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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)

One form of nucleotide excision repair (NER) is known to be functionally coupled to transcription, but the nature of this functional link in Escherichia coli is still unclear. Here we have employed the isolated membrane-associated nucleoids from E.coli to examine this issue. We show that the isolated nucleoid fraction is capable of excision of UV-induced pyrimidine dimers when reconstituted with a cytoplasmic fraction resolved by sucrose gradient fractionation. This excision activity by UvrABC is sensitive to rifampicin and is dependent on transcription. By using crosslinking and immunoprecipitation, the damage recognition protein, UvrA, was found to be specifically associated with the RNA polymerase beta subunit on the chromosomal DNA independent of DNA damage. It suggests that at least in one of the NER pathways the search for damage may be directly linked to RNA polymerase. In addition, the role of transcription in the unfolding of the nucleoid structure to allow repair enzymes to gain access to the damaged DNA is described. This study provides insight into the understanding of the transcription-repair coupling in vivo.
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PMID:Transcription coupled nucleotide excision repair by isolated Escherichia coli membrane-associated nucleoids. 949 Jul 93

Dihydrouracil (DHU) is a DNA base damage product produced in significant amounts by ionizing radiation damage to cytosine under anoxic conditions. DHU represents a model for pyrimidine base damage (ring saturation products) of the type recognized and repaired by Escherichia coli endonuclease III and its homologs in other species. We have built this lesion into synthetic oligonucleotides, with DHU placed at a single location downstream from an E.coli RNA polymerase promoter. This construct was used to determine the effect of DHU when encountered on a DNA template strand by either E.coli RNA or DNA polymerase (Klenow fragment). Single round transcription experiments or primer extension-type replication experiments were conducted in order to make a direct comparison between RNA and DNA polymerases with DHU placed within the same sequence context. Both DNA and RNA polymerase efficiently bypass DHU and insert adenine opposite this lesion. These results suggest that DHU is mutagenic with respect to both replication and transcription and have implications for DNA repair as well the routes leading to generation of mutant proteins in dividing and non-dividing cells.
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PMID:Escherichia coli RNA and DNA polymerase bypass of dihydrouracil: mutagenic potential via transcription and replication. 951 42

Oligonucleotide-directed triple helix formation offers a method for duplex DNA recognition, and has been proposed as an approach to the rational design of gene-specific repressors. Indeed, certain RNA and DNA oligonucleotides have previously been shown to bind duplex DNA and repress in vitro transcription by occluding the binding of transcription factors or RNA polymerase at target genes. While similar oligonucleotides have reportedly caused repression of target genes in cultured cells, physical evidence of triple helix formation in vivo is generally lacking. In the present study we wished to determine whether RNA transcripts could repress the activity of an Escherichia coli promoter in vivo by binding to the duplex promoter DNA. An in vivo genetic selection previously developed to identify DNA binding proteins was modified for this purpose. Using expression libraries encoding RNAs predisposed to forming triple helices with a DNA target site, we have selected RNA transcripts that confer survival to E.coli by disrupting transcriptional interference. Surprisingly, genetic and biochemical evidence shows that these RNAs do not form triple helices at the target promoter in vivo , despite the fact that they contain sequences capable of forming triple helices at the duplex DNA target in vitro . Rather, the selected RNAs appear to disrupt transcriptional interference via an antisense mechanism.
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PMID:Selection and characterization of RNAs that relieve transcriptional interference in Escherichia coli. 959 59

Plant chloroplasts contain transcription factors that functionally resemble bacterial sigma factors. We have cloned the full-length cDNA from mustard (Sinapis alba) for a 53 kDa derived polypeptide that contains similarity to regions 1.2-4.2 of sigma70-type factors. The amino acid sequence at the N-terminus has characteristics of a chloroplast transit peptide. An in vitro synthesized polypeptide containing this region was shown to be imported into the chloroplast and processed. The recombinant factor lacking the N-terminal extension was expressed in Escherichia coli and purified. It confers the ability on E.coli core RNA polymerase to bind specifically to a DNA fragment that contains the chloroplast psbA promoter. Transcription of the psbA template by E.coli core enzyme in the presence of recombinant SIG1 results in enhanced formation of transcripts of the size expected for correct initiation at the in vivo start site. Together, these data suggest that the mature protein acts as one of the chloroplast transcription factors in mustard. RNA gel blot hybridization reveals a transcript at approximately 1.8 kb, which is more abundant in light-grown than in dark-grown mustard seedlings.
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PMID:Sequence and expression characteristics of a nuclear-encoded chloroplast sigma factor from mustard (Sinapis alba). 959 64

The Bacillus subtilis alpha-amylase promoter amy P contains an essential TGTG motif (-16 region) upstream of the -10 region. Mutations of this region significantly reduced in vitro promoter strength. A -15 G-->C transversion eliminated transcription from amy P by both B.subtilis and Escherichia coli RNA polymerase (RNAP). A second alpha-amylase promoter ( amy P2) also required the -16 region for function. To determine conserved sequences in promoters containing -16 region elements, sequences of 64 B.subtilis promoters with the second TG motif of the -16 region were aligned and analyzed. Unlike the E.coli class of 'extended -10 promoters', with a similar TG motif but lacking a -35 region, the -16 region promoters contain highly conserved -35 regions. They also contain conserved A n and T n tracts upstream of the -35 region. In addition, we analyzed all available gram-positive bacterial promoter compilations to determine the generality of the -16 region. From this analysis, the -16 region TRTG motif (R = purine) appears to be a basic element found in a large portion of gram-positive bacterial promoters and is, in the case of at least the alpha-amylase promoters, necessary for transcription by the major form of B.subtilis and E.coli RNAP.
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PMID:The -16 region of Bacillus subtilis and other gram-positive bacterial promoters. 967 23

Conformational behaviour of T2 DNA during its complex formation with E.coli RNA polymerase was studied by spin label technique. T2 DNA was selectively modified at its readily melting AT-rich sites by bromoacetooxypiperidine-1-oxyl-radical. Specific conformational changes are induced in DNA structure by RNA polymerase attachment. The changes are observed under the conditions when open transcriptionally competent complexes are formed. The readily melting sites of T2 DNA were shown to be involved in the formation of functionally active promoters.
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PMID:[Conformational analysis of complexes of RNA polymerase from Escherichia coli with DNA]. 970 34

Periodicities in the position of E.coli RNA polymerase promoter contacts on several promoters (lacUV5, T7 A3, tetR, lambda cin, lambda c17, RNA1, and trp S.t.) were found by means of Fourier analysis. The comparison of the Fourier spectrum of core RNA polymerase contacts on the lacUV5 promoter and that of holoenzyme revealed a more prominent 7-periodicity in the Fourier spectrum of holoenzyme contacts. 6-, 7-, and 8-periodicities were found in the primary structure of the majority of E.coli promoters. It is shown that RNA polymerase recognizes specific periodic patterns in the promoter structure.
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PMID:[Periodicity in contacts of RNA-polymerase with promotors]. 1041 72

The monoclonal antibody Jel42 is specific for the Escherichia coli histidine-containing protein, HPr, which is an 85 amino acid phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. The binding domain (Fv) has been produced as a single chain Fv (scFv). The scFv gene was synthesized in vitro and coded for pelB leader peptide-heavy chain-linker-light chain-(His)(5) tail. The linker is three repeats from the C-terminal repetitive sequence of eukaryotic RNA polymerase II. This linker acts as a tag; it is the antigen for the monoclonal antibody Jel352. The codon usage was maximized for E.coli expression, and many unique restriction endonuclease sites were incorporated. The scFv gene incorporated into pT7-7 was highly expressed, yielding 10-30% of the cell protein as the scFv, which was found in inclusion bodies with the leader peptide cleaved. Jel42 scFv was purified by denaturation/renaturation yielding preparations with K(d) values from 20 to 175 nM. However, based upon an assessment of the amount of active refolded scFv, the binding dissociation constant was estimated to be 2.7 +/- 2.0 nM compared with 2.8 +/- 1.6 and 3.7 +/- 0.3 nM previously determined for the Jel42 antibody and Fab fragment respectively. The effect of mutation of the antigen HPr on the binding constant of the scFv was very similar to the properties determined for the antibody and the Fab fragment. It was concluded that the small percentage ( approximately 6%) of refolded scFv is a true mimic of the Jel42 binding domain and that the incorrectly folded scFv cannot be detected in the binding assay.
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PMID:Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs. 1043 89

PpLSU3, a mobile group I intron found in the ribo-somal RNA genes of Physarum polycephalum, encodes the I-PpoI homing endonuclease. This enzyme represents one of the rare cases in nature where a protein is expressed from an RNA polymerase I transcript. Our previous results showed that the full length intron, but not a further processed species, is the messenger for I-PpoI, implying a role of the untranslated region (UTR) in gene expression. To study the function of the 3'-UTR in expression of the endonuclease and in splicing of the intron, we replaced the I-PpoI gene in PpLSU3 with the gene for the alpha-fragment of Escherichia coli beta-galactosidase, and then integrated this chimeric intron into all the chromosomal rDNA repeats of yeast. The resulting cells synthesized functional alpha-fragment, as evidenced by a complementation assay analogous to that used in E.coli. The beta-galactosidase activity thus provides an unusual and potentially valuable readout for Pol I transcription from chromosomal rDNA. This is the first example in which a eucaryotic homing endonuclease gene has been successfully replaced by a heterologous gene. Using deletion mutagenesis and a novel randomization approach with the alpha-fragment as a reporter, we found that a small segment of the 3'-UTR dramatically influences both splicing and protein expression.
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PMID:Functional alpha-fragment of beta-galactosidase can be expressed from the mobile group I intron PpLSU3 embedded in yeast pre-ribosomal RNA derived from the chromosomal rDNA locus. 1068 39

It is possible to replace in a normal transcription reaction catalyzed by E.coli RNA polymerase one of the four precursors by the corresponding deoxynucleoside triphosphate. These deoxynucleotide-substituted RNA's offer interesting prospects for nucleotide sequence analysis. Indeed by the use of U(2)-RNase with dG-RNA, or pancreatic RNase with dC-RNA or dU-RNA, base specific cleavage can be obtained at any of the four residues. In this way overlap of at least six residues in length can be obtained for any site in the RNA. The technique offers also great benefit for solving the sequence of the more difficult T(1)-oligonucleotides. Some examples in the sequence analysis of SV40 DNA-Hind fragments are reported.
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PMID:Deoxynucleotide substitution: a new technique for sequence analysis of RNA. 1079 35


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