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

---

Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Through genetic studies, the fliA gene product has been shown to regulate positively gene expression in late operons of the flagellar regulon in Salmonella typhimurium. In the present study, the fliA gene was cloned and sequenced. The fliA coding region consisted of 717 nucleotides beginning with the GTG initiation codon and the conserved sequence specific to promoters for flagellar operons was found to exist upstream of the coding region. The fliA gene product deduced from the nucleotide sequence was a protein with 239 amino acid residues and the calculated molecular mass was 27,470 dalton. The deduced amino acid sequence was homologous with that of sigma 28, a flagellar specific sigma factor of Bacillus subtilis. The fliA gene product was identified as a protein of molecular mass 29 kDa in the in vitro transcription-translation system, while three proteins of 29 kDa, 31 kDa and 32 kDa were found in the products programmed by the fliA gene in minicells and in maxicells. The 29 kDa FliA protein was purified from the FliA overproducing strain which carried the ptac-fliA fusion. This protein activated the in vitro synthesis of flagellin, the fliC gene product. RNA polymerase containing the purified FliA protein was shown to transcribe the fliC gene. These results indicate that FliA protein functions as an alternative sigma factor specific for S. typhimurium flagellar operons.
...
PMID:Gene fliA encodes an alternative sigma factor specific for flagellar operons in Salmonella typhimurium. 219 28

Four flavonoids (i.e., baicalein, quercetin, quercetagetin, and myricetin), known to be inhibitors of HIV-reverse transcriptase, have been shown to be more or less inhibitory to the activities of various cellular DNA and RNA polymerases. The degree of the inhibition varied depending on the combination of the flavonoid and the enzyme species: baicalein was moderately inhibitory to DNA polymerase gamma and E. coli DNA polymerase I; quercetin was strongly inhibitory to DNA polymerase beta and E. coli RNA polymerase and moderately inhibitory to DNA polymerase I; quercetagetin was a potent inhibitor for all of DNA polymerases alpha, beta, gamma, and I and RNA polymerase; myricetin was a strong inhibitor of DNA polymerases alpha and I and RNA polymerase. However, terminal deoxynucleotidyltransferase was virtually insensitive to inhibition by these flavonoids. The inhibition by the flavonoids was due to competition with the template.primer in the case of the DNA polymerases, whereas the inhibition was due to competition with the triphosphate substrate (GTP) in the case of RNA polymerase. The Ki values of these flavonoid inhibitors for DNA and RNA polymerases was determined.
...
PMID:Mechanisms of inhibition of various cellular DNA and RNA polymerases by several flavonoids. 229 90

alpha-Sarcin is a cytotoxic protein that inactivates ribosomes by hydrolyzing a single phosphodiester bond on the 3' side of G-4325 in eukaryotic 28 S rRNA. We have examined the requirements for the recognition by alpha-sarcin of this domain using a synthetic oligoribonucleotide (35-mer) that reproduces the sequence and, we presume, the secondary structure (a stem, a bulged nucleotide, and a loop) at the site of modification. The wild type structure and a large number of variants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. Recognition of the substrate is strongly favored by a G at the position that corresponds to 4325. There is an absolute requirement for a helical stem; however, it can be reduced from the 7 base pairs in the natural structure to 3 without loss of specificity. The nature of the base pairs in the stem modifies but does not abolish recognition; whereas, the bulged nucleotide does not contribute to identification. Cleavage is materially affected by altering the nucleotides in the universal sequence surrounding G-4325 and changing the position in the loop of the tetranucleotide GAG(sarcin)A leads to loss of recognition by the toxin. We propose that the alpha-sarcin domain RNA participates in elongation factor catalyzed binding of aminoacyl-tRNA and of translocation; that translocation is driven by transitions in the structure of the alpha-sarcin domain RNA initiated by the binding of the factors, or the hydrolysis of GTP, or both; and that to toxin inactivates the ribosomes by preventing this transition.
...
PMID:RNA-protein interaction. An analysis with RNA oligonucleotides of the recognition by alpha-sarcin of a ribosomal domain critical for function. 229 46

Our understanding of the mechanism of RNA biosynthesis in archaebacteria is limited, due in part to the inability of purified RNA polymerases to transcribe purified genes accurately in vitro. In the present study, we show that cell extracts of Methanococcus vannielii and Methanococcus thermolithotrophicus purified by gradient centrifugation synthesize a distinct transcript from templates harboring a cloned homologous tRNA(Val) and tRNA(Arg) gene. The in vitro transcripts initiate with GTP at the same sites as in Methanococcus cells. About 60% of the sequence of the in vitro RNA products was analyzed by dideoxyterminated primer extension and found to be identical with that of the precursors of tRNA(Val) and tRNA(Arg). This finding indicates that this RNA polymerase fraction both initiates and terminates transcription faithfully in vitro. After purification of a cell-free extract (S-100) of M. thermolithotrophicus by phosphocellulose chromatography, the endogenous RNA polymerase has lost its ability to transcribe the tRNA(Val) gene accurately. The activity directing specific expression of this template was reconstituted by the addition of a protein-fraction devoid of RNA polymerase activity. Thus, a transcription factor appears to be required for accurate cell-free expression of tRNA genes from M. vannielii.
...
PMID:An archaebacterial cell-free transcription system. The expression of tRNA genes from Methanococcus vannielii is mediated by a transcription factor. 232 83

The NTP binding site of bacteriophage T7 DNA-dependent RNA polymerase was studied using GTP analogs. For four analogs the irreversible inhibition was demonstrated. The kinetic parameters for competitive (Ki) and irreversible (KI and k3) inhibition were determined. One of the analogs, 5'[2-hydroxy(4-iodoacetamido)benzoyl]guanosine, was shown to inactivate the enzyme rapidly due to the modification of SH-groups. Some suggestions on the structure of the RNA polymerase active site have been made.
...
PMID:[The study of nucleoside triphosphate-binding center of DNA-dependent RNA-polymerase of phage T7 using GTP analogs]. 239 73

Terbium (III) upon complexation with guanosine 5'-triphosphate showed remarkable enhancement of fluorescence emission at 488 and 545 nm when excited at 295 nm. Analysis of the binding data yielded a value for the mean Kd between Tb(III) and GTP of 0.2 microM, with three binding sites for Tb(III) on GTP. 31P and 1H NMR measurements revealed that Tb(III) mainly binds the phosphate moiety of GTP. Fluorescence titration of the emission signals of the TbGTP complex with varying concentrations of Escherichia coli RNA polymerase resulted in a Kd value of 4 microM between the TbGTP and the enzyme. It was observed that TbGTP can be incorporated in the place of GTP during E. coli RNA polymerase catalyzed abortive synthesis of dinucleotide tetraphosphate at T7A2 promoter. Both the substrate TbGTP and the inhibitor of the initiation of transcription rifampicin bind to the beta-subunit of E. coli RNA polymerase. This allows the measurement of the fluorescence excited-state energy transfer from the donor TbGTP-RNA polymerase to the acceptor rifampicin. Both emission bands of Tb(III) overlap with the rifampicin absorption, and the distances at 50% efficiency of energy transfer were calculated to be 28 and 24 A for the 488- and 545-nm emission bands, respectively. The distance between the substrate binding site and the rifampicin binding site on the beta-subunit of E. coli RNA polymerase was measured to be around 30 A. This suggests that the nature of inhibition of transcription by rifampicin is essentially noncompetitive with the substrate.
...
PMID:Resonance energy transfer study on the proximity relationship between the GTP binding site and the rifampicin binding site of Escherichia coli RNA polymerase. 240 99

Variation in the observed spin lattice relaxation rate (Robs) interpreted as proton exchange dominated in sequences corresponding to part of promoters where RNA polymerase initiates mRNA synthesis has been observed by both Patel et al. and Reid and co-workers. A higher Robs was also seen in the TA pair of the GTG/CAC in the sequence corresponding to the lambda phage cro repressor binding site by Kyogoku et al. As we pointed out in the introduction, the one case where a three-dimensional structure for a turn of a helix is known shows clear structural heterogeneity which has led to detailed consideration of geometry of regulatory regions. Nussinov and collaborators have generalized the details of the Dickerson dodecomer to note potential similarities in operators including the lac system and the enhancer sequences described above. Like the steric considerations of Calladine and Dickerson and nearest neighbor structure analysis of Bubienko et al., the focus is on the geometry of a sequence leading to base tilt angles and potential overlap since they are measurable parameters. With the observation that the DNA molecule is both structurally and dynamically flexible, there will no doubt be many new variables that can be made as a function of DNA sequence. Biophysical chemists in some ways are like the intoxicated person searching for a lost key at a site different from where it was lost because that is where the light is best. Thus, each physical method has its most convenient observable. It is hoped that the above discussion illustrates that a very large and diverse set of biochemical results are awaiting detailed explanation in molecular terms using the illumination of high resolution physical techniques.
...
PMID:A recurrent DNA sequence at sites of protein interaction. 242 83

To elucidate the mechanism of sigma release in the transcript by Escherichia coli DNA-dependent RNA polymerase, we obtained the time courses of sigma release and elongation of product RNA by a rapid kinetic technique; transcription was synchronously initiated from A1 promoter on T7 DNA by the addition of four substrates to a stoichiometric mixture of holoenzyme and template DNA, and then quenched by the addition of EDTA. The elongation rate was changed by limiting the concentration of one of four substrates, GTP. At reduced GTP concentration, elongation was decelerated, but the time course of sigma release was unchanged. No connection between sigma release and length of RNA product was found. The results lead to the conclusion that sigma is released depending only on time elapsed after initiation, not on the length of RNA product. We propose a two-step model for sigma release with a rapid triggering and a slow dissociation of about 5 s. This dissociation, the rate-determining step of sigma release, is independent of the rate of elongation.
...
PMID:Release of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase depends mainly on time elapsed after the start of initiation, not on length of product RNA. 242 13

Subunits of RNA polymerase II that come into contact with nascent RNA transcripts have been determined by photoaffinity labeling. Transcription was carried out in a cell-free extract in the presence of 4-thio-UTP, and utilized the major late promoter of adenovirus-2 DNA. The transcript length was limited by inclusion of the chain terminator 3'-O-methyl-GTP. Transcription complexes were irradiated with near-UV light (lambda greater than 300 nm) to specifically photoactivate 4-thio-uridine. After photocross-linking, radiolabeled proteins were separated by electrophoresis, blotted onto nitrocellulose, and visualized by autoradiography. Specific photoaffinity labeling of enzyme subunits IIo and IIc was observed. In some experiments, radiolabeled IIa was also detected. Based on the level of photoaffinity labeling of subunits IIo and IIa, relative to their concentration in the transcription reaction, the transcriptional activity of RNA polymerase IIO appears to be greater than 10 times that of IIA. RNA attached to subunits IIo and IIc was estimated to be 16-40 nucleotides in length, whereas RNA attached to subunit IIa was approximately 27-40 nucleotides long. Photoaffinity labeling was sensitive to alpha-amanitin, and required DNA containing an RNA polymerase II promoter.
...
PMID:RNA contacts subunits IIo and IIc in HeLa RNA polymerase II transcription complexes. 242 53

Transcription pausing is a key step in many prokaryotic transcription attenuation mechanisms. Pausing is thought to occur when an RNA hairpin forms near the 3' end of a growing transcript. We report here the isolation of the trp leader paused transcription complex containing a defined 92-nucleotide nascent transcript. Digestion of isolated paused complexes with RNase T1 suggests that the trp leader RNA hairpin designated 1:2 forms in the paused transcription complex. The transcription factor NusA alters the RNase T1 digestion pattern of the 92-nucleotide pause transcript in the complex but not the cleavage patterns of purified pause RNA, suggesting that NusA specifically affects the 1:2 hairpin in the paused transcription complex. The isolated paused transcription complex retains the ability to resume transcription. Kinetic studies on the resumption of elongation suggest that NusA is a non-competitive inhibitor of paused complex release and that the Ks for GTP is around 300 microM. RNA polymerase in the paused transcription complex protects approximately 30 base-pairs on both DNA strands from exonuclease digestion.
...
PMID:Isolation and structural analysis of the Escherichia coli trp leader paused transcription complex. 244 22


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---