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)

Xenopus transcription factor IIIA (TFIIIA) or TFIIIA mutants with internal deletions were expressed in E. coli utilizing a bacteriophage T7 RNA polymerase system. TFIIIA or deletion mutant TFIIIAs, isolated from E.coli cell extracts, were identified by SDS PAGE and immunoblotting with rabbit antiserum against native TFIIIA purified from Xenopus immature oocytes. Specific DNA binding of intact or internally deleted TFIIIA was compared by analyzing their abilities to protect the internal control gene (ICR) of the Xenopus 5S RNA gene from DNase I digestion. Intact protein, synthesized from a full-length TFIIIA cDNA, bound specifically to the entire ICR (+96 to +43) and promoted 5S RNA gene transcription in vitro. One TFIIIA deletion mutant, expressed from cDNA lacking the coding sequence for the putative fourth zinc finger (designated from the N-terminus, amino acids 103-132) protected the ICR from DNase I digestion from nucleotide positions +96 to +78. A second TFIIIA mutant resulting from fusion of putative zinc fingers 7 and 8 (deletion of amino acids 200-224) protected the 5S gene ICR from positions +96 to +63. The DNase I protection patterns of these mutant proteins are consistent with the formation of strong ICR contacts by those regions of the protein on the N-terminal side of the mutation but not by those regions on the C-terminal side of the mutation. The regions of the protein comprising the N-terminal 3 fingers and N-terminal six fingers appear to be in contact with approximately 18 and 33 bp of DNA respectively on the 3' side of the 5S gene ICR. These internal deletion mutants promoted 5S RNA synthesis in vitro and DNA renaturation.
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PMID:Internal deletion mutants of Xenopus transcription factor IIIA. 269 11

DNA-dependent RNA polymerase from Escherichia coli was purified further by elution through heparin-Sepharose CL-6B column after the enzyme was obtained, partially purified, using Burgess and Jendrisak's method [(1975)Biochemistry 14, 4634] The total yield of the pure protein was 10 mg from 50 g of E.coli cells. The method was found to be very reproducible and convenient. The enzyme preparation had 60% active molecules and the elongation rate of RNA synthesis by this enzyme was measured to be 11 bases/s over delta D111 T7 DNA.
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PMID:An improved method for the purification of DNA-dependent RNA polymerase from Escherichia coli. 328 88

The expression of the diphtheria tox228 gene encoding the nontoxic, serologically related CRM228 mutant diphtheria toxin has been analyzed in Corynebacterium diphtheriae and Escherichia coli. The diphtheria toxin promoter has been used to direct the expression of beta-galactosidase in E.coli, and the efficiency of promotion has been compared to that obtained with the lac promoter. Expression in C.diphtheriae is known to be dependent on the absence of iron, and we present for the first time direct evidence that this regulation occurs at the level of transcription. The 5' end of toxin mRNA maps at the same position in C.diphtheriae and E.coli, suggesting identical sequences to be recognized by C.diphtheriae and E.coli RNA polymerase. The diphtheria toxin promoter carries at position -34 a TTGATT sequence closely related to the E.coli -35 consensus sequence and in the -14 to -8 region a set of overlapping sequences with complete or partial homology to the E.coli -10 consensus sequence.
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PMID:Diphtheria toxin promoter function in Corynebacterium diphtheriae and Escherichia coli. 392 42

Kinetics of condensation of ribonucleotides to dinucleotides, leading to trinucleotide products formation, have been studied using wheat germ RNA polymerase II and poly(dAT). Assay conditions can be selected under which both ApUpA and UpApU are formed in catalytic amounts. The kinetic parameters associated with these reactions indicate that the rate of trinucleotide formation might be affected by DNA sequence, as reported for E.coli RNA polymerase. Kinetics of disappearance of ApUpA and UpApU were studied under experimental conditions allowing poly(rAU) synthesis. The results can be interpreted as if after formation of a phosphodiester bond, a slow isomerisation step of the ternary transcription complex could occur. During this step, transcription complexes could dissociate with a finite probability, releasing trinucleotides in an abortive pathway. The above results are discussed in the view that, under these experimental conditions, wheat germ RNA polymerase II catalyses poly(rAU) synthesis, as if it is a non-processive enzyme. Cordycepin triphosphate can be condensed to a dinucleotide primer, yielding ApUpA. However the ATP analogue cannot be incorporated into longer products than a trinucleotide. On the other hand 3'-dATP behaves as a very potent inhibitor of translocation, with an inhibition constant of 0.15 microM, a value which is two orders of magnitude smaller than the Km value corresponding to ATP utilization in poly(rAU) synthesis. Simple models are proposed which allow a comparison with E.coli RNA polymerase, for which the results are well documented.
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PMID:Poly(dAT) dependent trinucleotide synthesis catalysed by wheat germ RNA polymerase II. Effects of nucleotide substrates and cordycepin triphosphate. 404 41

Although the antibiotic rifampicin inhibits the transcription of poly[d(A-T)] by E.coli RNA polymerase, a series of short oligonucleotides is produced. It is claimed that the overall inhibition of RNA synthesis by rifampicin is caused by a destabilising effect on the binding of the intermediate oligonucleotides to the active enzyme-DNA complex. Rifampicin itself can only interact specifically with RNA polymerase if the enzyme is free or in a binary complex with DNA. However, the enzyme is not susceptible in a ternary complex, even if the "RNA" is as short as a trinucleotide.
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PMID:Rifampicin inhibition of RNA synthesis by destabilisation of DNA-RNA polymerase-oligonucleotide-complexes. 617 47

The genes encoding the beta and beta' subunits of RNA polymerase in E.coli, rpoB and rpoC, lie downstream of at least two ribosomal protein genes, rplJ (encoding L10) and rplL (L7/12), in a common operon. All four genes are served by promoter PL10, and an attenuator (partial terminator) of transcription, t1, lies between rplJL and rpoBC. Treatment of E.coli with rifampicin, under conditions producing partial inhibition of general RNA synthesis, can stimulate transcription of rpoBC. We have investigated the locus of this effect by fusing PL10 and t1 separately to galK, in suitable plasmids. Our studies of these fusions, and similar fusions involving transcriptional terminators derived from coliphage T7, indicate that low concentrations of rifampicin cause increased readthrough of several different transcriptional terminators in E.coli in vivo, including rpo t1. We discuss whether or not this unspecific mechanism is solely responsible for the observed stimulatory effects of the drug on rpoBC transcription.
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PMID:Evidence that rifampicin can stimulate readthrough of transcriptional terminators in Escherichia coli, including the attenuator of the rpoBC operon. 629 75

The genes encoding the beta and beta' subunits of RNA polymerase in E.coli lie downstream of at least two ribosomal protein genes in a single unit of transcription. Treatment of E.coli with rifampicin, under conditions producing partial inhibition of general RNA synthesis, can strongly stimulate transcription of the polymerase genes, while activating the neighbouring ribosomal genes only slightly. We have investigated the mechanism of this effect by fusing strong promoters, a weak internal promoter, and an attenuator of the polymerase operon to the lacZ gene, in derivatives of plasmid pMC81. Studies of these fusions confirm our conclusion, based on similar fusions to galK, that rifampicin can foster readthrough of transcriptional terminators. They also suggest the existence of extra terminators and anti-termination elements in the above transcription unit.
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PMID:Effect of rifampicin on expression of lacZ fused to promoters or terminators of the E.coli rpoBC operon. 629 76

Unusual guanosine nucleotides synthesised during amino acid or energy source starvation are thought to be the effectors of the stringent response. In vitro experiments suggest that the magic spot compounds alter transcription specificity of RNA polymerase by binding to the enzyme. However, there is no good in vivo evidence for such an interaction. We define sites on the beta-subunit of RNA polymerase which, when altered, yield E.coli mutants apparently insensitive to the presence of ppGpp.
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PMID:Relaxed mutants of Escherichia coli RNA polymerase. 635 28

The "alpha-operon" of E.coli is a unit of regulation comprising the following known genes, mostly encoding ribosomal proteins (in order of transcription, and with their products named in brackets): rpsM (S13), rpsK (S11), rpsD (S4), rpoA (alpha-subunit of RNA polymerase), rplQ (L17). There is evidence that S4 tightly regulates all of these genes, except rpoA, by repressing translation of the polycistronic mRNA. Binding of S4 to the S13 start-site is thought to regulate the first three genes. We have extended the 'rpsD-rpoA' sequences previously determined by others, to include all of rpoA and rplQ. The rpoA-rplQ intercistronic region shows strong primary, and potential secondary structural homologies with the S4-binding sites on 16S rRNA and S13 mRNA. We suggest that S4 represses L17 translation directly.
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PMID:Nucleotide sequence of the rpoA-rplQ DNA of Escherichia coli: a second regulatory binding site for protein S4? 637 5

We isolated a novel gene encoding a protein highly homologous to human FK506-binding protein 12kDa (hFKBP-12) from a human fetal brain cDNA library and determined the full-length cDNA sequence. The cDNA clone contained the open reading frame of 324 nucleotides encoding 108 amino acid and revealed 76% identity in DNA sequence and 88% identity in predicted amino acid sequence with hFKBP-12. The DNA and amino-acid sequence of this gene, designated OTK4, also had homology with other FKBPs in species ranging from humans to prokaryotes. Recombinant protein, produced in E.coli transformed by a pGEX2T expression vector containing the OTK4 cDNA and purified, showed peptidyl-prolyl cis-trans isomerase activity like other FKBP proteins. An alternatively spliced form of the transcript found in the cDNA library contained a 45-bp insertion which included a stop codon. Although the biological function of the truncated version of OTK4 is unknown, both transcripts were ubiquitously expressed in human tissues examined by the reverse-transcriptase PCR (RT-PCR) method.
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PMID:Molecular cloning and expression of a novel human gene that is highly homologous to human FK506-binding protein 12kDa (hFKBP-12) and characterization of two alternatively spliced transcripts. 751 96


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