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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The rate-limiting step during the refolding of S54G/P55N ribonuclease T1 is determined by the slow trans-->cis prolyl isomerisation of Pro39. We investigated the refolding of this variant by one-dimensional (1D) and two-dimensional (2D) real-time NMR spectroscopy, initiated by a tenfold dilution from 6 M guanidine hydrochloride at 10 degreesC. Two intermediates could be resolved with the 1D approach. The minor intermediate, which is only present early during refolding, is largely unfolded. The major intermediate, with an incorrect trans Pro39 peptide bond, is highly structured with 33 amide protons showing native chemical shifts and native NOE patterns. They could be assigned in a real-time 2D-NOESY (nuclear Overhauser enhancement spectroscopy) by using a new assignment strategy to generate positive and negative signal intensities for native and non-native NOE cross-peaks, respectively. Surprisingly, amide protons with non-native environments are located not only close to Tyr38-Pro39, but are spread throughout the entire protein, including the C-terminal part of the alpha-helix, beta-strands 3 and 4 and several loop regions. Native secondary and tertiary structure was found for the major intermediate in the N-terminal beta-strands 1 and 2 and the C terminus (connected by the disulfide bonds), the N-terminal part of the alpha-helix, and the loops between beta-strands 4/5 and 5/6. Implications of these native and non-native structure elements of the intermediate for the refolding of S54G/P55N ribonuclease T1 and for cis/trans isomerizations are discussed.
J Mol Biol 1999 Jan 15
PMID:A protein folding intermediate of ribonuclease T1 characterized at high resolution by 1D and 2D real-time NMR spectroscopy. 987 47

Single amino acid residue substitutions rarely destroy the structural integrity of proteins. Substitution of glycine residues, however, is among the few sorts of alterations that can have such an effect. Here, we seek to understand what accounts for the extreme functional impairment of the bacterial ribonuclease barnase upon substitution of Gly52 or Gly53. We find that inactivation is caused by overall disruption of the folded state that manifests itself in three ways: (1) dramatically reduced stability (by 5.2 to 8.4 kcal mol-1 for mutants showing inactivation in vivo); (2) progressive loss of folded-state activity with increasing temperature, indicating a less well formed fold; and (3) substantial proteolytic degradation of mutant enzymes in vivo. Examination of two deletion mutants, missing either Gly53 or Asp54, shows that the irregular beta-bulge formed by these two residues is of vital importance to the structural integrity of barnase. The parallel behaviour of mutants carrying replacements of either of the two glycine residues therefore appears to arise from a common mechanism: disruption of local structure at the beta-bulge. The importance of this structural element to the function of barnase raises the question of whether it may be present in other RNases. The Streptomyces enzymes RNase Sa and RNase St differ considerably from barnase in both sequence and structure, yet both show significant sequence similarity to barnase over a region beginning at Gly53. Structural comparison indicates that the Streptomyces enzymes do have the barnase-like irregular beta-bulge, making this an important characteristic feature of a group of bacterial ribonucleases. The sensitivity of this feature demonstrates that detailed aspects of local structure may have a major role in determining the overall structural and functional properties of an enzyme, even where no explanation for this role is readily apparent. If this is a general characteristic of the structure-function relationship, it may pose a formidable obstacle to the de novo design of new enzymes.
J Mol Biol 1999 Mar 12
PMID:An irregular beta-bulge common to a group of bacterial RNases is an important determinant of stability and function in barnase. 1006 10

Plasmids with whole genes for ribonucleases from B. intermedius (binase) and B. pumilis (RNase Bp) assembled with the whole gene of barstar, a specific intracellular inhibitor, are constructed. The resultant plasmids pMZ55 and pMZ56 effectively express binase and RNase Bp genes in B. subtilis cells. A medium for maximum expression of RNase genes by recombinant strains is developed. The expression of binase and RNase Bp genes in B. subtilis cells is negatively regulated by exogenic inorganic phosphate.
Mol Gen Mikrobiol Virusol 1999
PMID:[Expression of secreted guanyl-specific ribonuclease genes from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis cells]. 1019 Jan 4

Random mutations were generated in the sequence for the 5' untranslated region (5'UTR) of the Chlamydomonas reinhardtii chloroplast rps7 mRNA by PCR, the coding sequence for the mutant leaders fused upstream of the lacZ' reporter in pUC18, and transformed into Escherichia coli, and white colonies were selected. Twelve single base pair changes were found at different positions in the rps7 5'UTR in 207 white colonies examined. Seven of the 12 mutant leaders allowed accumulation of abundant lacZ' message. These mutant rps7 leaders were ligated into an aadA expression cassette and transformed into the chloroplast of C. reinhardtii and into E. coli. In vivo spectinomycin-resistant growth rates and in vitro aminoglycoside adenyltransferase enzyme activity varied considerably between different mutants but were remarkably similar for a given mutant expressed in the Chlamydomonas chloroplast and in E. coli. The variable effect of the mutants on aadA reporter expression and their complete abolition of lacZ' reporter expression in E. coli suggests differences in the interaction between the 5'UTR of rps7 and aadA or lacZ' coding regions. Several rps7 5'UTR mutations affected the predicted folding pattern of the 5'UTR by weakening the stability of stem structures. Site-directed secondary mutations generated to restore these structures in the second stem suppressed the loss of reporter activity caused by the original mutations. Additional site-directed mutations that were predicted to further strengthen (A-U-->G-C) or weaken (G-C-->A-U) the second stem of the rps7 leader both resulted in reduced reporter expression. This genetic evidence combined with differences between mutant and wild-type UV melting profiles and RNase T1 protection gel shifts further indicate that the predicted wild-type folding pattern in the 5'UTR is likely to play an essential role in translation initiation.
Mol Cell Biol 1999 Oct
PMID:Mutations altering the predicted secondary structure of a chloroplast 5' untranslated region affect its physical and biochemical properties as well as its ability to promote translation of reporter mRNAs both in the Chlamydomonas reinhardtii chloroplast and in Escherichia coli. 1049 Jun 35

The ribonuclease T1 variant 9/5 with a guanine recognition segment, altered from the wild-type amino acid sequence 41-KYNNYE-46 to 41-EFRNWQ-46, has been cocrystallised with the specific inhibitor 2'-GMP. The crystal structure has been refined to a crystallographic R factor of 0.198 at 2.3 A resolution. Despite a size reduction of the binding pocket, pushing the inhibitor outside by 1 A, 2'-GMP is fixed to the primary recognition site due to increased aromatic stacking interactions. The phosphate group of 2'-GMP is located about 4.2 A apart from its position in wild-type ribonuclease T1-2'-GMP complexes, allowing a Ca(2+), coordinating this phosphate group, to enter the binding pocket. The crystallographic data can be aligned with the kinetic characterisation of the variant, showing a reduction of both, guanine affinity and turnover rate. The presence of Ca(2+) was shown to inhibit variant 9/5 and wild-type enzyme to nearly the same extent.
J Mol Biol 1999 Dec 17
PMID:Structural analysis of an RNase T1 variant with an altered guanine binding segment. 1060 Mar 81

A second secreted ribonuclease, designated binase II, has been detected in Bacillus intermedius 7P, and its structural gene was cloned and sequenced. Unlike the well-known binase I, a 109-amino acid guanyl-specific enzyme, the 292-residue binase II is closely related to the B. subtilis nuclease Bsn, in structure and in its enzymatic properties. Binase II is also insensitive to inactivation by barstar, an inhibitor protein that is specific for guanyl-specific ribonucleases. While both B. intermedius enzymes are induced upon phosphate starvation, only the gene for binase I belongs to the pho regulon system and carries pho-box elements adjacent to its promoter sequence. The gene for binase II is similar to that for Bsn in lacking such elements. The birB gene coding for binase II appears to be located next to the 3'-end of a ferric ion transport operon, with which it convergently overlaps. This would allow attenuator control over binase II expression under conditions of starvation for ferric ions.
Mol Gen Genet 2000 May
PMID:A novel secreted ribonuclease from Bacillus intermedius: gene structure and regulatory control. 1085 77

The aim of this study was to gain a better understanding of the contribution of hydrogen bonds by tyrosine -OH groups to protein stability. The amino acid sequences of RNases Sa and Sa3 are 69 % identical and each contains eight Tyr residues with seven at equivalent structural positions. We have measured the stability of the 16 tyrosine to phenylalanine mutants. For two equivalent mutants, the stability increases by 0.3 kcal/mol (RNase Sa Y30F) and 0.5 kcal/mol (RNase Sa3 Y33F) (1 kcal=4.184 kJ). For all of the other mutants, the stability decreases with the greatest decrease being 3.6 kcal/mol for RNase Sa Y52F. Seven of the 16 tyrosine residues form intramolecular hydrogen bonds and the average decrease in stability for these is 2.0(+/-1.0) kcal/mol. For the nine tyrosine residues that do not form intramolecular hydrogen bonds, the average decrease in stability is 0.4(+/-0.6) kcal/mol. Thus, most tyrosine -OH groups contribute favorably to protein stability even if they do not form intramolecular hydrogen bonds. Generally, the stability changes for equivalent positions in the two proteins are remarkably similar. Crystal structures were determined for two of the tyrosine to phenylalanine mutants of RNase Sa: Y80F (1.2 A), and Y86F (1.7 A). The structures are very similar to that of wild-type RNase Sa, and the hydrogen bonding partners of the tyrosine residues always form intermolecular hydrogen bonds to water in the mutants. These results provide further evidence that the hydrogen bonding and van der Waals interactions of polar groups in the tightly packed interior of folded proteins are more favorable than similar interactions with water in the unfolded protein, and that polar group burial makes a substantial contribution to protein stability.
J Mol Biol 2001 Sep 14
PMID:Tyrosine hydrogen bonds make a large contribution to protein stability. 1155 95

Y-box proteins are major constituents of ribonucleoprotein particles (RNPs) which contain translationally silent mRNAs in gametic cells. We have recently shown that a sequence-specific RNA binding activity present in spermatogenic cells contains the two Y-box proteins MSY2 and MSY4. We show here that MSY2 and MSY4 bind a sequence, 5'-UCCAUCA-3', present in the 3' untranslated region of the translationally repressed protamine 1 (Prm1) mRNA. Using pre- and post-RNase T1-digested substrate RNAs, it was determined that MSY2 and MSY4 can bind an RNA of eight nucleotides containing the MSY2 and MSY4 binding site. Single nucleotide mutations in the sequence eliminated the binding of MSY2 and MSY4 in an electrophoretic mobility shift assay, and the resulting mutants failed to compete for binding in a competition assay. A consensus site of U(AC)C(A)CAU(C)CA(CU) (subscripts indicate nucleotides which do not disrupt YRS binding by MSY2 and MSY4), denoted the Y-box recognition site (YRS), was defined from this mutational analysis. These mutations in the YRS were further characterized in vivo using a novel application of the yeast three-hybrid system. Experiments with transgenic mice show that disruption of the YRS in vivo relieves Prm1-like repression of a reporter gene. The conservation of the RNA binding motifs among Y-box protein family members raises the possibility that other Y-box proteins may have previously unrecognized sequence-specific RNA binding activities.
Mol Cell Biol 2001 Oct
PMID:MSY2 and MSY4 bind a conserved sequence in the 3' untranslated region of protamine 1 mRNA in vitro and in vivo. 1156 83

The Escherichia coli endoribonucleases RNase E (Rne) and RNase G (Rng) have sequence similarity and broadly similar sequence specificity. Whereas the absence of Rne normally is lethal, we show here that E. coli bacteria that lack the rne gene can be made viable by overexpression of Rng. Rng-complemented cells accumulated precursors of 5S ribosomal RNA (rRNA) and the RNA component of RNase P (i.e. M1 RNA), indicating that normal processing of these Rne-cleaved RNAs was not restored by RNase G; additionally, neither 5S rRNA nor M1 RNA was generated from precursors by RNase G cleavage in vitro. Using DNA microarrays containing 4405 Escherichia coli open reading frames (ORFs), we identified mRNAs whose steady-state level was affected by Rne, Rng or the N-terminal catalytic domain of RNase E. Most transcript species affected by RNase E deficiency were also elevated in an rne deletion mutant complemented by Rng. However, approximately 100 mRNAs that accumulated in Rne-deficient cells were decreased by rng-complemention, thus identifying targets whose processing or degradation may be the basis for RNase E essentiality. Remarkably prominent in this group were mRNAs implicated in energy-generating pathways or in the synthesis or degradation of macromolecules.
Mol Microbiol 2002 Mar
PMID:RNase G complementation of rne null mutation identifies functional interrelationships with RNase E in Escherichia coli. 1195 97

Insulin directly down-regulates the gene expression of the rat CYP2E1 by altering its mRNA stability (De Waziers, I., Garlatti, M., Bouguet, J., Beaune, P. H., and Barouki, R. (1995) Mol. Pharmacol. 47, 474-479). Because the regulation of CYP mRNA stability was poorly understood, the molecular mechanisms involved in this regulation in the rat hepatoma H4IIEC3 cell line were studied. By using RNase T1 protection methods, the formation of a major CYP2E1 RNA-protein complex was observed. By competition experiments, the binding site of this complex was located on a 16-nucleotide sequence in the 5'-proximal region of the CYP2E1-coding sequence. Insulin did not modify the binding pattern of proteins to this sequence. and transfections of expression vectors or antisense oligonucleotides were undertaken to demonstrate the actual functionality of the 16-mer sequence. The insertion of this sequence in a luciferase gene was sufficient to render the chimeric mRNA sensitive to insulin. Furthermore, transfection of H4IIEC3 cells with antisense oligonucleotide complementary to this sequence blocked the insulin effect on the CYP2E1 mRNA expression, i.e. its rapid degradation. All these results demonstrate that this 16-nucleotide sequence is implicated in the CYP2E1 post-transcriptional regulation by insulin.
 ...
PMID:Identification of a 16-nucleotide sequence that mediates post-transcriptional regulation of rat CYP2E1 by insulin. 1227 Sep 35


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