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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)

Mu transcription occurs in three phases: early, middle, and late. Middle transcription occurs in the region of the C gene, which encodes the transactivator for late transcription. A middle promoter, Pm, was previously localized between 0.28 and 1.2 kilobase pairs upstream of C. We used S1 nuclease mapping with both unlabeled and radiolabeled capped RNAs from induced lysogens to characterize C transcription and identify its promoter. The C transcription initiation site was localized to a 4-base-pair region, approximately 740 base pairs upstream of C within the region containing Pm. Transcription of C was activated between 4 and 8 min after induction of cts and Cam lysogens and increased throughout the lytic cycle. Significant C transcription did not occur in replication-defective Aam lysogens. These kinetic and regulatory characteristics identify the C transcript as a middle RNA species and demonstrate that Pm is the C promoter. DNA sequence analysis of the Pm region showed a good -10, but poor -35, site homology to the Escherichia coli RNA polymerase consensus sequence. In addition, the sequence demonstrated that C is the distal gene in a middle operon containing several open reading frames. S1 mapping also showed an upstream transcript with a 3' end in the Pm region at a sequence strongly resembling a Rho-independent terminator. The regulatory characteristics of this RNA are consistent with this terminator, t9.2, being the early operon terminator.
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PMID:Characterization of the C operon transcript of bacteriophage Mu. 213 35

A catabolite-sensitive promoter was found to be involved in transcription of the heat shock regulatory gene rpoH encoding the sigma 32 protein. Expression of lacZ from the operon fusion, rpoHp-lacZ, was partially inhibited by glucose added to the broth medium. Dissection of the rpoH promoter region allowed us to localize the glucose-sensitive promoter to the 110-base-pair (bp) segment directly upstream of the rpoH coding region. Experiments on lacZ expression from the set of fusions in cya (adenylate cyclase) and crp (cyclic AMP [cAMP] receptor protein) mutants also supported the involvement of a catabolite-sensitive promoter. Analysis of rpoH mRNAs by S1 nuclease protection experiments led us to identify a novel promoter, designated P5, that is regulated by cAMP and the cAMP receptor protein. Studies of rpoH transcription in vitro demonstrated that RNA polymerase-sigma 70 can transcribe from the P5 promoter only in the presence of cAMP and its receptor protein. The 5' ends of P5 transcripts obtained in vivo and in vitro were found to be at 61 to 62 bp upstream of the initiation codon, and a putative binding sequence for the cAMP receptor protein was found at 38 to 39 bp further upstream. Transcription from the P5 promoter is increased by the addition of ethanol to the growth medium; however, the increase is greater in the presence of glucose than in its absence. These results add a new dimension to the transcriptional control of rpoH and to the regulation of the heat shock response in Escherichia coli.
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PMID:Transcriptional regulation of the heat shock regulatory gene rpoH in Escherichia coli: involvement of a novel catabolite-sensitive promoter. 213 50

The rpoD gene of Myxococcus xanthus was used as a probe to isolate three Streptomyces coelicolor genes, hrdB, hrdC, and hrdD, which appear to encode RNA polymerase sigma factors extremely similar to the sigma 70 polypeptide of Escherichia coli. Gene disruption experiments suggested that hrdB is essential in S. coelicolor A3(2) but showed that hrdC and hrdD mutants are viable and are apparently unaffected in differentiation, gross morphology, and antibiotic production. S1 nuclease mapping showed that hrdB and hrdD, but not hrdC, were transcribed in liquid culture. The most upstream of two hrdD promoters is internal to an open reading frame (ORF X) on the opposite strand. The predicted product of this gene is homologous to the phosphinothricin acetyltransferases of Streptomyces hygroscopicus and Streptomyces viridochromogenes. The possible significance of the overlapping and divergent transcription of hrdD and ORF X is discussed. A general method for in vivo gene replacement was developed that allowed a positive selection for the desired mutants even in the absence of a mutant phenotype; it was used to isolate a stable hrdC mutant.
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PMID:Cloning, disruption, and transcriptional analysis of three RNA polymerase sigma factor genes of Streptomyces coelicolor A3(2). 216 Sep 42

The transcriptional activity of the topA gene which codes for topoisomerase I was examined. An in vitro assay determined that the P1 promoter was dependent on the sigma 32 subunit of RNA polymerase. The transcriptional activity of the four topA promoters was examined by nuclease S1 mapping of the transcripts during a heat shock. This sigma 32-dependent promoter was shown to function as a heat shock promoter, although topoisomerase I is not a heat shock protein. A possible method of compensation of transcription activity by the other promoters to maintain the level of topoisomerase I during heat shock is proposed.
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PMID:Identification of a heat shock promoter in the topA gene of Escherichia coli. 217 62

Antibody was raised against purified vaccinia virus RNA polymerase and used to screen a recombinant vaccinia virus-lambda gt11 library. The DNA from several immunopositive clones was shown by Southern hybridization to originate from the vaccinia virus HindIII E fragment. The nucleotide sequence of the RNA polymerase subunit gene predicts a polypeptide 287 amino acids in length and 30,000 daltons in mass. An early transcript with a 5' terminus just upstream of the putative initiation codon was identified by S1 nuclease protection and primer extension analyses, demonstrating that this RNA polymerase subunit is expressed as an early viral gene product. The RNA polymerase subunit was synthesized by a bacterial expression vector to demonstrate that it corresponds to the previously described 37,000-dalton RNA polymerase subunit.
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PMID:Vaccinia virus gene encoding a 30-kilodalton subunit of the viral DNA-dependent RNA polymerase. 221 20

The identities of two cloned, arabinose-inducible promoters were tested by hybridizing promoter DNA fragments with restriction digests of chromosomal DNA containing Mudlac phage inserted in either araFGH or in araE transport operons. One promoter, thought to be araE, is within 10(3) base-pairs of a Mudlac insertion in the araE gene. The second promoter was not found within several thousand base-pairs of either of the known transport genes. This promoter is now named araPJ (araJ). The DNA sequence of the fragment containing the araFGH promoter was determined. The start site of transcription in vivo was located to within +/- 1 base-pair (bp) by S1 nuclease mapping. DNase 1 footprinting revealed that, in comparison with the araBAD and araE promoters, the locations of the AraC and cyclic AMP receptor protein (CRP) binding sites are reversed with CRP lying between AraC and RNA polymerase. The central location of the CRP binding site may explain why the araFGH promoter is more catabolite sensitive than the other ara promoters. AraC and CRP were both required for maximal transcription in vitro, although a low level of transcription was detected with CRP alone. S1 nuclease mapping of mRNA-DNA hybrids from the araJ promoter located the transcription start point to within #/- 3 bp, and demonstrates that the promoter is dependent upon AraC protein and CRP in vivo. DNase footprinting showed that the location of the AraC protein binding site on araJ is adjacent to the RNA polymerase site, as seen at the araBAD and araE promoters. Two CRP sites were observed; one is upstream from the AraC site and one is downstream from the transcription start site.
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PMID:Characterization of the Escherichia coli araFGH and araJ promoters. 223 17

The 5' and 3' ends of the lux mRNA of Vibrio harveyi, which extends over 8 kilobases, have been mapped, and two new genes, luxG and luxH, were identified at the 3' end of the lux operon. Both S1 nuclease and primer extension mapping demonstrated that the start site for the lux mRNA was 26 bases before the initiation codon of the first gene, luxC. The promoter region contained a typical -10 but not a recognizable -35 consensus sequence. By using S1 nuclease mapping the mRNA was found to be induced in a cell density- and arginine-dependent manner. The DNA downstream of the five known V. harveyi lux genes, luxCDABE, was sequenced and found to contain coding regions for two new genes, designated luxG and luxH, followed by a classical rho-independent termination signal for RNA polymerase. luxG codes for a protein of 233 amino acids with a molecular weight of 26,108, and luxH codes for a protein of 230 amino acids with a molecular weight of 25,326. The termination signal is active in vivo as demonstrated by 3' S1 nuclease mapping, confirming that the two genes are part of the V. harveyi lux operon. Comparison of the luxG amino acid sequence with coding regions immediately downstream from luxE in other luminescent bacteria has demonstrated that this gene may be a common component of the luminescent systems in different marine bacteria.
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PMID:Delineation of the transcriptional boundaries of the lux operon of Vibrio harveyi demonstrates the presence of two new lux genes. 230 59

Matrix attachment regions (MARs) are DNA elements that dissect the genome into topologically separated domains by binding to a chromosomal skeleton. This study explored the putative influence of the MAR located 5' of the chicken lysozyme gene on expression of heterologous genes in heterologous cell systems. Expression of a construct with the chloramphenicol acetyltransferase (CAT) indicator gene controlled by the herpes simplex virus thymidine kinase promoter (TC) and a construct in which the same transcriptional unit is flanked by chicken lysozyme 5' MARs (MTCM) was assayed after stable transfection into rat fibroblasts. Median CAT activity per copy number in MTCM transfectants was elevated approximately 10-fold relative to that in TC transfectants. Total variation in normalized CAT activity decreased from more than 100-fold among TC transfectants to nearly 6-fold among MTCM transfectants. The steady-state level of transcripts and the relative rate of transcription were increased in MTCM transfectants, as shown by S1 nuclease and run-on transcription assays, respectively. The chicken lysozyme 5' MAR thus can confer elevated, less position-dependent expression on a heterologous promoter in cells of a different species by increasing the density of transcribing RNA polymerase molecules. MAR-mediated transcriptional enhancement suggests that MARs are important for gene expression and not just for DNA packaging.
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PMID:The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes. 232 53

The DNA-directed RNA polymerase from the extremely thermophilic eubacterium Thermus thermophilus HB8 was purified employing a new and rapid method. The subunit pattern of the enzyme, analyzed by SDS gel electrophoresis, was interpreted as: 140 kDa and 170 kDa for beta and beta', 40 kDa for alpha and 92 kDa for sigma. The RNA polymerase is active at elevated temperatures (65 degrees C). Kinetic data provide evidence for the existence of two NTP binding sites with very strong cooperativity. The promoter site specificity of the isolated enzyme has been proven by in vitro transcription employing two T. thermophilus templates whose in vivo starts of transcription were characterized by nuclease S1 mapping.
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PMID:Isolation and physical properties of the DNA-directed RNA polymerase from Thermus thermophilus HB8. 238 94

Binding of Escherichia coli RNA polymerase and the abortive initiation of transcription at the A2 promoter of bacteriophage T7, separately cloned in pBR322, was found to be strongly dependent on the degree of supercoiling of the plasmid. Supercoiling does not seem to play any role in the initiation of transcription at the T7A1 promoter under identical conditions. Plasmid containing T7A2 promoter was found to be less amenable to S1 nuclease in comparison to that having T7A1. Sequence comparison reveals a high G/C content upstream to the -35 region of T7A2 which by extra duplex stability probably renders the initiation of transcription more dependent on the state of supercoiling of the template.
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PMID:Correlation between the DNA supercoiling and the initiation of transcription by Escherichia coli RNA polymerase in vitro: role of the sequences upstream of the promoter region. 240 1


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