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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)

Washed mature spermatozoa from bulls incorporate ribonucleoside triphosphates into RNA using an endogenous template. Maximum incorporation was observed at 31 degrees C in the presence of MgCl2, all four ribonucleoside triphosphates, beta-mercaptoethanol, and glycine sodium hydroxide buffer at pH 9.0. The amount of synthesis was linearly dependent upon the concentration of spermatozoa and continued for at least 4 h. Digestion studies revealed the RNA to be present in a protected (intracellular?) location in the spermatozoa. The RNA synthesis was inhibited by ethidium bromide, rifampicin, acriflavine, actinomycin D, and caffeine, but not by alpha-amanitine or rifamycin SV. Fractionation of the spermatozoa by sonication and separation of the heads and tails by centrifugation through a discontinuous gradient revealed that more than half of the total RNA polymerase activity was associated with the tail fraction.
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PMID:RNA polymerase activity in bovine spermatozoa. 2 Apr 46

Brain RNA polymerase isolated from rats treated with pemoline and magnesium hydroxide (Cylert) was not more active than enzyme from control animals. The drug did not increase enzymic activity in vitro. Pemoline did not significantly affect either RNA or protein synthesis in suspensions of Ehrlich ascites carcinoma cells.
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PMID:Pemoline and magnesium hydroxide: lack of effect on RNA and protein synthesis. 438 48

The regulatory protein CbbR, which activates the transcription of the duplicate, chromosomally and megaplasmid pHG1-borne cbb CO2 assimilation operons of Alcaligenes eutrophus H16, was purified to homogeneity from Escherichia coli after heterologous expression of the cloned cbbR gene. The pure protein occurred as either a 63-kDa dimer at room temperature or a 125-kDa tetramer at 4 degrees C. CbbR bound to the 167-bp cbb control region separating the divergently oriented cbbR gene (defective copy on pHG1) from the cbb operon. DNase I footprinting revealed binding of the protein between position -29 and -74 relative to the transcriptional start point of the cbb operon, with a hypersensitive site at positions -47 and -48, suggesting potential DNA bending. Hydroxyl radical footprinting disclosed the same central binding region. The region was found to consist of two subsites to which the activator apparently bound in a cooperative manner. At higher CbbR concentrations, the binding region extended to position +13. The overlapping arrangement of the operon promoter and CbbR-binding region (operator) suggests an interaction between CbbR and RNA polymerase to cause transcription activation. Transcriptional fusions with fragments carrying 1- or 2-bp insertions within the central region showed no operon promoter activity, although CbbR binding was not prevented by these mutations. Dissection of the central region enabled the differentiation of two apparently independent binding subsites. Strongly increased cbbR promoter activity originating from a fragment that contained only a part of the central region indicated negative autoregulation of cbbR transcription.
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PMID:Operator binding of the CbbR protein, which activates the duplicate cbb CO2 assimilation operons of Alcaligenes eutrophus. 759 35

Hydroxyl radical footprinting has been used to study different open complexes between Escherichia coli RNA polymerase and the galactose operon regulatory region, which contains two overlapping promoters, P1 and P2. Complexes at P1 were studied by exploiting a P2- mutant and complexes at P2 were studied with a P1-mutant. We have identified the precise location of alpha binding in both binary RNA polymerase-galP1 and RNA polymerase-P2 complexes from the effects of deletion of the C-terminal domain of the RNA polymerase alpha subunit: alpha binds to different sites at the upstream end of each complex. Transcription initiation at galP1 can be activated by the cyclic AMP receptor protein (CRP). Addition of CRP to the RNA polymerase-galP1 complex displaces the C-terminal domain of alpha, which then binds to a different site upstream of CRP in the ternary CRP-RNA polymerase-galP1 complex. Thus, the C-terminal domain of alpha can occupy three different sites at the gal operon regulatory region. We have also examined the effect of disrupting the Activating Region of CRP on interactions between CRP and the C-terminal domain of alpha in ternary CRP-RNA polymerase-galP1 complexes. Footprinting experiments show that these substitutions interfere with the contact between CRP and alpha but do not affect the position of alpha binding to its site upstream of bound CRP.
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PMID:Location of the C-terminal domain of the RNA polymerase alpha subunit in different open complexes at the Escherichia coli galactose operon regulatory region. 871 Apr 92

Transcription initiation is accompanied with iterative synthesis and release of short transcripts. The molar ratio of enzyme to template was found to be critical for the amounts and distribution of the abortive products synthesized by Escherichia coli RNA polymerase from several promoters. At a high ratio abortive synthesis of 4-8 nt were enhanced at thelambda P R promoter. Removing excess RNA polymerase just before initiation, achieved by washing immobilized transcription complexes, prevented this enhancement. At this high ratio synthesis of an unexpected 6 nt transcript was enhanced when the enzyme stalled at position +32, but not when it stalled at position +73. This transcript had misincorporations at its fifth and sixth positions, probably due to slippage. Hydroxyl radical footprinting of the complex stalled at +32 in the presence of excess enzyme showed that more than one molecule of RNA polymerase was tandemly bound to the same DNA. These results suggest that: (i) when RNA polymerase molecules are tandemly transcribing the same DNA, transient collisions enhance abortive synthesis by the trailing molecule; (ii) when the leading polymerase stalled in the initially transcribed region blocks progression of the trailing polymerase, the latter can commit misincorporations, probably due to slippage synthesis.
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PMID:Physical interference between escherichia coli RNA polymerase molecules transcribing in tandem enhances abortive synthesis and misincorporation. 918 76

A reversible target capture viral RNA extraction procedure was combined with a reverse-transcriptase nested polymerase chain reaction (PCR) to develop a capture PCR assay providing a rapid and safe prediction method for circulating bluetongue virus in infected ruminants. This new assay was compared with virus isolation and a recently developed antigen-capture enzyme-linked immunosorbent assay (ELISA) for the detection of bluetongue virus. Eight Warhill crossbred sheep were inoculated subcutaneously with bluetongue virus serotype 10, and blood samples were taken sequentially over a period of 28 days. The capture PCR detected the peak of viremia, as determined by virus isolation and antigen-capture ELISA, from day 5 to day 14 after challenge. The results indicate that the rapid-capture bluetongue virus PCR provides a rapid indicator of samples in which virus can be isolated. In addition, this capture bluetongue virus PCR procedure does not require a lengthy phenol extraction or the use of the highly toxic methyl mercury hydroxide denaturant.
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PMID:Bluetongue virus detection: a safer reverse-transcriptase polymerase chain reaction for prediction of viremia in sheep. 921 Dec 28

The RNA polymerase III factor TFIIIB forms a stable complex with DNA and can promote multiple rounds of initiation by polymerase. TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIB-related factor (BRF), and B". Chemical footprinting, as well as mutagenesis of TBP, BRF, and promoter DNA, was used to probe the architecture of TFIIIB subunits bound to DNA. BRF bound to TBP-DNA through the nonconserved C-terminal region and required 15 bp downstream of the TATA box and as little as 1 bp upstream of the TATA box for stable complex formation. In contrast, formation of complete TFIIIB complexes required 15 bp both upstream and downstream of the TATA box. Hydroxyl radical footprinting of TFIIIB complexes and modeling the results to the TBP-DNA structure suggest that BRF and B" surround TBP on both faces of the TBP-DNA complex and provide an explanation for the exceptional stability of this complex. Competition for binding to TBP by BRF and either TFIIB or TFIIA suggests that BRF binds on the opposite face of the TBP-DNA complex from TFIIB and that the binding sites for TFIIA and BRF overlap. The positions of TBP mutations which are defective in binding BRF suggest that BRF binds to the top and N-terminal leg of TBP. One mutation on the N-terminal leg of TBP specifically affects the binding of the B" subunit.
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PMID:Architecture of protein and DNA contacts within the TFIIIB-DNA complex. 948 85

We have constructed a family of promoters carrying tandem DNA sites for the Escherichia coli cyclic AMP receptor protein (CRP), with one of the sites centred between base-pairs 41 and 42 upstream from the transcription start site, and the second site located further upstream. In vivo activity measurements show that the activity of these promoters is completely dependent on CRP and that, depending on the precise location, CRP bound at the upstream site increases transcription activation. Hydroxyl radical footprinting was exploited to investigate the binding of CRP and RNA polymerase holoenzyme (RNAP) to these promoters. The study shows that the C-terminal domains of the RNAP alpha subunits bind adjacent to the upstream CRP and that their precise positioning depends on the location of upstream-bound CRP. The C-terminal domains of the RNAP alpha subunits interact with both the upstream and downstream-bound CRP via activating region 1 of CRP.
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PMID:Transcription activation at promoters carrying tandem DNA sites for the Escherichia coli cyclic AMP receptor protein: organisation of the RNA polymerase alpha subunits. 954 73

The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which leads to the synthesis of the competence transcription factor (CTF). Previous studies suggested that CTF is encoded by comK. ComK is required for the transcription of comK itself, as well as of the late competence genes encoding the DNA uptake machinery and of genes required for homologous recombination. Here, we used purified ComK to study its role in transcription and to determine the DNA recognition sequence for ComK. In vitro transcription from the comG promoter, which depends on ComK in vivo, was observed on the addition of purified ComK together with Bacillus subtilis RNA polymerase, proving that ComK is CTF. To determine the DNA sequences involved in ComK recognition, footprinting analysis was performed with promoter fragments of the CTF-dependent genes: comC, comE, comF, comG, comK, and addAB. The ComK binding sites determined by DNase I protection experiments were unusually long, with average lengths of approximately 65 bp, and displayed only weak sequence similarities. Hydroxy-radical footprinting, performed with the addAB promoter, revealed a unique arrangement of four short A/T-rich sequences. Gel retardation experiments indicated that four molecules of ComK bound the addAB promoter and the dyad symmetrical arrangement of the four A/T-rich sequences implied that ComK functions as a tetramer composed of two dimers each recognizing the motif AAAAN5TTTT. Comparable A/T-rich sequences were identified in all six DNase I footprints and could be used to predict ComK targets in the B. subtilis genome. On the basis of the variability in distance between the ComK-dimer binding sites, ComK-regulated promoters could be divided into three classes, demonstrating a remarkable flexibility in the binding of ComK. The pattern of hydroxy-radical protections suggested that ComK binds at one face of the DNA helix through the minor groove. This inference was strengthened by the observation that minor groove binding drugs inhibited the binding of ComK.
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PMID:The competence transcription factor of Bacillus subtilis recognizes short A/T-rich sequences arranged in a unique, flexible pattern along the DNA helix. 958 13

A cysteine-tethered DNA cleavage agent has been used to locate the position of region 2.5 of sigma70 in transcriptionally competent complexes between Escherichia coli RNA polymerase and promoters. In this study we have engineered sigma70 to introduce a unique cysteine residue at a number of positions in region 2.5. Mutant proteins were purified, and in each case, the single cysteine residue used as the target for covalent coupling of the DNA cleavage agent p-bromoacetamidobenzyl-EDTA.Fe (FeBABE). RNA polymerase core reconstituted with tagged sigma derivatives was shown to be transcriptionally active. Hydroxyl radical-based DNA cleavage mediated by tethered FeBABE was observed for each derivative of RNA polymerase in the open complex. Our results show that region 2.5 is in close proximity to promoter DNA just upstream of the -10 hexamer. This positioning is independent of promoter sequence. A model for the interaction of this region of sigma with promoter DNA is discussed.
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PMID:Organization of open complexes at Escherichia coli promoters. Location of promoter DNA sites close to region 2.5 of the sigma70 subunit of RNA polymerase. 989 Sep 89


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