Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

DNA fragments from Mycobacterium paratuberculosis were cloned in the promoter probe plasmid pKO1. Of 957 recombinant DNA clones, 24 induced synthesis of galactokinase (the reporter gene) when these plasmids were transformed into an Escherichia coli strain deficient for the enzyme. A DNA insert from one putative promoter-containing plasmid, designated pAG5, was sequenced and shown to contain, a characteristic RNA polymerase binding site, a probable ribosomal binding site and a putative open reading frame.
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PMID:Molecular cloning and characterization of Mycobacterium paratuberculosis promoters in Escherichia coli. 145 29

We used two different approaches to study the requirement for Escherichia coli Nus factors for the activity of bacteriophage lambda late antiterminator Q. Using an in vitro coupled transcription-translation assay, based on Q-dependent synthesis of galactokinase from a pR'-tR'-galK template, we showed that mutations in the host nusB and nusE genes do not affect Q activity. A mutation in nusA (nusA1) only partially affects Q action at all temperatures tested. Defective Q function in the nusA1 mutant extract could be restored by the addition of pure NusA but not by excess Q. In a pure transcription system, measurement of the run-off transcript produced by Q-mediated suppression of tR' revealed that NusA is greatly stimulatory to Q activity, whereas NusB and S10, in the presence or absence of NusA, have no effect. Unidentified E. coli factor(s) present in an S30 extract efficiently suppress the natural pausing by RNA polymerase at +15, +16 of pR' without affecting Q activity. These results show that NusA is the only host protein that directly participates in Q function.
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PMID:An analysis of the role of host factors in transcription antitermination in vitro by the Q protein of coliphage lambda. 214 85

Promoters which function in Gram-positive organisms show, with few exceptions, remarkable conservation of sequences identical with those in Escherichia coli. An E. coli system was tested to select putative promoters of two anaerobes, the Gram-positive Clostridium absonum and the Gram-negative Bacteroides thetaiotaomicron. Random restriction fragments of chromosomal DNA from these organisms were fused to the galactokinase (galK) gene of E. coli within a plasmid vector. Approximately 10% of these fragments functioned as promoters in E. coli, and a broad range of activities was evident. A single 88 base pair (bp) C. absonum DNA fragment yielded, in the E. coli plasmid vector, approximately the same high activity as that provided by the E. coli galK promoter. Sequence analysis of this fragment showed typical -35 and -10 sequences, with about five -10-like sequences closely flanking each other, some overlapping, and this appears to result in multiple start sites for transcription. The transcriptions of E. coli plasmid fragments in vitro with both E. coli RNA polymerase and C. absonum RNA polymerase showed pairs of transcripts corresponding to two start sites. By colony hybridization with the 88 bp fragment, radioactively labelled, as a probe, a 4.2 kilobase segment of C. absonum chromosomal DNA containing the 88 bp fragment was isolated. About 375 bp of this fragment was sequenced. A putative Shine-Dalgarno sequence and ATG start site were detected, followed by an opening reading frame. Using a sequence about 100 bp downstream from the 88 bp sequence, a 17-base oligonucleotide was synthesized to serve as a primer. With C. absonum RNA as a template, a reverse transcriptase primer extension assay located a pair of transcription start sites just downstream from the 88 bp sequence, proving that the 88 bp sequence functions as a promoter in C. absonum.
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PMID:Isolation of promoters from two anaerobic bacteria. 245 83

The rifampicin (Rif)-resistant RNA polymerase of phage T7 has proved invaluable for the exclusive over-expression, in Escherichia coli, of genes cloned downstream from the T7 phi 10 promoter [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 82 (1985) 1074-1078]. Here, we demonstrate that the system can be extended to Gram-negative bacteria other than E. coli, by the use of compatible wide host range plasmids. As an example, the Rif-resistant in vivo synthesis and specific radiolabelling of E. coli galactokinase in Pseudomonas ATCC19151, is demonstrated. The incidental observation that 30 min after treatment with Rif, two polypeptides continue to be synthesized in plasmid-free Pseudomonas ATCC19151, indicates that these proteins are produced by very stable mRNA species.
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PMID:Bacteriophage T7 RNA polymerase-controlled specific gene expression in Pseudomonas. 268 92

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

We have characterized the functional attributes of a 211-base pair region containing the Escherichia coli tryptophan operon rho-dependent terminator trp t', utilizing a series of constructs that alter the orientation, location, or extent of the trp t' sequences with respect to the trp promoter. In each instance, the extent of the rho-dependent response was monitored in vivo by read-through expression into a distal galactokinase gene and was compared with the results of transcription assays in vitro. As expected, transcription termination in vivo is dependent on the proper orientation of the terminator, and a tandem repeat of the terminator increases termination proportionally. Placing the terminator fragment only 14 nucleotides from the promoter does not affect termination significantly, supporting the belief that sequences outside of the 211-base pair fragment itself are dispensable. One construct, which lacks 116 base pairs, including the region encoding the normal RNA end points, still reduces galK activity in vivo and terminates transcription in vitro. Our results indicate that the rho response depends primarily on sequences in this 95-base pair segment, causing RNA polymerase to terminate transcription in a region 15-45 nucleotides further downstream.
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PMID:Signals sufficient for rho-dependent transcription termination at trp t' span a region centered 60 base pairs upstream of the earliest 3' end point. 327 76

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

Bacterial promoters are the sites at the 5' end of each gene that bind RNA polymerase and direct the initiation of transcription. The functional elements of Escherichia coli promoters are two highly conserved sequences, each about six nucleotides long, usually centred at sites -10 and -35, +1 being the initiating nucleotide. We have been interested in the structure of promoters of genes that are subject to stringent control, that is whose expression is reduced in conditions of amino acid shortage, such as rRNA and tRNA genes. We have therefore mapped the sequences involved in promoting in vivo transcription of a bacterial tRNATyr (tyrT) gene by fusing the tyrT promoter region to a galactokinase (galK) gene, and using in vivo expression of galactokinase activity to measure promoter strength. We show here that efficient expression from the tyrT promoter requires specific sequences upstream of the canonical promoter elements, and we suggest that these sequences constitute an extended promoter structure.
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PMID:Requirement for an upstream element for optimal transcription of a bacterial tRNA gene. 635 Aug 94

Prokaryotic gene control signals can be isolated, compared, and characterized by precise fusion in vitro to the Escherichia coli galactokinase gene (galK), which provides both a simple assay and genetic selection. This recombinant galK fusion vector system was applied to the study of promoters and terminators recognized by the Escherichia coli RNA polymerase. Three promoters created by mutation from DNA sequences having no promoter function were characterized. Mutations that inactivate promoter function were selected, structurally defined, and functionally analyzed. Similarly, transcription termination was examined, and mutations affecting terminator function were isolated and characterized.
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PMID:Studying promoters and terminators by gene fusion. 635 55

The terminator tI is located approx. 280 nucleotides beyond the int gene of bacteriophage lambda. Besides its role as a transcription terminator, tI may confer stability to the int message by protecting it from 3' exonucleolytic degradation. In order to study the role of the tI sequence in transcription termination and RNA stability, three different point mutations tI1, tI2, and tI3 were isolated and characterized. All the tI mutations map in the G + C-rich region of dyad symmetry in the terminator and decrease the transcriptional termination of tI in vivo from 99% for the wild type terminator to 81-93% as determined by galactokinase activity and in vitro from 80% for the wild type terminator to 8-12% using the E. coli RNA polymerase. Additionally, the tI mutations cause upstream transcript instability in vivo. This instability defect caused by tI mutations is compensated by the host mutant deficient in polynucleotide phosphorylase resulting in increased steady state levels of these mutant transcripts. The results show that the intact hairpin of tI is essential for efficient transcription termination and for maintaining mRNA stability by blocking the 3' to 5' exonucleolytic activity of polynucleotide phosphorylase.
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PMID:Point mutations in a transcription terminator, lambda tI, that affect both transcription termination and RNA stability. 897 20


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