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)

We investigated HutP-dependent transcription antitermination of the Bacillus subtilis hut operon. In vitro transcription assays with the B. subtilissigmaA-containing RNA polymerase indicated that HutP inhibits transcription termination at the internal terminator by binding to the antiterminator on hut mRNA in the presence of histidine. Ethylnitrosourea modification interference assays and mutational analyses of the interference sites showed that interaction of HutP with a region containing three UAG trinucleotide sequences, which is located on top of the antiterminator structure, is critical for hut antitermination in vivo. Results from kinetic analysis of binding of HutP to RNA containing various portions of the antiterminator sequences indicated that secondary structure is required for binding of HutP to the region containing three UAG triplets in the antiterminator. The in vivo HutP antiterminator activity was reduced by the mutations in the N-terminal region of HutP. The HutP variants with H4A, R7A, I9A and Q26A mutations exhibited reduced binding affinities to the antiterminator RNA in vitro. A 25-mer peptide consisting of amino acid residues 2-26 of HutP bound to the antiterminator RNA. These results indicated that the N-terminus of HutP is involved in binding of HutP to the antiterminator RNA.
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PMID:Analysis of HutP-dependent transcription antitermination in the Bacillus subtilis hut operon: identification of HutP binding sites on hut antiterminator RNA and the involvement of the N-terminus of HutP in binding of HutP to the antiterminator RNA. 1476 87

Bacteriophage T7 lysozyme binds to T7 RNA polymerase and inhibits transcription initiation and the transition from initiation to elongation. We have investigated each step of transcription initiation to determine where T7 lysozyme has the most effect. Stopped flow and equilibrium DNA binding studies indicate that T7 lysozyme does not inhibit the formation of the preinitiation open complex (open complex in the absence of initiating nucleotide). T7 lysozyme, however, does prevent the formation of a fully open initiation complex (open complex in the presence of the initiating nucleotide). This is consistent with the results that in the presence of T7 lysozyme the rate of G ladder RNA synthesis is about 5-fold slower and the GTP Kd is about 2-fold higher, but T7 lysozyme does not inhibit the initial rate of RNA synthesis with a premelted bulge-6 promoter (bubble from -4 to +2). Neither the RNA synthesis rate nor the extent of promoter opening is restored by increasing the initiating nucleotide concentration, indicating that T7 lysozyme represses transcription by interfering with the formation of a stable and a fully open initiation bubble or by altering the structure of the DNA in the initiation complex. As a consequence of the unstable initiation bubble and/or the inhibition of the conformational changes in the N-terminal domain of T7 RNAP, T7 lysozyme causes an increased production of abortive products from 2- to 5-mer that delays the transition from the initiation to the elongation phase.
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PMID:T7 lysozyme represses T7 RNA polymerase transcription by destabilizing the open complex during initiation. 1476 84

A gel mobility-shift assay was used to demonstrate the binding of the Sindbis virus transcriptase to the promoter for the synthesis of subgenomic (SG) RNA. The assay made use of a P15 fraction (the cell fraction that is pelleted at 15,000 x g) from cells infected with recombinant vaccinia virions expressing various Sindbis virus nonstructural proteins (nsPs) and a (32)P-labeled 24-mer oligoribonucleotide representing the minimal sequence with SG promoter activity. By itself, nsP4, the viral RNA-dependent RNA polymerase, did not bind to the SG promoter; rather, all four nsPs were required for the binding of the transcriptase to the promoter. UV crosslinking of the transcriptase to a thiouridine-containing SG promoter, followed by V8 protease digestion of the complex, generated a peptide fragment that was bound to the SG promoter. This peptide fragment contained a sequence that corresponded to residues 329-334 of nsP4. This peptide may be in the fingers domain of nsP4. The peptide that was identified contained Arg residues at positions 331 and 332. Another Arg is present at position 327. By changing each of the Arg residues to Ala, we demonstrated that only the Arg residues at positions 331 and 332 were required for binding nsP4 to the SG promoter.
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PMID:Identification of the amino acid sequence in Sindbis virus nsP4 that binds to the promoter for the synthesis of the subgenomic RNA. 1519 79

T7 RNA polymerase recognizes a small promoter, binds DNA, and begins the process of transcription by synthesizing short RNA products without releasing promoter contacts. To determine whether the promoter contact must be released to make longer RNA products and at what position the promoter must be released, a mutant RNA polymerase was designed that allows cross-linking to a modified promoter via a covalent disulfide bond. The modifications individually have no measurable effect on transcription. Under oxidizing conditions that produce the protein-DNA cross-link, the complex is able to synthesize short RNA products, strongly supporting a model in which promoter contacts are not lost on translocation through at least position +6. However, cross-linked complexes are impaired in promoter escape in that only about one in four can escape to make full-length RNA. The remainder release 12- and 13-mer RNA transcripts, suggesting an increased energetic barrier in the transition from an initial transcribing complex to a fully competent elongation complex. The results are discussed in the context of a model in which promoter release helps drive initial collapse of the upstream edge of the bubble, which, in turn, drives initial displacement of the 5'-end of the RNA.
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PMID:Cross-linking of promoter DNA to T7 RNA polymerase does not prevent formation of a stable elongation complex. 1530 97

The hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase required for replication of the HCV RNA genome. We have identified a peptide that most closely resembles a short region of the protein kinase C-related kinase 2 (PRK2) by screening of a random 12-mer peptide library displayed on the surface of the M13 bacteriophage with NS5B proteins immobilized on microwell plates. Competitive phage enzyme-linked immunosorbent assay with a synthetic peptide showed that the phage clone displaying this peptide could bind HCV RNA polymerase with a high affinity. Coimmunoprecipitation and colocalization studies demonstrated in vivo interaction of NS5B with PRK2. In vitro kinase assays demonstrated that PRK2 specifically phosphorylates NS5B by interaction with the N-terminal finger domain of NS5B (amino acids 1-187). Consistent with the in vitro NS5B-phosphorylating activity of PRK2, we detected the phosphorylated form of NS5B by metabolic cell labeling. Furthermore, HCV NS5B immunoprecipitated from HCV subgenomic replicon cells was specifically recognized by an antiphosphoserine antibody. Knock-down of the endogenous PRK2 expression using a PRK2-specific small interfering RNA inhibited HCV RNA replication. In contrast, PRK2 overexpression, which was accompanied by an increase of in the level of its active form, dramatically enhanced HCV RNA replication. Altogether, our results indicate that HCV RNA replication is regulated by NS5B phosphorylation by PRK2.
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PMID:Protein kinase C-related kinase 2 regulates hepatitis C virus RNA polymerase function by phosphorylation. 1536 41

Single-stranded long oligonucleotide-based (50- to 70-mer) microarrays offer several advantages over conventional cDNA microarrays. These include the easy preparation of the probes, low cost of array production, and low cross-contamination during probe handling. However, the application of oligonucleotide microarrays for the analysis of global gene expression with small amounts of total RNA using the conventional oligo(dT)-T7 promoter-based amplification is hampered by the single-stranded nature (sense strand) of oligonucleotide probes in microarrays. In this report, we describe modified RNA amplification methods generating antisense-labeled cDNA targets and a successful application for oligonucleotide microarray gene expression analysis. In the first round, mRNA was amplified linearly with oligo(dT)24T7-primed reverse transcription and in vitro transcription by T7 RNA polymerase. In the second round, random 9-mer T3 primers and T3 RNA polymerase were used to generate sense-strand amplified RNA (aRNA). Fluorescently labeled cDNA targets were generated from the aRNA and hybridized to the oligonucleotide microarrays. Our data show that the amplification provides highly reproducible results, as evidenced by a significant correlation between the amplified and nonamplified samples. We also demonstrate that amplification of RNA derived from laser-microdissected tumor samples reproduced the gene expression profiles that were obtained from total RNA isolated from the same samples.
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PMID:Oligonucleotide microarray analysis of aminoallyl-labeled cDNA targets from linear RNA amplification. 1551 70

Transcription of RNA molecules from synthetic DNA templates with T7 RNA polymerase is a common procedure for the preparation of long RNA molecules. However, enzymatic synthesis does not allow for site-specific incorporation of modified nucleotides. RNA synthesis by chemical methods on the other side can satisfy this purpose, but it is limited to RNA fragments of about 80 nucleotides at maximum. We aimed to combine both chemical and enzymatic procedures to synthesise RNA molecules by RNA primed transcription with T7 RNA polymerase. To this end we have chemically synthesised a fluorescein labelled RNA primer and studied elongation of this primer by T7 RNA polymerase on a single-stranded DNA template. We show that the enzyme is capable of extending the primer to the full-length product. The 34-mer RNA that has been synthesised by RNA primed transcription served as substrate for a twin ribozyme and was successfully cleaved in the expected manner.
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PMID:[RNA synthesis by T7 RNA polymerase supported primer extension]. 1555 83

The distribution of unusual mercury resistance transposons, Tn5044 and Tn5070, was examined. A characteristic feature of Tn5044 is temperature sensitivity of its mercury operon and the presence in the mer operon of the gene homologous to RNA polymerase a subunit. Structural organization of mercury operon Tn5070, containing minimum gene set (merRTPA), differs from mer operons of both Gram-negative and Gram-positive bacteria. None of more than two thousand environmental bacterial strains displaying mercury resistance and isolated from the samples selected from different geographical regions hybridized to Tn5040- and Tn5070-specific probes. A concept on the existence of cosmopolite, endemic, and rare transposons in environmental bacterial populations was formulated.
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PMID:[Distribution of transposons Tn5044 and Tn5070 with unusual mer operons in environmental bacterial populations]. 1564 57

A concise synthetic way has been developed for the preparation of guanosine monophosphate derivatives carrying a decaethylene glycol spacer at their 5'-oxygen to which are attached a range of organic substrates. The four different compounds, prepared via a convergent synthetic strategy, carry a tethered benzylallyl ether residue (1a), an anthracene (1b), a benzyl carbamate residue (1c), or a primary amino group (1d), respectively. All four compounds have been successfully incorporated at the 5'-end of a 25-mer long RNA transcript via T7 RNA polymerase, and no inhibition of chain elongation could be observed. Under proper conditions, 1a and 1b can be incorporated up to 90-95% and 1c up to 68%. The amino-terminated initiator 1d is incorporated less efficiently although still up to 49%. These results show that the more hydrophobic the guanosine monophosphate derivative is, the higher is its enzymatic incorporation.
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PMID:Efficient preparation of organic substrate-RNA conjugates via in vitro transcription. 1596 9

The propensity of RNA to fold into higher-order structures poses a major barrier to the use of short probes (<15 nucleotides) by preventing their accessibility. Introduction of the pseudo-complementary bases 2-aminoadenine (nA) and 2-thiouracil (sU) and the destabilizing base 7-deazaguanine (cG) into RNA provides a partial solution to this problem. While complementary in hydrogen bonding groups, nA and sU cannot form a stable base pair due to steric hindrance, and are thus pseudo-complementary. Each, however, recognizes the regular T/U and A complements, allowing pairing with oligonucleotides. Short pseudo-complementary RNAs can be prepared by in vitro transcription. Relative to standard transcripts, the modified transcripts possess reduced secondary structure and increased accessibility to short (8-mer) probes in the locked nucleic acid (LNA) configuration. They also hybridize to complementary probes with increased specificity and thermostability. Practical application of this strategy to oligonucleotide-based hybridization assays will require engineering of RNA polymerase for more efficient utilization of pseudo-complementary nucleoside triphosphates.
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PMID:Unrestricted accessibility of short oligonucleotides to RNA. 1612 Aug 34


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