Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have investigated in detail the secondary and tertiary structures of E. coli 16S rRNA binding site of protein S15 using a variety of enzymatic and chemical probes. RNase T1 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with 1-cyclohexyl-3 (2-(1-methylmorpholino)-ethyl)-carboiimide-p- toluenesulfonate (at U(N-3) and G(N-1)) and with diethylpyrocarbonate (at A(N-7)). The RNA region corresponding to nucleotides 652 to 753 was tested within: (1) the complete 16S rRNA molecule; (2) a 16S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S15-16S rRNA complex; (4) the S15-fragment complex. Cleavage and modification sites were detected by primer extension with reverse transcriptase. Our results show that: (1) The synthetized fragment folds into the same overall secondary structure as in the complete 16S rRNA, with the exception of the large asymmetrical internal loop (nucleotides 673-676/714-733) which is fully accessible in the fragment while it appears conformationally heterogeneous in the 16S rRNA; (2) the reactivity patterns of the S15-16S rRNA and S15-fragment complexes are identical; (3) the protein protects defined RNA regions, located in the large interior loop and in the 3'-end strand of helix [655-672]-[734-751]; (4) the protein also causes enhanced chemical reactivity and enzyme accessibility interpreted as resulting from a local conformational rearrangement, induced by S15 binding.
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PMID:The E. coli 16S rRNA binding site of ribosomal protein S15: higher-order structure in the absence and in the presence of the protein. 245 25

The cytosolic untransformed molybdate-stabilized glucocorticoid-receptor complex from rat liver was eluted as a heterogenous peak containing two components with Stokes radii (Rs) of 8.3 nm and 7.1 nm when analyzed by size-exclusion HPLC even in the absence of molybdate. In contrast, the highly purified glucocorticoid receptor yielded a sharp symmetrical peak of Rs = 7.1 nm. We demonstrate that the 7.1-nm component could not result from a proteolytic degradation of the 8.3-nm receptor form. The same receptor heterogeneity was observed in thymus cytosol which contains less proteases than liver. After labeling with [3H]dexamethasone 21-mesylate and SDS/PAGE the same 94-kDa receptor band was revealed in both the 8.3-nm and 7.1-nm forms. Immunoblotting experiments showed that both the 94-kDa hormone-binding subunit and the 90-kDa heat-shock protein were present in the two different receptor forms. The 8.3-nm receptor form was converted to the 7.1-nm receptor form after treatment by ribonuclease A in the presence of molybdate and this effect was dose-dependent, being completely prevented by placental ribonuclease inhibitor (RNasin). In contrast, in the presence of molybdate, the 7.1-nm receptor form was ribonuclease-insensitive. Treatment of cytosol with RNase A in the absence of molybdate, partially shifted the untransformed receptor towards the 5.2-nm transformed receptor form. This effect was abolished by placental ribonuclease inhibitor. RNase S protein, an enzymatically inactive proteolytic fragment of RNase A, or S1 nuclease, which is specific for single-stranded nucleic acids, were ineffective when used instead of RNase A. In contrast, cobra venom endonuclease, which preferentially attacks double-stranded regions of small RNAs, caused a complete conversion of the 7-8-nm untransformed receptor to the 5.2-nm transformed receptor form. These results were not observed in the presence of molybdate. Addition of RNasin prior to heating cytosol in the absence of molybdate did not prevent the receptor from dissociating to the 5.2-nm form, suggesting that an endogenous RNase is not involved in the transformation process. The 7.1-nm receptor form was shifted to a 9.2-nm complex when incubated with an excess of GR 49 antireceptor antibody, whereas the 8.3-nm receptor form did not bind to the antibody.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:RNA binding to the untransformed glucocorticoid receptor. Sensitivity to substrate-specific ribonucleases and characterization of a ribonucleic acid associated with the purified receptor. 246 3

The processed gene L32', a member of the mouse gene family for ribosomal protein L32, could encode a 135 amino acid protein nearly identical to L32. The 5'-flanking region of the gene contains CAAT and TATA sites at positions commonly found in expressed genes. The L32' gene lies within highly methylated, DNase I-insensitive chromatin of mouse L1210 cells. Although S1 nuclease digestion studies suggested that an L32' transcript might be produced, an oligonucleotide probe specific for L32' mRNA, and RNase digestion of a cRNA probe to L32', indicated fewer than 0.1 L32' transcripts/cell. These results demonstrate that extreme caution is required when measuring transcription from related genes.
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PMID:Analysis of potential expression of highly related members of the ribosomal protein L32 gene family. 246 15

Translational initiation factor 3 (IF3) is an RNA helix destabilizing protein which interacts with strongly conserved sequences in 16S rRNA, one at the 3' terminus and one in the central domain. It was therefore of interest to identify particular residues whose exposure changes upon IF3 binding. Chemical and enzymatic probing of central domain nucleotides of 16S rRNA in 30S ribosomal subunits was carried out in the presence and absence of IF3. Bases were probed with dimethyl sulfate (DMS), at A(N-1), C(N-3), and G(N-7), and with N-cyclohexyl-N'-[2-(N-methyl-4-morpholinio)ethyl] carbodiimide p-toluenesulfonate (CMCT), at G(N-1) and U(N-3). RNase T1 and nuclease S1 were used to probe unpaired nucleotides, and RNase V1 was used to monitor base-paired or stacked nucleotides. 30S subunits in physiological buffers were probed in the presence and absence of IF3. The sites of cleavage and modification were detected by primer extension. IF3 binding to 30S subunits was found to reduce the chemical reactivity and enzymatic accessibility of some sites and to enhance attack at other sites in the conserved central domain of 16S rRNA, residues 690-850. IF3 decreased CMCT attack at U701 and U793 and V1 attack at G722, G737, and C764; IF3 enhanced DMS attack at A814 and V1 attack at U697, G833, G847, and G849. Many of these central domain sites are strongly conserved and with the conserved 3'-terminal site define a binding domain for IF3 which correlates with a predicted cleft in two independent models of the 30S ribosomal subunit.
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PMID:Escherichia coli initiation factor 3 protein binding to 30S ribosomal subunits alters the accessibility of nucleotides within the conserved central region of 16S rRNA. 251 87

Murine complement protein H is encoded by a 100-kb gene on chromosome 1. A 3.2-kb fragment of the 5' flanking region of the H gene was sequenced, and two transcription start sites for this gene were identified by RNase protection and S1 nuclease analyses, each of which had upstream TATA and CAAT boxes. This region shares sequence homology with known regulatory elements, including the SV40 enhancer consensus, the Sp1 binding site, and two glucocorticoid-responsive core elements (GRE). Tissue and cell-line specificity has been examined by Northern analysis, and the 4.4-kb full-length H messenger RNA was identified in liver, kidney, spleen, thymus, liver cell line 1469, and L cells. IFN-gamma did not induce H mRNA expression in the macrophage cell line P388D.1 but had a positive effect on both the mRNA and protein levels of H in L cells. PMA, LPS, and vitamin D did not increase H mRNA levels in L cells. Pursuant to the discovery of two GRE in the 5' regulatory region of the H gene, we examined the effects of glucocorticoids on H mRNA expression. Dexamethasone (10(-7) M) was found to increase markedly the levels of H mRNA and protein after 24 h of incubation, and the effect on the mRNA was detectable by 30 min. The fact that H is a down-regulator of complement activation is consistent with the known immunosuppressive role of glucocorticoids. To our knowledge, this is the first time that dexamethasone has been shown to increase the levels of a complement protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Analysis of complement factor H mRNA expression: dexamethasone and IFN-gamma increase the level of H in L cells. 253 12

To determine the pattern of alternative splicing at the 5' end of class I genes, the 3' splice sites bordering exon 2 of the H-2Dd and H-2Kd genes were mutated from AG to GG (H-2Dd) or CG (H-2Kd). The mutant genes were transfected into L cells, and RNA from clones expressing these Ag was used for analysis by RNase and S1 nuclease mapping techniques. The first intervening sequence of both class I genes contains several potential 3' splice acceptor sites. However, a clear preference for only one site was detected in each of the H-2Dd and H-2Kd mRNA. Examination of the endogenous H-Dd and H-2Kd class I transcripts in normal murine tissues and in tumors demonstrated that the alternatively spliced mRNAs were produced, but at a low frequency. Infection of transfected L cells or tumor lines with vesicular stomatitis virus altered the level of differentially spliced message in these cells.
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PMID:Mutation of 3' splice sites in two different class I genes results in different usage of cryptic splice sites. 254 75

Mutations that define the ctaA gene of Bacillus subtilis block cytochrome aa3 formation and sporulation. We have recently described the isolation and initial characterization of the ctaA locus. Analysis of in vivo mRNA transcripts by RNase protection experiments located the 5' and 3' termini of the ctaA transcript, confirming a monocistronic structure. By using a nuclease protection assay, an increase in the abundance of steady-state ctaA mRNA was observed during the initiation of sporulation, followed by a decrease during subsequent stages. Transcripts originating from the ctaA gene were most abundant 2.0 h after the end of exponential growth. This pattern of ctaA mRNA accumulation was confirmed by coupling the transcription of the ctaA gene to lacZ in an integrative plasmid vector. Expression of ctaA was not repressed by glucose and was independent of the spoOA and spoOH (sigH) gene products. Postexponential expression was found to be dependent on the product of the strC gene. The expression of ctaA appears to be regulated in a growth stage-specific manner. The transcriptional start site, identified by high-resolution S1 nuclease protection experiments, was preceded by a single sigma A-dependent promoter sequence.
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PMID:Structure and expression of the cytochrome aa3 regulatory gene ctaA of Bacillus subtilis. 254 7

Degradation intermediates of the estrogen-regulated apolipoprotein (apo) II mRNA were identified by S1 nuclease mapping and primer extension analysis. S1 mapping of poly(A)-RNA detected a series of mRNAs truncated at specific sites in the 3'-noncoding region. Many of these sites were also detected by primer extension analysis indicating that truncated molecules resulted from endonucleolytic cleavage in the 3'-noncoding region. Identical cleavage sites were seen with RNA from estrogen-treated animals or from animals withdrawn from hormone under conditions where apoII mRNA degraded in the slow (t1/2 = 13 h) or rapid (t1/2 = 1.5 h) decay mode. No differences were seen in poly(A) tail length or heterogeneity among these conditions. These results indicate that the estrogen-induced alteration in apoII mRNA turnover does not involve a new pathway of degradation, but, more likely, involves an increased targeting of the mRNA for degradation by a preexisting pathway. These data are consistent with a mechanism in which the initial step in apoII mRNA degradation is an endonucleolytic cleavage in the 3'-noncoding region without prior removal of the poly(A) tail. The endonucleolytic cleavage sites occurred predominantly at 5'-AAU-3' or 5'-UAA-3' trinucleotides found in single-stranded domains in a secondary structure model of the naked mRNA (Hwang, S-P. L., Eisenberg, M., Binder, R., Shelness, G. S., and Williams, D. L. (1989) J. Biol. Chem. 264, 8410-8418). The structure of the 3'-noncoding region in polyribosomal messenger ribonucleoprotein was examined by titrations of liver homogenates with dimethyl sulfate and cobra venom RNase. The results suggest that the typical cleavage site is a 5'-AAU-3' or 5'-UAA-3' trinucleotide in an accessible single-stranded loop domain. Single-stranded domains alone or accessible domains alone are not sufficient for cleavage. Similarly, 5'-AAU-3' or 5'-UAA-3' trinucleotides alone are not sufficient for cleavage. Localization of these trinucleotides to accessible single-stranded domains in the polyribosomal messenger ribonucleoprotein may provide the specificity for cleavage during targeted degradation.
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PMID:Degradation of apolipoprotein II mRNA occurs via endonucleolytic cleavage at 5'-AAU-3'/5'-UAA-3' elements in single-stranded loop domains of the 3'-noncoding region. 255 Apr 65

Total RNA from Ehrlich ascites mitochondria pretreated with RNase-free DNase was capped in vitro with [alpha-32P]GTP and guanylyl transferase. The cappable RNAs representing the primary transcripts show a heterogeneous size distribution with four major species of 46, 63, 94, and 152 nucleotides and four minor species of 19, 24, 104, and 790 nucleotides in size. Hybridization with the D-loop DNA probes shows that the 19-nucleotide-long capped RNA is coded by the H-strand of mitochondrial DNA while the rest are coded by the L-strand. S1 nuclease mapping and primer extension analyses suggest the occurrence of a transcription initiation of H-strand at about 19 nucleotides upstream from the start of the tRNA(Phe) gene. All of the L-strand cappable RNAs have a common 5' end mapping to nucleotide 16,183 +/- 5 of the genome. The 3' ends of four major cappable RNA species line up to the conserved sequence boxes, putative start sites of DH-DNA; and in fact about 2% of these cappable species are found to exist as DNA-linked RNA under steady-state conditions. The 3' end of the 790-nucleotide cappable RNA lies close to the start of the tRNA(Pro) gene, suggesting that it may be the true precursor of L-strand transcript endonucleolytically processed at the 3' end. The level of L-strand-coded cappable RNAs varies markedly under different growth conditions. Treatment with cycloheximide results in a reduction while chloramphenicol caused over 3-fold induction, suggesting that these "primer" RNAs may have an additional regulatory function.
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PMID:Characterization of primary transcripts and identification of transcription initiation sites on the heavy and light strands of mouse mitochondrial DNA. 271 42

Human rRNA precursor from normal or stressed HeLa cells were studied by S1 nuclease mapping of unlabeled RNA and by antisense RNase mapping of RNA from cells that had been labeled in vivo with [32P]PO4. Heating cells to 43 degrees C decreased the amount of newly synthesized rRNA to less than 5% of the control level and led to greater than 95% inhibition of transcription termination at a region 355 to 362 nucleotides downstream of the 3' end of 28S rRNA, with readthrough continuing into the next transcription unit. Heating of cells to 42 degrees C led to 60% inhibition of termination at this site; 50% of transcripts that extended into the nontranscribed spacer ended in a region 200 to 210 nucleotides upstream of the polymerase I (Pol I) initiation site. This is presumed to be the human upstream transcription termination site because of the absence of RNAs with a 5' end corresponding to this region, the location relative to the Pol I initiation site (which is similar to the location of upstream terminators in other species), and the fact that it is 15 to 25 nucleotides upstream of the sequence GGGTTGACC, which has an 8-of-9 base identity with the sequence 3' of the downstream termination site. Surprisingly, treatment of cells with sodium arsenite, which also leads to the induction of a stress response, did not inhibit termination. Pol I initiation was decreased to the same extent as termination, which lends support to the hypothesis that termination and initiation are coupled. Although termination was almost completely inhibited at 43 degrees C, the majority of the recently synthesized rRNAs were processed to have the correct 3' end of 28S. This finding suggests that 3'-end formation can involve an endonucleolytic cut and is not solely dependent on exonucleolytic trimming of correctly terminated rRNAs.
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PMID:Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription. 276 37


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