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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two sets of deletions produced in vitro by S1 nuclease were used to study the structure and function of promoters lacPUV5 and lacP115. The upstream boundary of the RNA polymerase binding site in lacPUV5 was determined by comparing the levels of beta-galactoside expression in vivo programmed from a set of deletions progressively extending into the -35 region of the lacPUV5 promoter. Sequences upstream from base-pair -37 are not necessary for the full functioning of lacPUV5. A deletion that removes base-pair -37 retains only half of the promoter activity. Deletion of the first T X A base-pair of the consensus -35 region sequence, 5' T-T-T-A-C-A 3', leads to a sixfold reduction of promoter activity. Deletion of the whole -35 region of lacPUV5 leads to at least a 20-fold reduction of its promoter activity. Abortive initiation assays were performed on the fully functional lacPUV5 and two lacPUV5 deletions, which removed part of the -35 consensus sequence, to study their effect on the kinetics of RNA polymerase-promoter open complex formation. These two deletions show a 3.5 to 7-fold reduction in KB. Analysis in vivo of lacP115 showed that sequence information upstream from the -35 region is important for the full functioning of lacP115. A deletion removing sequences upstream from -41 caused a three- to fourfold reduction in promoter activity, apparently due to reduced transcription initiation. lacP115 has a much lower k2 value than lacPUV5; its KB value is about threefold higher than that of lacPUV5.
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PMID:Deletion analysis of RNA polymerase interaction sites in the Escherichia coli lactose operon regulatory region. 242 57

A 166-bp DNA fragment from the Streptomyces multicopy plasmid pIJ101 with in vivo promoter activity both in Streptomyces lividans and in Escherichia coli was isolated. The start point of the RNA transcribed from this fragment, determined by high resolution S1 nuclease mapping, was the same in S. lividans and in E. coli. This suggests that the E. coli RNA polymerase recognizes the same sequence determinants and chooses the point of initiation of RNA synthesis in the same way as the corresponding S. lividans enzyme. The putative promoter sequence shows good homology to the E. coli promoter consensus sequence in the '-35' region but poor homology in the '-10' region.
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PMID:Expression of a Streptomyces plasmid promoter in Escherichia coli. 242 88

To determine whether RNA polymerase pauses during transcription in vivo, we have examined transcripts of the trp operon leader regions of Serratia marcescens and Escherichia coli. Labeled RNAs synthesized in E. coli strains containing plasmids bearing wild-type or mutant trp leader regions of S. marcescens or E. coli were isolated by hybridization and analyzed by polyacrylamide gel electrophoresis. The labeled RNAs synthesized in vivo on the S. marcescens wild-type and deletion mutant plasmids were the same size as the in vitro pause and leader transcripts. Hybridization of the presumed in vivo pause RNAs, and control in vitro pause RNAs, to M13 phage DNA containing a trp leader region deletion followed by treatment with S1 nuclease produced identical protected RNA species, proving that the in vitro and in vivo RNAs were identical. The amount of labeled pause RNAs relative to leader RNAs decreased following a chase with unlabeled uridine. E. coli RNAs identical to the previously characterized in vitro pause and leader transcripts were demonstrated by electrophoretic band position and fingerprint analysis. The finding that transcription pausing occurs in vivo is consistent with the view that transcription pausing and ribosome release of paused transcription complexes are responsible for the coupling of translation with transcription that is crucial to attenuation.
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PMID:Detection of transcription-pausing in vivo in the trp operon leader region. 243 19

Transcripts from the rplKAJL-rpoBC ribosomal protein-RNA polymerase gene cluster have been quantified and their ends mapped using RNA-DNA hybridization, sucrose density-gradient sedimentation, Northern hybridization and S1 nuclease protection. The results indicate that the most abundant transcript is the 2600 nucleotide tetracistronic L11-L1-L10-L12 mRNA initiated at the upstream major PL11 promoter and terminated at the transcription attenuator in the L12-beta intergenic space. Somewhat less abundant 1300 nucleotide L11-L1 and L10-L12 bicistronic transcripts were observed. The 3' ends of the L11-L1 transcripts were heterogeneous; most of the ends were localized to three sites within a 110 base-pair region in the L1-L10 intergenic space. This intergenic space encodes also the major PL10 promoter and the mRNA binding site for the L10 translational control protein. Two 5' ends were observed for L10-L12 bicistronic mRNA, one at the PL10 promoter and the other 150 nucleotides further downstream in a region in which promoter activity has not been detected. It is suggested that this second downstream 5' end is generated by processing of the transcripts initiated at the major PL10 promoter. No transcript initiation in the L10-L12 intergenic space was detected. About 80% of the transcripts reading through the L12 gene were terminated in the vicinity of the transcription attenuator that is responsible for the reduction in the expression of the downstream RNA polymerase genes. Transcripts reading through the attenuator were partially processed by RNase III within a potential hairpin structure in the RNA transcript. Processing appears to produce 3' and 5' transcript end sites separated by about ten nucleotides. No other major 5' ends were observed in the L12-beta intergenic space. These results indicate that the two major promoters, PL11 and PL10, are both utilized to drive the interrelated transcriptional expression of this ribosomal protein-RNA polymerase gene cluster.
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PMID:Transcription products from the rplKAJL-rpoBC gene cluster. 244 6

In Mycoplasma hyopneumoniae, the single genes for 16S and 23S rRNAs are clustered in one operon from which the 5S rRNA gene is separated by more than 4 kbp. This operon, gene 2413 and gene X from M. hyopneumoniae and the 5' ends of both rRNA operons from M. capricolum were cloned and used for analysis of the following gene expression signals: promoters and terminators of the DNA-directed RNA polymerase, ribosomal binding sites of mRNA, and sites for processing of precursor rRNA. The analyses were performed by nuclease S1 protection experiments, the primer extension technique, and DNA sequencing. From these studies we conclude that putative promoter sequences in M. hyopneumoniae deviate significantly from those in Escherichia coli and Bacillus subtilis in having its -35 consensus sequence replaced by A-T rich sequences, whereas the characterized M. capricolum promoter resembles more closely a typical E. coli promoter. The other expression signals show sequences or structures similar to those found in other eubacteria, indicating related underlying principles.
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PMID:Gene expression signals in Mycoplasma hyopneumoniae and Mycoplasma capricolum. 244 58

The single RNA operon of Mycoplasma pneumoniae was cloned into a lambda vector and subcloned into pBR322. This was carried out in order to enable the analysis of the transcription control regions of this operon. S1 nuclease mapping was used to locate the 5' ends of RNA transcripts synthesized from the operon. The 5' ends of the 23S, 16S, and a precursor RNA synthesized in vivo in M. pneumoniae were mapped on the DNA template. Preliminary in vitro transcription experiments using RNA polymerase of Escherichia coli led to the conclusion that E. coli recognizes one promoter in the 5' region of the M. pneumoniae rRNA operon. The startsite of the in vitro transcript seems to lie downstream from the 5' end of the M. pneumoniae precursor transcript. Preliminary sequencing of the 5' regions of the M. pneumoniae rRNA operon and of the M. capricolum rRNA B operon enabled their comparison to each other and to known sequences from other organisms.
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PMID:Transcription control elements of the Mycoplasma pneumoniae rRNA operon. 244 63

Promoters recognized by RNA polymerase III were used to direct synthesis of RNAs of opposite polarity to the 5' end of the mRNA for the large T-antigen of SV40. A construct was made utilizing the adenovirus (human type II) VA1 gene promoter linked to 163 bp of SV40 DNA sequences cloned in antisense orientation relative to the promoter. The SV40 sequence corresponds to the 5' end of the large T-antigen gene. In addition to the antisense constructs control plasmids were utilized which either lacked both promoter and SV40 elements, lacked RNA polymerase III promoter elements but contained SV40 sequences or contained the VA1 gene promoter fused to SV40 sequences in the sense orientation. The function of the various gene fusions was demonstrated in an in vitro transcription system and in vivo by S1 nuclease 5' end mapping following transfection into COS1 cells. Cotransfection of COS1 cells with the 'antisense' gene and a plasmid containing an SV40 origin of replication resulted in a substantial transient inhibition of SV40-replicon function when compared to control determinations (50% to nearly complete inhibition of large T-antigen dependent DNA replication for 18-36 h). These results show that an antisense RNA generated by RNA polymerase III can effectively block expression of a chromosomally located gene.
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PMID:Inhibition of SV40 replicon function by engineered antisense RNA transcribed by RNA polymerase III. 244 65

In order to investigate the molecular mechanisms of the regulation of immunoglobulin (Ig) gene transcription, a cell-free system was developed in which a cloned mouse Ig mu heavy-chain gene was transcribed using nuclear extracts prepared from a mouse B cell hybridoma line. To monitor transcription, an RNA.RNA hybridization assay was developed in which a 32P-labeled, SP6-synthesized RNA probe complementary to Ig mu RNA was hybridized to unlabeled RNA transcribed in the nuclear extract. Accurate initiation of transcription, which resulted in the protection of the RNA probe from digestion with nuclease S1, was detected by the separation of the products on denaturing polyacrylamide gels, followed by autoradiography. Using this assay, an in-vitro-synthesized RNA was detected. The 5' end of the in-vitro-transcribed Ig mu RNA maps exactly to the same position as the 5' end of the corresponding in vivo mRNA and its formation was sensitive to the addition of low levels of alpha-amanitin (1 microgram/ml), indicating transcription by RNA polymerase II. It was shown by competition experiments with oligonucleotides containing the 'decamer recognition site' that this sequence interacts with (a) decamer-binding factor(s) and plays a positive role in transcription. The competition effects of the decamer-containing oligonucleotide appeared to be restricted to the decamer motif present in the promoter region. No effects of the enhancer region were detectable in vitro. Little or no transcriptional activity was found in transcription experiments using the Ig mu promoter and nuclear extracts prepared from HeLa cells. This suggests that tissue-specific factors involved in Ig mu heavy-chain gene transcription are present in the mouse B cell extracts.
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PMID:A homologous in vitro system to analyze transcription of a mouse immunoglobulin mu heavy-chain gene. 245 Jul 48

The sporulation-essential gene spoIIG of the Gram-positive bacterium Bacillus subtilis encodes the sporulation-specific sigma factor sigma 29(sigma E). We report here the initial characterization of a gene, referred to as ORF3, located immediately downstream of the spoIIG gene. The results indicate that ORF3 encodes a sigma homolog, whose expression is highly regulated during development. Analysis of the ORF3 nucleotide sequence reveals an open reading frame encoding a polypeptide of 260 amino acid residues (molecular mass of 30.1 kDa). Its predicted amino acid sequence shows significant similarity to that of other RNA polymerase sigma factor sequences. S1 nuclease mapping experiments indicate that ORF3 is initially cotranscribed with spoIIG from about 1 to 4 hr into the sporulation process and that later on ORF3 is transcribed independently from a new site located between spoIIG and ORF3. The role of ORF3 was investigated by constructing a deletion mutation in its structural gene. The mutant exhibits normal growth but is unable to produce heat-resistant spores. We propose that the ORF3 gene product is a sigma factor or a related peptide essential for sporulation at a late stage of development.
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PMID:Two developmental genes encoding sigma factor homologs are arranged in tandem in Bacillus subtilis. 245 11

The nucleotide sequence of 2.5 kb of the Streptomyces coelicolor A3(2) rRNA gene set rrnD, extending from upstream of the 16S rRNA gene to the putative 5' end of the 23S rRNA gene, has been determined (Baylis and Bibb, 1987; this paper). In addition to locating the 5' end of the 16S rRNA gene, nuclease S1 mapping identified seven RNA 5' end-points upstream of the 16S rRNA gene; four of these were coincident with transcriptional initiation points for S. coelicolor A3(2) RNA polymerase in vitro and were consequently regarded as in vivo transcription start points for promoters p1 to p4. One end-point identified by nuclease S1 mapping localized a putative processing site analogous to those found upstream of 16S rRNA genes in other eubacteria. Sequence motifs similar to those discovered in low G+C Gram-positive bacteria were found associated with two of the promoters and the processing site. A probable protein coding region was observed upstream of the promoter region.
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PMID:Transcriptional analysis of the 16S rRNA gene of the rrnD gene set of Streptomyces coelicolor A3(2). 246 Jul 16


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