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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)

Promoters were isolated at random from the genome of Saccharomyces cerevisiae by using a plasmid that contains a divergently arrayed pair of promoterless reporter genes. A comprehensive library was constructed by inserting random (DNase I-generated) fragments into the intergenic region upstream from the reporter genes. Simple in vivo assays for either reporter gene product (alcohol dehydrogenase or beta-galactosidase) allowed the rapid identification of promoters from among these random fragments. Poly(dA-dT) homopolymer tracts were present in three of five randomly cloned promoters. With two exceptions, each RNA start site detected was 40 to 100 base pairs downstream from a TATA element. All of the randomly cloned promoters were capable of activating reporter gene transcription bidirectionally. Interestingly, one of the promoter fragments originated in a region of the S. cerevisiae rDNA spacer; regulated divergent transcription (presumably by RNA polymerase II) initiated in the same region.
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PMID:Properties of promoters cloned randomly from the Saccharomyces cerevisiae genome. 284 31

We have constructed two new multi-purpose cloning vectors, pJKKmf(-) and pJKSp/Smf(-), that carry resistance to kanamycin (Km) and spectinomycin/streptomycin (Sp/Sm), respectively. These plasmids are based on pGEM3Zf(-) and therefore contain a pUC-vector-derived multiple cloning site for 13 restriction sites flanked by T7 and SP6 RNA polymerase promoters, the alpha-peptide coding region of beta-galactosidase for blue/white colony screening and the bacteriophage f1 origin of replication for production of single-stranded DNA in the presence of a helper phage. We have used these vectors to reclone sequences from a maize genomic library, to produce radiolabeled RNA probes and to make single-stranded DNA.
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PMID:Two new tools: multi-purpose cloning vectors that carry kanamycin or spectinomycin/streptomycin resistance markers. 285 90

We have constructed the PRM promoter of phage lambda and eight variants, which represents intermediates in the conversion of this promoter to one that has complete homology to the consensus sequences in the -10 and -35 regions. The in vivo activity of these promoters was determined from the beta-galactosidase or galactokinase activities in cells harboring plasmids, in which the cloned promoters were driving the expression of these genes. Additionally, the kinetics of the interaction of Escherichia coli RNA polymerase with the same series of promoters was measured as a function of RNA polymerase concentration. This allowed the overall rate of functional or open complex formation to be dissected into the equilibrium constant for binding of the polymerase to form a closed promoter complex and the rate of subsequent isomerization to yield the open complex. The following conclusions can be drawn from the data presented: (1) The consensus sequence is optimal for promoter function both in vivo and in vitro. (2) Alterations of the -10 and -35 regions have similar effects on the kinetics of RNA polymerase binding in vitro; with one exception, the same holds for promoter activity in vivo. (3) The in vitro rate of RNA polymerase binding to a promoter is solely determined by the number of positions at which its -10 and -35 regions match the consensus promoter sequence. The functional importance of a match does not appear to be determined by the sequence conservation at the particular position. (4) The extent to which a particular base change affects the kinetic parameters depends on the sequence of the promoter into which it is introduced.
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PMID:Promoter recognition by Escherichia coli RNA polymerase: effects of base substitutions in the -10 and -35 regions. 296 67

Antisera were raised in rabbits against fusion proteins consisting of beta-galactosidase and partial amino acid sequences of Semliki Forest virus (SFV)-specific non-structural proteins nsP1, nsP2, nsP3 and nsP4. The antisera were specific since each of them precipitated only one labelled protein of a size expected for nsP1, nsP2, nsP3 or nsP4 from lysates of [35S]methionine-labelled SFV-infected BHK-21 cells. The specific antisera also precipitated p220 (with sequences of nsP1, nsP2 and nsP3), p155 (nsP1 and nsP2) and p135 (nsP3 and nsP4) which have been previously shown to be cleavage products of the polyprotein precursor of the non-structural proteins. nsP1, nsP4 and most of nsP3, together with the virus-specific RNA polymerase activity, were in the mitochondrial pellet (P15) fraction of infected BHK-21 cells whereas nsP2 was evenly distributed between P15 and the supernatant fraction (S15). Only antisera directed against nsP3 sequences precipitated a labelled protein from cells incubated with [32P]orthophosphate during SFV infection. Treatment of the immunoprecipitate with calf alkaline intestinal phosphatase reduced the amount of labelled nsP3 considerably. Immunoprecipitated 32P-labelled nsP3, isolated by SDS-PAGE, was subjected to acid hydrolysis. Both phosphoserine and phosphothreonine but not phosphotyrosine could be identified in the hydrolysate. Approximately twice as much [32P]serine as [32P]threonine was detected in nsP3. P15 and S15 fractions were prepared from [35S]methionine- and 32P-labelled SFV-infected cells and the 35S/32P ratio of nsP3 was determined after immunoprecipitation and SDS-PAGE. The nsP3 in S15 was less heavily phosphorylated (about 50%) than P15-associated nsP3. Anti-nsP3 serum revealed large cytoplasmic vesicles in SFV-infected cells in indirect immunofluorescence microscopy.
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PMID:Semliki Forest virus-specific non-structural protein nsP3 is a phosphoprotein. 297 May 23

The Escherichia coli lac promoter has been shown to contain an RNA polymerase binding site (P2) that overlaps with, and is shifted 22 base-pairs upstream from the normal lac promoter (P1). In this paper, we provide RNA polymerase protection data obtained in vitro that show that, in the absence of CAP-cAMP, in vitro P2 is the preferred polymerase binding site on the P+ template. In the presence of CAP-cAMP, polymerase binding to P2 is reduced and more polymerase is bound at P1. Two lac P1 "-35 region" mutations, L157 and 4, which increase the homology between this region and the consensus "-10 region" sequence, are both shown to have an increased affinity for polymerase binding at P2. CAP-cAMP is also able to decrease the amount of polymerase bound to P2 and to increase the amount bound to P1 on these mutant promoter fragments. P2 does not initiate transcription efficiently in vivo. Nuclease S1 mapping experiments detect only a low level of transcription from one of the P2 "up" mutations, but no beta-galactosidase synthesis is directed by this mutant. Mutations such as L157 and 4, which alter the P2-10 region, also alter lac P sensitivity to CAP-cAMP in vivo, suggesting that the P2 sequence plays a role in CAP-cAMP regulation of lac P. Possible roles for P2 in vivo are discussed.
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PMID:Properties of lac P2 in vivo and in vitro. An overlapping RNA polymerase binding site within the lactose promoter. 299 53

ompC expression in Escherichia coli K-12 is known to be regulated by the ompB locus, comprising the ompR and envZ genes, and the OmpR protein is believed to act as a positive transcriptional factor. We examined the transcriptional capability of the ompC gene in vitro and found that RNA polymerase could transcribe ompC without a requirement for other transcriptional factors. Furthermore, transcripts from three tandem promoters in ompC were identified in vitro. We employed oligonucleotide-directed site-specific mutagenesis to dissect the promoter region of the gene and assayed the promoters separately for transcriptional ability using fusions to the lacZ gene. The levels of beta-galactosidase indicate that ompC expression in vivo is dependent on the function of at least one of the upstream promoters. The function of OmpR appears to be the enhancement of a basal level of ompC expression. From the results of our experiments, the site of action of OmpR was deduced to be in the vicinity of the upstream promoters of ompC.
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PMID:Regulation of the ompC gene of Escherichia coli. Involvement of three tandem promoters. 301 84

The purE operon of Escherichia coli has been cloned and localized to a 1.7-kb HpaI fragment. The operon has been further characterized by subcloning into the lac fusion vector, pMC1403, and by the construction of BAL 31-generated deletions. The purE regulation region has been identified by assay of beta-galactosidase produced by pur-lac fusion plasmids and by RNA polymerase binding to end-labelled restriction fragments. Two purE promoters have been identified; one strong that is regulated by purines, the other weaker which is not regulated. The latter may be internal to the purE1 structural gene.
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PMID:Molecular cloning and characterization of the purE operon of Escherichia coli. 302 90

Previous studies have attributed two activities to the NusA protein of Escherichia coli; namely, termination and antitermination of transcription. To examine these activities, we isolated a temperature-sensitive mutant of the nusA gene (nusAts11). The mutant cells produce a thermolabile NusA protein and grow at 32 degrees C, but not at 42 degrees C. At 42 degrees C, nusAts11 is recessive to nusA+ and nusA1, indicating the absence of its active gene product at that temperature. In the mutant, the efficiency of termination at the lambda tR1 terminator decreases, resulting in an increased expression of distal gene(s). On the other hand, the synthesis of the beta-galactosidase and beta beta' subunits of RNA polymerase is reduced in the mutant. This mimics effects seen in vitro when NusA protein is removed from a coupled transcription-translation system. These results suggest that the NusA protein plays both negative and positive modulator roles in vivo. The mutation nusAts11, unlike nusA1, does not block lambda phage growth at non-permissive temperatures, suggesting that NusA protein is not required for N antitermination in the mutant. Besides, the nusAts11 allows lambda Nam7Nam53byp phage growth under sup0 conditions, indicating that the N antitermination function is dispensable (at least partly) in this mutant.
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PMID:Regulatory defects of a conditionally lethal nusAts mutant of Escherichia coli. Positive and negative modulator roles of NusA protein in vivo. 302 18

The first gene of the Bacillus subtilis RNA polymerase sigma 43 operon, P23, has a protein-coding capacity of 23,000 daltons. Sequence analysis revealed three potential translational initiation sites within the same reading frame, which could encode proteins of 23,000 (P23), 19,000 (P19), and 9,000 (P9) daltons, respectively. An internal promoter (P3), which is expressed only during the sporulation stage, is located between the second and the third translational start sites. By protein fusion to the Escherichia coli beta-galactosidase gene, we showed that all three translational initiation sites of the P23 gene are used in vivo in both E. coli and B. subtilis, and regulation for differential expression of the three proteins during the development of B. subtilis is coupled to the transcriptional promoter switching mechanism. The physiological function of these multiple gene products is unknown and is currently under investigation.
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PMID:Developmental expression of three proteins from the first gene of the RNA polymerase sigma 43 operon of Bacillus subtilis. 304 Jun 82

Expression plasmids containing the E. coli lacZ coding region preceded by a set of different ribosome-binding sites and put under transcriptional control of the leftward promoter of phage lambda (PL) were used to study the synthesis of lacZ mRNA. In a normal host the steady state level of full-length lacZ mRNA varied 100-fold with the different synthesis levels of beta-galactosidase, whereas in a host expressing the antitermination protein N of phage lambda, all vectors synthesized the same amount of full-length lacZ mRNA, while maintaining the differences in beta-galactosidase expression. We present evidence for a causal relationship between the rate of ribosome loading and the continuation of transcription across the lacZ gene. We suggest that extended spacing between the RNA polymerase and the elongating ribosome causes transcriptional polarity by increasing the extent of premature termination. The conditional character of the termination event can best be explained by invoking termination factor Rho.
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PMID:Inefficient translation initiation causes premature transcription termination in the lacZ gene. 308 Dec 64


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