced form it is unfolded in the presence of urea, but native-like folded when > or = 1.5 M NaCl is present. The influence of a folded conformation on the individual thiol/disulfide exchange reactions between the protein and glutathione could thus be studied in oxidative folding by varying the urea and NaCl concentrations. When the reduced protein was folded native-like the initial formation of the mixed disulfide between the protein and glutathione was decelerated about fourfold. The attachment of a glutathionyl moiety in this step destabilizes the protein by about 5 kJ mol-1 and led to a local unfolding near the two Cys residues. The reacting thiol groups still remained in close proximity for the subsequent intramolecular thiol/disulfide exchange reaction, but an increase in the energy of the transition state (e.g. by a hydrophobic environment or by steric strain) could be avoided. As a consequence the formation of the protein disulfide in this reaction was 100-fold faster when the mixed-disulfide species was in this ordered conformation. These results illustrate the importance of a low stability and a high flexibility of folding intermediates.
J Mol Biol 1995 Aug 04
PMID:Influence of protein conformation on disulfide bond formation in the oxidative folding of ribonuclease T1. 764 82

Electrostatic interactions between charged residues and the helix dipole in a protein were investigated by protein engineering methods. In ribonuclease T1, two surface-exposed acidic residues (Glu28 and Asp29) are located near the carboxyl terminus of the alpha-helix between residues 13 and 29. They were replaced, individually and in concert, by the uncharged amides Gln28 and Asn29, and the stabilities of the wild-type protein and its variants were determined as a function of pH. The effects of the two mutations are additive. Either one leads to a marginal destabilization by 0.7 kJ/mol at pH 2 but to a strong stabilization by about 3.2 kJ/mol at pH 7. This suggests that the deprotonations of Glu28 and Asp29 reduce the free energy of stabilization of folded ribonuclease T1 by about 4 kJ/mol each. This destabilization is probably caused by unfavorable electrostatic interactions of Glu28 and Asp29 with the negative end of the helix dipole. The activation energies for the unfolding of the different variants of ribonuclease T1 change in parallel with the differences in the thermodynamic stability when the pH is varied. This indicates that the unfavorable electrostatic interactions of Glu28 and Asp29 are lost very early in unfolding, and are not present in the activated state of unfolding.
J Mol Biol 1995 Sep 08
PMID:Destabilization of a protein helix by electrostatic interactions. 766 25

An activity from Saccharomyces cerevisiae mitochondria was identified that specifically bound to a 12-nucleotide sequence, AAUAA(U/C)AUUCUU, that is a site for processing of pre-mRNAs so as to generate the mature 3' ends of mRNAs. Because processing occurs 3' to the end of the dodecamer site, all mRNAs in yeast mitochondria terminate with that sequence. RNase T1 digestion fragments which terminated precisely at their 3' ends with the dodecamer sequence bound the activity, indicating that mRNAs in vivo would be capable of binding. Gel mobility shift analyses using RNA oligonucleotides showed that binding was reduced by a U-to-A substitution at position 3 of the dodecamer sequence; a C-to-A substitution at position 10 eliminated binding. UV cross-linking identified three polypeptides with approximate molecular masses of 19, 60, and 70 kDa as constituents of the binding activity. These estimates included the contribution of the 32P-labeled RNA oligonucleotide used to tag these polypeptides. An oligonucleotide with a UA-->AU substitution at positions 3 and 4 of the dodecamer site formed complexes deficient in the 19-kDa species, suggesting that binding specificity was inherent to the higher-molecular-weight polypeptides. Assembly of the complex at a dodecamer site on an RNA protected sequences located 5' to the dodecamer site from digestion by a nucleoside triphosphate-dependent 3' exoribonuclease found in yeast mitochondria. Since mitochondrial mRNAs terminate with an intact dodecamer sequence, the binding activity may function in the stabilization of mRNAs in addition to 3'-end formation of mRNAs.
Mol Cell Biol 1993 Jul
PMID:Identification of a protein complex that binds to a dodecamer sequence found at the 3' ends of yeast mitochondrial mRNAs. 768 18

An approach is described for extending free energy calculations to take into account the pH dependence of the relative binding of ligands to an enzyme or other receptor protein. The method is based on the calculation of the free energy difference for a single protonation state via the thermodynamic cycle simulation approach followed by inclusion of all possible protonation states of the enzyme and the inhibitor by use of a macroscopic continuum dielectric (Poisson-Boltzmann) model. A detailed formulation of the combined model is presented. It involves solution of the multiple equilibrium problem and makes use of the calculated pKa values of all titrating groups on both enzyme and ligand. The method is illustrated by calculations of the pH dependence of the differential binding of the inhibitors 2'GMP and 3'GMP to ribonuclease T1. A free energy simulation of the differential binding is made for a given protonation state of the enzyme and inhibitor. Although only qualitative agreement with experiment is obtained, the results provide insights concerning the interactions involved. The pH dependence of the binding is calculated by using the protonation state of the residues from the free energy simulation as the standard state for a Poisson-Boltzmann calculation. Information is obtained concerning the pKa values of the titrating amino acids in the free, 2'GMP and 3'GMP bound enzyme forms of RNase T1 and the difference in the pH dependence of the binding of 2'GMP and 3'GMP to RNase T1. The contributions of different types of interactions (e.g. protein residues versus solvent) to the free energy differences are examined. A free energy simulation of the pKa shift of Glu58 shows that it is important to consider both carboxyl oxygen atoms as possible protonation sites since they may behave very differently in a protein. It is found in the protein that the interactions with the solvent favor the neutral (protonated) state of Glu58. This contrasts sharply with the solution behavior, where the solvent favors the charged state. Analysis of the results shows that the interactions of bound water with other protein residues leads to the observed effect. Comparisons are made with a continuum calculation that uses the charged state employed in the free energy simulation.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1995 Apr 07
PMID:pH dependence of binding reactions from free energy simulations and macroscopic continuum electrostatic calculations: application to 2'GMP/3'GMP binding to ribonuclease T1 and implications for catalysis. 772 31

In wild-type ribonuclease T1 the peptide bond between Tyr38 and Pro39 is in the cis conformation. When Pro39 is replaced by an alanine this cis conformation is retained, and a non-prolyl cis Tyr38-Ala39 peptide bond is generated. We employed a stopped-flow double-mixing technique to investigate the kinetics of the cis-->trans isomerization of this peptide bond in the unfolding and the trans-->cis isomerization in the refolding of Pro39Ala-ribonuclease T1. In 6.0 M GdmCl (pH 1.6) and 25 degrees C the protein unfolds rapidly with a time constant of 20 ms, followed by Tyr38-Ala39 cis-->trans isomerization. This reaction shows a time constant of 730 ms and is about 60-fold faster than the isomerization of the Tyr38-Pro39 bond in the wild-type protein. Unfolded molecules with the Tyr38-Ala39 bond still in the native-like cis conformation accumulate transiently for a short time after unfolding is initiated, and they can refold very rapidly to the native state with a time constant of 290 ms (in 1.0 M GdmCl, pH 4.6, 25 degrees C). After more than three seconds of unfolding virtually all protein molecules contain an incorrect trans Tyr38-Ala39 bond and refolding is decelerated approximately 1000-fold, because Tyr38-Ala39 trans-->cis re-isomerization is very slow and, with its time constant of 480 s, determines the overall rate of refolding. Due to the coupling of the cis-trans equilibrium with protein folding it was possible to measure the kinetic parameters of the isomerization of a non-prolyl peptide bond in a protein. Previously this could not be accomplished, because the trans isomer is strongly preferred for unsubstituted peptide bonds in oligopeptides under virtually all conditions. Our data indicate that the kinetics of Tyr38-Pro39 and of Tyr38-Ala39 isomerization differ predominantly in the rate of the cis-->trans, rather than of the trans-->cis reaction. The rate of the trans-->cis reaction is, however, measured during refolding and may be influenced by the formation of ordered protein structure.
J Mol Biol 1995 Jan 06
PMID:Non-prolyl cis-trans peptide bond isomerization as a rate-determining step in protein unfolding and refolding. 782 21

The transcript of the Saccharomyces cerevisiae MER2 gene is spliced efficiently during meiosis but not during vegetative growth. Efficient splicing of the wild-type MER2 transcript requires the Mer1 protein, which is produced only in meiotic cells. Analysis of deletion and substitution mutations in the MER2 5' exon demonstrates that the unusually large size of this exon plays an important role in splicing regulation. The cis-acting sequences essential for Mer1-dependent splicing of MER2 RNA were determined by the analysis of MER2 deletion mutants and hybrid genes. The 80-base MER2 intron is sufficient for Mer1-dependent splicing in vivo, but sequences in the 5' exon enhance splicing efficiency. The Mer1 protein contains the KH motif found in some RNA-binding proteins, and RNA gel mobility shift assays demonstrate that Mer1 binds specifically to MER2 RNA. Both the transcript derived from the intronless MER2 gene and the transcript consisting only of the intron are able to bind to Mer1 in vitro, but neither has as high affinity for the protein as the intact substrate. RNase T1 footprinting indicates that the Mer1 protein contacts MER2 RNA at several points in the 5' exon and in the intron. Thus, Mer1 interacts directly with a regulatory element in MER2 RNA and promotes splicing.
Mol Cell Biol 1995 Apr
PMID:Binding of a cell-type-specific RNA splicing factor to its target regulatory sequence. 789 89

In chloroplasts, the 3' untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3'-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3' IR-containing RNA (3' IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b6/f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T1 digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3' IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3' untranslated region, only a single copy, which we have termed box II, appears to be essential for in vitro protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3'-end processing and/or influence the stability of petD mRNA in chloroplasts.
Mol Cell Biol 1995 Apr
PMID:An AU-rich element in the 3' untranslated region of the spinach chloroplast petD gene participates in sequence-specific RNA-protein complex formation. 789 96


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>