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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 bacteriophage T4 Alt gene product is a component of the phage head and enters the host cell in the process of infection together with the phage DNA. It immediately ADP-ribosylates host RNA polymerase, presumably at only one of the two alpha-subunits. Transcription from T4 "early" promoters, therefore, might be catalyzed, at least in part, by an altered RNA polymerase. The T4 alt gene was cloned into the expression vector pBluescript. E. coli cells, transformed with this recombinant vector, overexpressed the 76 kDa Alt gene product, which was purified to homogeneity. The purified enzyme not only ADP-ribosylates the alpha-subunit of RNA polymerase, but also subunits beta and beta', as well as the sigma 70-factor. The recombinant enzyme behaved like the native enzyme isolated from mature phage particles. The effect of the ribosylation reaction on the transcription activity of host RNA polymerase was investigated in vivo. It results in a modulation of T4 "early" promoter strengths, presumably, in a number of cases, leading to an overexpression of T4 "early" genes. The degree of overexpression, in some cases, should reach 50%, and seems to be well dosed for each promoter, controlling an individual transcription unit.
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PMID:Overexpression, purification, and characterization of the ADP-ribosyltransferase (gpAlt) of bacteriophage T4: ADP-ribosylation of E. coli RNA polymerase modulates T4 "early" transcription. 778 17

Many activator proteins generate their positive control of transcription through interactions with the COOH-terminal domain of the Escherichia coli RNA polymerase alpha subunit. We have examined the participation of this alpha-domain in transcriptional enhancement and suppression at bacteriophage T4 late promoters. Enhancement is generated by the T4 gene 45 protein, which is the DNA-tracking processivity factor of viral DNA replication; suppression of unenhanced transcription is generated by the RNA polymerase-binding co-activator T4 gene 33 protein. Enhanced and unenhanced transcription by RNA polymerase reconstituted with intact and truncated alpha subunits and by RNA polymerase containing ADP-ribosylated alpha has been compared; the internal structures of transcription complexes formed with these RNA polymerases have also been analyzed by footprinting and photocross-linking. Comparison of these structural and functional analyses suggests that enhancement of T4 late transcription by gp45 is not compatible with any significant role of the COOH-terminal domain of the RNA polymerase core alpha subunit in transcriptional initiation. Suppression of unenhanced T4 late transcription by the gene 33 protein also does not require the COOH-terminal domain of alpha.
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PMID:The COOH-terminal domain of the RNA polymerase alpha subunit in transcriptional enhancement and deactivation at the bacteriophage T4 late promoter. 779 94

In this study, we describe the molecular and antigenic characteristics of a cloned enterotoxin from Salmonella typhimurium strain Q1. The full length Salmonella enterotoxin gene (stn), localized on a 2.8 kb ClaI/PstI DNA fragment, was cloned from a genomic library of Salmonella. Based on nucleotide sequence analysis, the stn gene contained 749 bp that would encode a protein having a molecular size of 29,073. The most unusual feature of the stn gene was the presence of a rare initiation codon (TTG) in lieu of the typical ATG codon, which required site-directed mutagenesis to confirm the precise initiation site. The expression of the stn gene in a bacteriophage T7 RNA polymerase/promoter system was enhanced by introducing a typical ATG start codon and an optimal Shine-Dalgarno sequence upstream of the stn gene by site-directed mutagenesis. The stn gene was located opposite the hydHG operon that regulates labile hydrogenase activity in Salmonella species and Escherichia coli. The overall amino acid sequence of the enterotoxin was quite dissimilar to any other published sequence, including cholera toxin or other adenylate cyclase-activating proteins. However, an intriguing similarity in a small region of the amino acid sequence of Stn was observed with portions of the amino acid sequences from several other protein toxins known to ADP-ribosylate host cell proteins. This region of homology may indicate a conserved motif, within the active site, that is involved in the stimulation of adenylate cyclase activity.
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PMID:Molecular characterization of an enterotoxin from Salmonella typhimurium. 804 4

The Alt gene product is a component of the T4 phage head. Upon infection of the host cell, approximately 40 copies of the Alt protein enter the cell together with the viral DNA molecule. The Alt protein then ADP-ribosylates one of the two alpha-subunits of host RNA polymerase. A restriction fragment harboring the ADP-ribosyltransferase gene of bacteriophage T4 was cloned into the plasmid vector pBluescript, the nucleotide sequence was determined, and the reading frame was identified. Two M13 clone libraries, established with DNA isolated from bacteriophages T2 and T6, then were screened for the corresponding genes. The nucleotide sequences of the three alt genes and the deduced amino acid sequences were compared. Secondary structure predictions and NAD-binding studies resulted in the location of the substrate-binding site in the NH2-terminal regions of the enzymes.
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PMID:The ADP-ribosyltransferases (gpAlt) of bacteriophages T2, T4, and T6: sequencing of the genes and comparison of their products. 805 53

In vitro specific transcription by the Rickettsia prowazekii RNA polymerase was investigated. The purified rickettsial RNA polymerase, in striking contrast to that of Escherichia coli, could specifically transcribe two R. prowazekii genes (ATP/ADP translocase and citrate synthase genes) and one E. coli gene (RNA-I) on negative supercoiled plasmids but not the same genes on linear plasmids. Following the specific binding of the rickettsial RNA polymerase to the translocase gene promoter on a linear plasmid, there was no detectable open complex formation. Both the E. coli and the R. prowazekii RNA polymerases worked well when poly(dA-dT).poly(dA-dT) or poly(dI-dC).poly-(dI-dC) was used as template for generalized transcription. However, the rickettsial RNA polymerase, in contrast to the E. coli enzyme, had little activity on poly(dG-dC).poly(dG-dC), a template with a larger number of hydrogen bonds. These data indicate that the rickettsial RNA polymerase is weak, at least relative to E. coli, in the function required for the opening of DNA duplex. It appears that this operation in R. prowazekii is aided by the negative supercoiling and the high 72% AT composition of the rickettsial genome.
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PMID:Characterization of the DNA-melting function of the Rickettsia prowazekii RNA polymerase. 844 Jun 83

S100 extract prepared from rapidly growing mouse FM3A cells (approx. 5 x 10(5) cells/ml) transcribed ribosomal RNA gene (rDNA) much more actively in vitro than that from stationary phase cells (1-2 x 10(6) cells/ml). When the inactive S100 extract was preincubated with NAD+, rDNA transcriptional activity was restored almost to the level of the active extract. The extract activated with NAD+ exhibited a gel-shift band in the gel mobility shift assay and enhancement of protection of the sequence between -44 and -8 nt from the initiation site from exonuclease III digestion. Such an extract labeled with [32P]NAD+ was analyzed by immunoprecipitation with anti-RNA polymerase I (pol I) antibody; a protein with M(r) 130 kDa was detected. In contrast, the polypeptide was hardly labeled in the active extract. 3-Aminobenzamide, a specific inhibitor of poly ADP-ribosylation, did not inhibit the activation by NAD+. These results suggest that the activation by NAD+ is due to enhancement of the formation of initiation complex by mono ADP-ribosylation of the second-largest subunit (130 kDa) of pol I.
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PMID:Transcription of mouse ribosomal RNA gene with inactive extracts is activated by NAD+ in vitro. 845 71

The chaperone system formed by DnaK, DnaJ and GrpE mediates stress-dependent negative modulation of the Escherichia coli heat shock response, probably through association with the heat shock promoter-specific sigma32 subunit of RNA polymerase. Interactions of the DnaK system with sigma32 were analysed. DnaJ and DnaK bind free, but not RNA polymerase-bound, sigma32 with dissociation constants of 20 nM and 5 muM respectively. Association and dissociation rates of DnaJ-sigma32 complexes are 5900- and 20-fold higher respectively than those of DnaK-sigma32 complexes in the absence of ATP. ATP destabilizes DnaK-sigma32 interactions. DnaJ, through rapid association with sigma32 and stimulation of hydrolysis of DnaK-bound ATP, mediates efficient binding of DnaK to sigma32 in the presence of ATP, resulting in DnaK-DnaJ-sigma32 complexes containing ADP. GrpE binding to these complexes stimulates nucleotide release and subsequent complex dissociation by ATP. We propose that the principles of this cycle also operate in other chaperone activities of the DnaK system. DnaK and DnaJ cooperatively inhibit sigma32 activity in heat shock gene transcription and GrpE partially reverses this inhibition. These data indicate that reversible inhibition of sigma32 activity through transient association of DnaK and DnaJ is a central regulatory element of the heat shock response.
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PMID:A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the Escherichia coli heat shock transcription factor sigma32. 859 44

The events that take place at the prokaryotic enhancer of the Pu promoter of Pseudomonas putida prior to the engagement of the sigma 54-RNA polymerase (sigma 54-RNAP) have been studied in vitro. ATP hydrolysis by XylR, the cognate regulator of the system, is preceded by the multimerization of XylR at the enhancer, which is itself triggered by the sole allosteric effect of ATP binding to the protein. Since ADP is unable to support multimerization, ATP hydrolysis might be followed by a return to the nonmultimerized state. This notion is supported further by the properties of mutant proteins that seem to be frozen, in either the nonmultimerized or the multimerized state, respectively. These results support a cyclic mechanism of ATP-dependent association/dissociation of XylR at the promoter UAS that precedes any involvement of the polymerase in transcription initiation.
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PMID:ATP binding to the sigma 54-dependent activator XylR triggers a protein multimerization cycle catalyzed by UAS DNA. 870 37

Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two cell types that are formed by an asymmetric cell division. Major changes in the pattern of transcription during sporulation are brought about by the synthesis of new sigma factors (sigma), which are subunits of RNA polymerase that determine promoter specificity. Transcription in the smaller prespore cell type is initiated by a sigma factor called sigma F, the activity of which is subject to tight spatial and temporal control. It is negatively regulated by an anti-sigma factor, SpoIIAB, which is in turn controlled by an anti-anti-sigma factor, SpoIIAA. SpoIIAA and SpoIIAB participate in two contrasting reactions in vitro. In the presence of ATP, the proteins interact transiently and SpoIIAA is inactivated by phosphorylation on a specific serine residue; SpoIIAA then remains free to inhibit sigma F. In the presence of ADP, SpoIIAA binds tightly to SpoIIAB, and sigma F is set free. Release of sigma F activity in vivo might thus be effected by a prespore-specific reduction in the ATP/ADP ratio. Genetic experiments have implicated a fourth protein, called SpoIIE, in this system. It now appears that SpoIIE has two important and independent functions in the establishment of the prespore-specific transcription by sigma F. First it regulates sigma F activity, probably acting as a phosphatase to regenerate the active, non-phosphorylated form of SpoIIAA. Second it controls the formation of the septum that generates the prespore compartment. Combination of these two functions in a single polypeptide may provide a means of coupling gene expression with morphogenesis.
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PMID:Control of the cell-specificity of sigma F activity in Bacillus subtilis. 873 76

We have investigated the over-production of seven membrane proteins in an Escherichia coli-bacteriophage T7 RNA polymerase expression system. In all seven cases, when expression of the target membrane protein was induced, most of the BL21(DE3) host cells died. Similar effects were also observed with expression vectors for ten globular proteins. Therefore, protein over-production in this expression system is either limited or prevented by bacterial cell death. From the few survivors of BL21(DE3) expressing the oxoglutarate-malate carrier protein from mitochondrial membranes, a mutant host C41(DE3) was selected that grew to high saturation cell density, and produced the protein as inclusion bodies at an elevated level without toxic effect. Some proteins that were expressed poorly in BL21(DE3), and others where the toxicity of the expression plasmids prevented transformation into this host, were also over-produced successfully in C41(DE3). The examples include globular proteins as well as membrane proteins, and therefore, strain C41(DE3) is generally superior to BL21(DE3) as a host for protein over-expression. However, the toxicity of over-expression of some of the membrane proteins persisted partially in strain C41(DE3). Therefore, a double mutant host C43(DE3) was selected from C41(DE3) cells containing the expression plasmid for subunit b of bacterial F-ATPase. In strain C43(DE3), both subunits b and c of the F-ATPase, an alanine-H(+) symporter, and the ADP/ATP and the phosphate carriers from mitochondria were all over-produced. The transcription of the gene for the OGCP and subunit b was lower in C41(DE3) and C43(DE3), respectively, than in BL21(DE3). In C43(DE3), the onset of transcription of the gene for subunit b was delayed after induction, and the over-produced protein was incorporated into the membrane. The procedure used for selection of C41(DE3) and C43(DE3) could be employed to tailor expression hosts in order to overcome other toxic effects associated with over-expression.
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PMID:Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. 875 92


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