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)

The promoter region preceding the hutUH operon in Klebsiella aerogenes contains two oppositely oriented, overlapping promoters. In the absence of catabolite gene activator protein-cyclic AMP (CAP-cAMP), transcription proceeds primarily from the backward-oriented promoter (Pc), whose function has not yet been determined, and only very weakly from the forward hutUH promoter, hutUp. In the presence of CAP-cAMP, Pc is repressed and transcription from hutUp is favored. Two protein components required for this in vitro transcription system, RNA polymerase (RNAP) and CAP, were purified from K. aerogenes and were shown to be functionally interchangeable with the corresponding proteins from Escherichia coli, suggesting that E. coli RNAP could be used to study some aspects of hut transcription. We showed that a gradual activation of hutUp (by increasing concentrations of CAP, cAMP, or glycerol) resulted in a parallel repression of Pc, arguing in favor of a direct competition between the two promoters. The presence of a DNA sequence resembling the consensus for CAP-binding sites and centered at nucleotide -82 (relative to hutUp) initially suggested that a primary role of CAP was to repress Pc, thereby indirectly activating hutUp. However, the relatively slow formation of open complexes at Pc, even in the absence of CAP-cAMP, showed that Pc is a weak promoter and likely to be a poor competitor for RNAP. The observed dominance of Pc over hutUp suggested that the latter is an even weaker promoter. Thus, repression of Pc would not be sufficient to cause the observed increase in hutUp activity, and the CAP-cAMP complex must play a direct role in the activation of hutUp.
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PMID:In vitro transcription of the histidine utilization (hutUH) operon from Klebsiella aerogenes. 184 33

Previous studies of the structure and expression of the ribosome-releasing factor (RRF) cistron (frr) have suggested that an efficient promoter region is located in the RRF cistron. We report here on the nucleotide sequence and in vivo function of the RRF promoter. The transcriptional start site was determined by primer extension to be 58 bp upstream of the translational initiation codon of frr. The location of the RRF promoter region was confirmed by means of (i) deletion analysis of the 5' proximal sequences of frr fused to the chloramphenicol acetyltransferase reporter gene, (ii) analysis of RRF produced in vivo from the deletion derivatives of frr cloned into pUC19, and (iii) gel retardation analysis with Escherichia coli RNA polymerase. The -35 and -10 regions were TTacCc and TATAcT, respectively. The strength of the RRF promoter was similar to that of the lac promoter, as determined by in vivo expression of chloramphenicol acetyltransferase activity. However, the RRF promoter was not affected by the intracellular cyclic AMP level despite the presence of a cyclic AMP receptor protein binding site downstream of the RRF promoter.
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PMID:Identification of the promoter region of the ribosome-releasing factor cistron (frr). 186 Aug 27

The major surface antigens of Trypanosoma brucei are the VSG (variant surface glycoprotein) at the bloodstream stage, and procyclin at the procyclic stage. Variation in the VSG allows the parasite to escape the antibody response of its mammalian host. This occurs through either DNA rearrangement in the telomeric VSG gene expression site, or alternate activation, without DNA rearrangement, of different telomeric expression sites. The VSG and procyclin genes each belong to large, polycistronic transcription units. Although the promoters of these units are both active at the two main stages of the parasite life cycle, stage-specific controls operating at the level of RNA elongation and processing lead to strictly differential expression of the end products of the two units. Despite their mutually exclusive control of expression, the VSG and procyclin transcription units share common characteristics. Both contain a similar gene, and both are transcribed by the same type of RNA polymerase, unusually resistant to alpha-amanitin. Among the eight genes present in the VSG transcription unit, two may be involved in the synthesis of cyclic AMP. The function of the other genes is unknown.
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PMID:Genetics of antigenic variation in African trypanosomes. 196 83

The lactose promoter-operator region of Escherichia coli contains two binding sites for cyclic AMP receptor protein (CAP), two for the lactose repressor, and two for RNA polymerase. The high density of binding sites makes cooperative interactions between these proteins likely. In this study, we used the gel electrophoresis mobility shift assay and binding partition analysis techniques to determine whether the secondary CAP site influences the binding of CAP to the principal CAP site in the lactose promoter when both are present on a linear DNA molecule. Such an effect could occur through the formation of a bridged DNA-CAP-DNA structure, through the interaction of CAP molecules bound to each of the sites, or through allosteric effects caused by CAP-mediated DNA bending. We found, however, that the interaction of CAP with these sites was not cooperative, indicating that CAP sites 1 and 2 bind CAP in an independent manner.
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PMID:The binding of cyclic AMP receptor protein to two lactose promoter sites is not cooperative in vitro. 198 34

We report the results of photo-cross-linking of RNA polymerase and the cyclic AMP receptor protein (CRP) to the lac UV5 promoter region carried on either a linear fragment or a supercoiled plasmid. We have devised a protocol that allows the localisation of bases in contact with the protein. RNA polymerase makes contacts within the -10 and -35 regions of the promoter, essentially on the non-template strand. The CRP contact points found in a binary complex are affected by the formation of the ternary complex containing RNA polymerase. Supercoiling has no effect on the position of contacts in any of the complexes. These conclusions were derived from experiments performed using a generally applicable, non-interfering technique that reveals direct contacts between proteins and nucleic acids in nucleoprotein complexes.
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PMID:Protein-DNA cross-linking at the lac promoter. 201 66

The amiloride-sensitive, growth factor-activatable Na/H exchanger (NHE-1) is a ubiquitous mammalian protein that is involved in the regulation of intracellular pH and cell volume. We have determined the intron/exon boundaries and the transcription initiation sites and have characterized a portion of the 5'-flanking regulatory region of the human NHE-1 gene. The Na/H exchanger gene spans approximately 70 kilobases. The coding region is divided into 12 exons and 11 introns, one of which is 41.5 kilobases in length. The first exon contains the entire 5'-noncoding region, which is 786 bases long, and 352 bases of the coding sequence. Primer extension identified two discrete start sites for RNA polymerase. 1377 bases of the 5'-regulatory region were sequenced. The promoter/enhancer region is characterized by a TATA box, four GC boxes, two CAAT boxes, five CACCC boxes, three Ap-1 sites, a cyclic AMP response element, and four partial glucocorticoid response elements. Promoter activities of a 313- and a 1441-base pair fragment containing the TATA box were demonstrated by their ability to direct chloramphenicol acetyltransferase expression when transiently expressed in fibroblasts.
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PMID:Structure of the 5'-flanking regulatory region and gene for the human growth factor-activatable Na/H exchanger NHE-1. 204 Jun 1

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 cyclic AMP receptor protein-cAMP complex (CRP-cAMP) binds at a variety of distances upstream of several E. coli promoters and activates transcription. We have constructed a model system in which a consensus CRP binding site is placed at different distances upstream of the melR promoter. CRP-cAMP activates transcription from melR when bound at a number of positions, all of which lie on the same face of the DNA helix. The two distances at which transcription is strongly activated correspond exactly to those at which CRP-cAMP binds upstream of the well-studied galP1 and lac promoters. Footprinting of the synthetic promoters reveals that RNA polymerase makes identical contacts with their -10 regions even though CRP-cAMP binds at a different distance in each case. Kinetic analysis in vitro indicates that CRP-cAMP activates transcription from these promoters in similar but distinct ways. A model is proposed to explain this two-position activation.
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PMID:Stringent spacing requirements for transcription activation by CRP. 216 78

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

RNA polymerase II from human placenta was affinity labelled in crude preparation using two-step technique, which includes treatment of the enzyme with an aldehyde-containing reactive analogue of ATP, ADP or AMP in the presence of poly[d(A-T)] followed (after borohydride reduction) by the elongation of the attached label with [alpha-32P]UTP. A polypeptide of the molecular mass ca. 140 kDa proved to be the labelling target. No labelling was observed in the absence of poly[d(A-T)] or the reagent or in the presence of alpha-amanitin. All the results suggest the attachment of the affinity reagents to the second-largest subunit of the human RNA polymerase II, which therefore takes part in the initiation substrate's binding.
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PMID:[Highly selective affinity labeling of DNA-dependent RNA-polymerase II from human placenta]. 228 30


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