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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)

The termination of transcription in the dnaA gene of E. coli was analyzed using transcriptional fusions to the galactokinase gene, S1 nuclease mapping and quantification of translation products by Western blots. The majority of transcripts originating from dnaA promoters terminated at several positions within a 200 bp region inside the dnaA reading frame.
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PMID:Transcription termination in the dnaA gene. 176 43

DNA sequences affecting the transcription of the Escherichia coli rnpB transcript encoding the catalytic M1 RNA subunit of RNase P have been analyzed. Previous work (Motamedi, H., Lee, Y., and Schmidt, F.J.) (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3959-3963) identified S1 nuclease protection products corresponding to transcripts originating upstream of the M1 RNA gene. Sequence analysis of the upstream region of rnpB identified three regions homologous to the E. coli consensus promoter sequence. In the present work, analysis of in vitro transcription products by S1 nuclease mapping indicated that all three promoter homologies were capable of directing transcription. The nearest promoter, P-1, was approximately 100 times more active than either of the upstream homologies P-2 and vivo experiments, wherein the three promoter homologies preceding rnpB were cloned into the galactokinase (GalK) expression vector pKO100. The promoter homology nearest to the M1 RNA gene directed the synthesis of GalK above background. The upstream promoter homologies did not direct the synthesis of GalK at a level greater than 1% of transcription from P-1. Deletion of the upstream homologies did not affect transcription from P-1. It was concluded that P-1 is responsible for essentially all M1 RNA transcription in vivo. Single-round transcription experiments in vitro detected strong NusA-independent transcriptional pausing at nucleotides +118 and +121 of the rnpB transcript, with a half-life of 27 s when concentrations of NTPs were near the average Km for elongation. Pausing at these points was eliminated by substitution of ITP for GTP in the transcription mixture. This suggests that pausing is dependent on transcript secondary structure. The position of pausing corresponds to that of a dual stem and loop structure of M1 RNA which has recently been proposed on the basis of phylogenetic sequence analysis.
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PMID:Sites of initiation and pausing in the Escherichia coli rnpB (M1 RNA) transcript. 246 43

The fumarate reductase enzyme complex allows Escherichia coli to grow anaerobically with fumarate as a terminal electron acceptor for oxidative phosphorylation when the preferred compounds oxygen and nitrate are not available. We used the pKO promoter test vectors to identify a single promoter for the frdABCD genes which encode fumarate reductase. Expression of galactokinase from the frd promoter-galK operon fusion plasmid was repressed by oxygen and by nitrate and was induced by fumarate, indicating that frd gene expression is regulated at the transcriptional level by these terminal electron acceptors. S1 nuclease analysis, using a single-stranded DNA probe from the frd promoter region and mRNA isolated from a fumarate reductase-induced culture, revealed that the frd mRNA transcript initiates with an adenine residue 93 bases prior to the start of frdA translation. No promoters internal to the frd genes were revealed with the plasmid promoter screening system. S1 nuclease analysis revealed that the frd mRNA terminates in a uridine-rich region centered at 46 bases after the last codon of frdD. A stem and loop structure previously described as the growth rate-dependent attenuator for the linked ampC gene precedes the frd mRNA terminus. This result confirms the proposal that the stem and loop structure serves the dual role of a frd terminator anaerobically and an ampC attenuator aerobically. The four frd genes encoding the subunits of the fumarate reductase complex thus comprise an operon which is regulated at the transcriptional level in response to the cellular availability of the alternate electron acceptors oxygen, nitrate, and fumarate.
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PMID:Transcription of the Escherichia coli fumarate reductase genes (frdABCD) and their coordinate regulation by oxygen, nitrate, and fumarate. 299 70

The promoter region of Bacillus subtilis subtilisin E was found to be composed of two overlapping promoters with their transcription starting sites separated from each other by 15 base pairs (Wong, S.-L., Price, C. W., Goldfarb, D. S., and Doi, R. H. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1184-1188). At least one of the promoters is transcribed by a minor form of B. subtilis RNA polymerase with a sigma factor of 37,000 daltons. In vitro transcription analyses and in vivo studies with promoter probe plasmids pKO-1 and pCED-6 demonstrated that Escherichia coli RNA polymerase was able to initiate transcription from the subtilisin promoter cluster. S1 nuclease-mapping studies with both in vivo and in vitro transcribed RNA from E. coli and B. subtilis illustrate that E. coli can initiate transcription from both promoters with the same transcription start points as B. subtilis. The promoter strength of this promoter cluster in E. coli, as expressed in terms of galactokinase units, was 64 units and represents weak promoter activity in the E. coli system. These data indicate that either the single E. coli RNA polymerase is able to recognize the minor sigma 37 promoter or E. coli contains a hitherto unrecognized minor RNA polymerase holoenzyme which is capable of recognizing a B. subtilis sigma 37 promoter. On the other hand the B. subtilis RNA polymerase holoenzymes have been quite promoter-specific in our experiments to date.
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PMID:Utilization of a Bacillus subtilis sigma 37 promoter by Escherichia coli RNA polymerase in vivo. 608 47

We have isolated the in vivo messenger RNA encoding the lactose repressor protein of Escherichia coli by hybridization to lacI gene DNA, and have verified its identity by characterizing specific fragments derived from its 5' and 3' ends. The 3' end points of the RNA, as shown by oligonucleotide analysis and S1 nuclease-mapping data, are clustered in the lac control region, within 70 nucleotides beyond the end of the repressor protein coding sequence. DNA fragments from this region, when inserted between the E. coli gal operon promoter and galactokinase (galK) gene, effect an 80% reduction in downstream galK expression. These results are indicative that sequences at the gene end function in vivo to terminate lacI gene transcription.
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PMID:Lac repressor mRNA transcription terminates in vivo in the lac control region. 630 41

Regions of bacteriophages phi X174 and S13 that contain putative promoter sequences were amplified by the polymerase chain reaction (PCR) and cloned into the reporter vector pKO-1. Assays of galactokinase activity revealed in vivo promoter activity in those constructs containing the promoter sequences with transcription initiation (+1) sites at nucleotide positions 45, 982, 1823, and 5211. These were identical in location to sequences with in vitro promoter activity and to the three known promoters PA, PB, and PD. P5211 is the location of a new, fourth, promoter. A site with a +1 position at nucleotide 4876, previously shown to initiate RNA synthesis in an in vitro run-off transcription assay, had no in vivo promoter activity. To investigate whether flanking sequences had effects on promoter activity, restriction fragments of phi X174 and S13 that encompass the in vivo promoters were cloned into the reporter vector pKO-1. The PA and P5211 promoter constructs showed dramatic effects with increases in activity of up to 7 times that shown with the PCR-generated promoter constructs. The phi X174 PB promoter construct had a 50% decrease in activity compared with the PCR-generated PB clone. While the data showed that in most instances promoter activity is affected by the flanking sequences in which the promoter is embedded, no general pattern correlating flanking sequences and promoter activity could be discerned. Additional evidence that the promoter sequence regions were active in vivo promoters was obtained by S1 nuclease mapping experiments. Initiation of RNA synthesis was shown at positions 45, 982, and 5211.
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PMID:Identification of the in vivo promoters of bacteriophages S13 and phi X174 and measurement of their relative activities. 1009 83