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Query: UNIPROT:P06889 (Mol)
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We have studied the effects of protein mutations on the higher order structure of 16 S rRNA in Escherichia coli ribosomes, using a set of structure-sensitive chemical probes. Ten mutant strains were studied, which contained alterations in ribosomal proteins S4 and S12, including double mutants containing both altered S4 and S12. Two ribosomal ambiguity (ram) S4 mutant strains, four streptomycin resistant (SmR) S12 mutant strains, one streptomycin pseudodependent (SmP) S12 mutant strain, one streptomycin dependent (SmD) S12 mutant strain and two streptomycin independent (Sm1) double mutants (containing both-SmD and ram mutations) were probed and compared to an isogenic wild-type strain. In ribosomes from strains containing S4 ram mutations, nucleotides A8 and A26 become more reactive to dimethyl sulfate (DMS) at their N-1 positions. In ribosomes from strains bearing the SmD allele, A908, A909, A1413 and G1487 are significantly less reactive to chemical probes. These same effects are observed when the S4 and S12 mutations are present simultaneously in the double mutants. An interesting correlation is found between the reactivity of A908 and the miscoding potential of SmR, SmD, SmP and wild-type ribosomes; the reactivity of A908 increases as the translational error frequency of the ribosomes increases. In the case of ram ribosomes, the reactivity of A908 resembles that of wild-type, unless tRNA is bound, in which case it becomes hyper-reactive. Similarly, streptomycin has little effect on A908 in wild-type ribosomes unless tRNA is bound, in which case its reactivity increases to resemble that of ram ribosomes with bound tRNA. Finally, interaction of streptomycin with SmP and SmD ribosomes causes the reactivity of A908 to increase to near-wild-type levels. A simple model is proposed, in which the reactivity of A908 reflects the position of an equilibrium between two conformational states of the 30 S subunit, one of which is DMS-reactive, and the other DMS-unreactive. In this model, the balance between these two states would be influenced by proteins S4 and S12. Mutations in S12 generally cause a shift toward the unreactive conformer, and in the case of SmD and SmP ribosomes, this shift can be suppressed phenotypically by streptomycin, ram mutations in protein S4 cause a shift toward the reactive conformer, but only when tRNA is bound. This suggests that the opposing effects of these two classes of mutations influence the proof-reading process by somewhat different mechanisms.
J Mol Biol 1989 Aug 05
PMID:Mutations in ribosomal proteins S4 and S12 influence the higher order structure of 16 S ribosomal RNA. 247 54

The DNase I protection pattern of E sigma 32 was assayed on three heat shock promoters, the E sigma 32 promoter for the groESL operon, P2 of the dnaKJ operon, and rpoD PHS, the E sigma 32 promoter upstream from rpoD. E sigma 32 protected each of these promoters from DNase I digestion from around -60 to around +20. Protection from dimethyl sulfate methylation was assayed at the groE promoter. E sigma 32 binding altered the sensitivity to methylation of bases in the vicinity of both the -10 and -35 regions. The DNase I footprints for the E sigma 32 promoters were very similar to the DNase I footprint of E sigma 70 on the lacUV5 promoter. After analyzing the DNase I footprints by taking into account the contacts predicted to be made by DNase I, it appeared that E sigma 32, like E sigma 70, contacts the DNA primarily on one face of the helix in the -35 region and on both faces in the -10 region.
J Mol Biol 1989 Dec 05
PMID:Interaction of Escherichia coli RNA polymerase holoenzyme containing sigma 32 with heat shock promoters. DNase I footprinting and methylation protection. 269 36

The interaction of E sigma 32 with the groE promoter at temperatures between 0 degrees C and 37 degrees C was studied using DNase I footprinting and dimethyl sulfate methylation. Three distinct complexes were observed. At 0 degrees C E sigma 32 fully protected sequences between -60 and -5 from DNase I digestion on the top (non-template) strand of the promoter. At 16 degrees C the majority of the E sigma 32 promoter complexes had a DNase I footprint almost identical with that seen at 37 degrees C, protecting the DNA from about -60 to +20; however, little DNA strand separation had occurred, and the changes in sensitivity of guanine residues to dimethyl sulfate methylation caused by E sigma 32 differed from those seen at 37 degrees C. DNA strand separation, and changes in the pattern of protections from and enhancements of methylation by dimethyl sulfate to those characteristic of the open complex, occurred at temperatures between 16 degrees C and 27 degrees C. It is plausible to assume that these temperature-dependent isomerizations are analogous to the time-dependent sequence of intermediates on the pathway to open complex formation at 37 degrees C. Therefore we propose that the formation of an open complex by E sigma 32 at the groE promoter involves three classes of steps: E sigma 32 initially binds to the promoter in a closed complex (RPC1) in which the enzyme interacts with a smaller region of the DNA than in the open complex. E sigma 32 then isomerizes to form a second closed complex (RPC2) in which the enzyme interacts with the same region of the DNA as in the open complex. Finally, a process of local DNA denaturation (strand opening) leads to formation of the open complex (RPO).
J Mol Biol 1989 Dec 05
PMID:Intermediates in the formation of the open complex by RNA polymerase holoenzyme containing the sigma factor sigma 32 at the groE promoter. 269 37

The FLP recombinase interacts with its target sequence with the formation of three distinct DNA-protein complexes. The first complex leaves neither a DNase footprint nor is the DNA protected from methylation by dimethyl sulfate. We have found, however, that the FLP protein is bound predominantly to only one of the three 13 base-pair (bp) symmetry elements. This asymmetric loading of the FLP site seems to require the presence of an adjacent directly repeated 13 bp element. We speculate that this asymmetric filling of the target site may be accompanied by the unique order of cleavage and exchange of DNA strands.
J Mol Biol 1988 Nov 20
PMID:The mechanism of loading of the FLP recombinase onto its DNA target sequence. 285 60

The murine immunoglobulin kappa gene enhancer has previously been found to coincide with a region of altered chromatin structure reflected in a DNase I hypersensitivity site detectable on Southern blots of B-cell DNA. We examined the chromatin structure of the homologous region of human DNA using the high-resolution electroblotting method originally developed for genomic sequence analysis by G. Church and W. Gilbert (Proc. Natl. Acad. Sci. USA 81:1991-1995, 1984). Analysis of DNA isolated from cells treated in vivo with dimethyl sulfate revealed two B-cell-specific sites of enhanced guanine methylation. Both sites are located within perfect inverted repeats theoretically capable of forming cruciform structures; one of these repeats overlaps an enhancer core sequence. No enhancement or protection of guanine methylation was observed within sequences similar to sites of altered methylation previously described in the immunoglobulin heavy-chain enhancer. Treatment of isolated nuclei with DNase I or a variety of restriction endonucleases defined a B-cell-specific approximately 0.25-kilobase region of enhanced nuclease susceptibility similar to that observed in the murine kappa enhancer. The 130-base-pair DNA segment that shows high sequence conservation between human, mouse, and rabbit DNAs lies at the 5' end of the nuclease-susceptible region.
Mol Cell Biol 1987 Jan
PMID:Human immunoglobulin kappa gene enhancer: chromatin structure analysis at high resolution. 303 54

Proteins capable of interacting with the enhancer of the immunoglobulin kappa gene in vitro have been detected in extracts of nuclei from human B cells and from human, mouse, and rabbit spleens. The experiments, based on an exonuclease protection technique, demonstrate nuclear protein factors binding to a 30- to 35-base-pair domain containing both the simian virus 40 enhancer core element (TTTCCA) and the octamer CAGGTGGC that was previously identified as the consensus sequence for protein-binding sites in the murine immunoglobulin heavy-chain enhancer. This 30- to 35-base-pair domain in the human kappa enhancer is homologous to a site of protein binding detected in the murine kappa enhancer by other investigators using a gel retardation assay. Our results complement in vivo dimethyl sulfate footprinting studies of the human immunoglobulin kappa enhancer which demonstrated B cell-specific changes in guanine reactivity immediately 5' to the consensus octamer. Together, these findings suggest that DNA-binding proteins in B-cell nuclei interact with the 5' portion of the human kappa-gene enhancer. Such proteins could play a role in the B cell-specific transcription of the human immunoglobulin kappa gene.
Mol Cell Biol 1987 May
PMID:B-cell nuclear proteins binding in vitro to the human immunoglobulin kappa enhancer: localization by exonuclease protection. 303 35

Escherichia coli RNA polymerase contacts promoter DNA at two regions (the -10 and -35 regions) which are separated by a segment of spacer DNA. Previously we showed that base substitutions in the spacer DNA can affect promoter strength both in vitro and in vivo; these results were interpreted to reflect altered structural properties of the substituted DNAs. Here we provide experimental support for this interpretation. The pattern of cleavage of the promoters with Neurospora crassa endonuclease and the reactivity of their guanine residues with dimethyl sulfate (DMS) suggest that the structures of the spacer DNAs in the promoters with altered transcriptional activities are distinct. In addition, the binding of RNA polymerase to the latter promoters induces characteristic enhancements in the extent to which specific guanine residues in the spacer DNAs react with DMS. We propose that for these promoters the substitutions in the spacer DNAs have affected the relative orientation of the -10 and -35 regions. The observed differences in promoter activity then would reflect the requirement for realignment of these regions during the process of open complex formation; we postulate that two such realignments occur.
J Mol Biol 1988 Aug 05
PMID:Promoter recognition by Escherichia coli RNA polymerase. Influence of DNA structure in the spacer separating the -10 and -35 regions. 305 Jan 26

The nucleotide sequence of a 6.5 kilobasepair chromosomal DNA fragment encoding the anaerobic dimethylsulphoxide (DMSO) reductase operon of Escherichia coli has been determined. The DMSO reductase structural operon was shown to consist of three open reading frames, namely dmsABC, encoding polypeptides with predicted molecular weights of 87,350, 23,070, and 30,789 Daltons, respectively. The DMS A polypeptide displayed a high degree of amino acid sequence homology with the single-subunit enzyme, biotin sulphoxide reductase (bisC) and with formate dehydrogenase (fdhF), suggesting that the active site and molybdopterin cofactor binding site that is common to these enzymes is located in the DMS A subunit. A comparison of the predicted N-terminal amino acids of the dmsA gene product to those of the 82,600 subunit of purified DMSO reductase indicated that post-translational processing of a 16 amino acid peptide at the amino terminus of DMS A had occurred. The DMS B polypeptide contains 16 cysteine residues organized in four clusters, two of which are typical of 4Fe-4S binding domains. The DMS C polypeptide is composed of eight segments of hydrophobic amino acids of appropriate length to cross the cytoplasmic membrane, suggesting that this subunit functions to anchor the enzyme to the membrane.
Mol Microbiol 1988 Nov
PMID:Nucleotide sequence of the dmsABC operon encoding the anaerobic dimethylsulphoxide reductase of Escherichia coli. 306 12

The estrogen-dependent binding of a protein to the upstream region of the chicken vitellogenin gene was detected by using in vivo dimethyl sulfate, genomic DNase I, and in vitro exonuclease III footprinting. The site is located between base pairs -848 and -824, and its sequence resembles that of the nuclear factor I binding site. The results suggest that a nuclear factor binding to this site is involved in the regulation of the vitellogenin gene.
Mol Cell Biol 1988 Oct
PMID:Estrogen-inducible binding of a nuclear factor to the vitellogenin upstream region. 318 60

The rhaC gene, whose product is the positive activator of the genes required for L-rhamnose utilization, has been cloned along with the rhamnose structural genes. The rhaC sequence shows two partially overlapping reading frames, encoding two proteins of molecular weight 32,000 and 35,000 RhaS and RhaR. Both proteins show significant homology to AraC, the positive activator of the arabinose operon. S1 mapping located transcriptional start points and showed that RhaR, and possibly RhaS, positively regulate transcription from the structural gene promoters as well as transcription from their own promoter. In-vivo dimethyl sulfate footprinting and DNase I footprinting indicate that the RhaR protein may bind to DNA elements upstream from its RNA polymerase binding site.
J Mol Biol 1987 Aug 20
PMID:Positive regulation of the Escherichia coli L-rhamnose operon is mediated by the products of tandemly repeated regulatory genes. 331 63


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