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)

Poliovirus RNA polymerase and infectious virus particles were synthesized by translation of virion RNA in vitro in HeLa S10 extracts. The in vitro translation reactions were optimized for the synthesis of the viral proteins found in infected cells and in particular the synthesis of the viral polymerase 3Dpol. There was a linear increase in the amount of labeled protein synthesized during the first 6 h of the reaction. The appearance of 3Dpol in the translation products was delayed because of the additional time required for the proteolytic processing of precursor proteins. 3Dpol was first observed at 1 h in polyacrylamide gels, with significant amounts being detected at 6 h and later. Initial attempts to assay for polymerase activity directly in the translation reaction were not successful. Polymerase activity, however, was easily detected by adding a small amount (3 microliters) of translation products to a standard polymerase assay containing poliovirion RNA. Full-length minus-strand RNA was synthesized in the presence of an oligo(U) primer. In the absence of oligo(U), product RNA about twice the size of virion RNA was synthesized in these reactions. RNA stability studies and plaque assays indicated that a significant fraction of the input virion RNA in the translation reactions was very stable and remained intact for 20 h or more. Plaque assays indicated that infectious virus was synthesized in the in vitro translation reactions. Under optimal conditions, the titer of infectious virus produced in the in vitro translation reactions was greater than 100,000 PFU/ml. Virus was first detected at 6 h and increased to maximum levels by 12 h. Overall, the kinetics of poliovirus replication (protein synthesis, polymerase activity, and virus production) observed in the HeLa S10-initiation factor in vitro translation reactions were similar to those observed in infected cells.
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PMID:Coupled translation and replication of poliovirus RNA in vitro: synthesis of functional 3D polymerase and infectious virus. 838 Apr 67

We used chromosomal walking methods to isolate a 10.8-kb region from the major ribosomal protein (r-protein) gene cluster of Bacillus subtilis (Bs). The gene order in this region, given by gene product, was r-proteins L16-L29-S17-L14-L24-L5-S14-S8-L6-L18-S5-L30-L15-SecY-adenylate kinase (Adk)-methionine aminopeptidase (Map)-initiation factor 1 (IF1)-L36-S13-S11-alpha subunit of RNA polymerase-L17. The region cloned, therefore, contains the homologues for the last three genes of the Escherichia coli (Ec) S10 operon, together with entire spc and alpha operons. This Bs organization differs from the corresponding region in Ec by the inclusion of the genes encoding Adk, Map and IF1 between the genes encoding SecY and L36. Plasmid integration experiments indicated that all 22 genes comprise a single large transcriptional unit controlled from a major promoter which lies upstream from the gene encoding r-protein L16. Promoter probe experiments located lesser activities internal to this large transcriptional unit, the secY and map promoters. The secY promoter region (psecY) contained two activities, each principally functioning in the stationary growth phase when high protein export is required. Thus, the Bs S10-spc-alpha region differs from its Ec counterpart in both genetic and transcriptional organization. Given this difference in transcriptional organization, the mechanisms coordinating expression of the translational apparatus are also likely to differ between Ec and Bs.
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PMID:Genetic and transcriptional organization of the Bacillus subtilis spc-alpha region. 863 44

The structure of a large gene cluster containing 22 ribosomal protein (r-protein) genes of the cyanobacterium Synechococcus sp. strain PCC6301 is presented. Based on DNA and protein sequence analyses, genes encoding r-proteins L3, L4, L23, L2, S19, L22, S3, L16, L29, S17, L14, L24, L5, S8, L6, L18, S5, L15, L36, S13, S11, L17, SecY, adenylate kinase (AK) and the alpha subunit of RNA polymerase were identified. The gene order is similar to that of the E. coli S10, spc and alpha operons. Unlike the corresponding E. coli operons, the genes for r-proteins S4, S10, S14 and L30 are not present in this cluster. The organization of Synechococcus r-protein genes also resembles that of chloroplast (cp) r-protein genes of red and brown algal species. This strongly supports the endosymbiotic theory that the cp genome evolved from an ancient photosynthetic bacterium.
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PMID:Organization of a large gene cluster encoding ribosomal proteins in the cyanobacterium Synechococcus sp. strain PCC 6301: comparison of gene clusters among cyanobacteria, eubacteria and chloroplast genomes. 930 Aug 23

The association of the transcriptional antitermination protein N of bacteriophage lambda with Escherichia coli RNA polymerase depends on nut site RNA (boxA + boxB) in the nascent transcript and the host protein, NusA. This ribonucleoprotein complex can transcribe through Rho-dependent and intrinsic termination sites located up to several hundred base pairs downstream of nut. For antitermination to occur farther downstream, this core antitermination complex must be stabilized by the host proteins NusB, NusG, and ribosomal protein S10. Here, we show that the assembly of NusB, NusG, and S10 onto the core complex involves nucleotides 2-7 of lambda boxA (CGCUCUUACACA) and is a fully cooperative process that depends on the presence of all three proteins. This assembly of NusB, NusG, and S10 also requires the carboxyl-terminal region (amino acids 73-107) of N, which interacts directly with RNA polymerase. NusB and S10 assemble in the absence of NusG when lambda boxA is altered at nucleotides 8 and 9 to create a consensus version of boxA (CGCUCUUUAACA). These experiments suggest that multiple protein-protein and protein-RNA interactions are required to convert a core antitermination complex into a complete complex.
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PMID:Involvement of boxA nucleotides in the formation of a stable ribonucleoprotein complex containing the bacteriophage lambda N protein. 946 9

Forty-one open reading frames (ORFs) were identified in a 32-kb DNA fragment of alkaliphilic Bacillus sp. C-125. A similarity search using the BSORF database found 37 ORFs with significant sequence similarity to B. subtilis RNA polymerase subunits, elongation factor G, elongation factor Tu, and ribosomal proteins. Each ORF product showed more than 70% identity to those of B. subtilis. Gene organization in the region of str, S10, spc, and the alpha cluster was highly conserved among three strains, C-125, B. subtilis, and B. stearothermophilus.
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PMID:Sequence analysis of a 32-kb region including the major ribosomal protein gene clusters from alkaliphilic Bacillus sp. strain C-125. 1019 28

The phylogenetic placement of the Aquifex and Thermotoga lineages has been inferred from (i) the concatenated ribosomal proteins S10, L3, L4, L23, L2, S19, L22, and S3 encoded in the S10 operon (833 aa positions); (ii) the joint sequences of the elongation factors Tu(1alpha) and G(2) coded by the str operon tuf and fus genes (733 aa positions); and (iii) the joint RNA polymerase beta- and beta'-type subunits encoded in the rpoBC operon (1130 aa positions). Phylogenies of r-protein and EF sequences support with moderate (r-proteins) to high statistical confidence (EFs) the placement of the two hyperthermophiles at the base of the bacterial clade in agreement with phylogenies of rRNA sequences. In the more robust EF-based phylogenies, the branching of Aquifex and Thermotoga below the successive bacterial lineages is given at bootstrap proportions of 82% (maximum likelihood; ML) and 85% (maximum parsimony; MP), in contrast to the trees inferred from the separate EF-Tu(1alpha) and EF-G(2) data sets, which lack both resolution and statistical robustness. In the EF analysis MP outperforms ML in discriminating (at the 0.05 level) trees having A. pyrophilus and T. maritima as the most basal lineages from competing alternatives that have (i) mesophiles, or the Thermus genus, as the deepest bacterial radiation and (ii) a monophyletic A. pyrophilus-T. maritima cluster situated at the base of the bacterial clade. RNAP-based phylogenies are equivocal with respect to the Aquifex and Thermotoga placements. The two hyperthermophiles fall basal to all other bacterial phyla when potential artifacts contributed by the compositionally biased and fast-evolving Mycoplasma genitalium and Mycoplasma pneumoniae sequences are eschewed. However, the branching order of the phyla is tenuously supported in ML trees inferred by the exhaustive search method and is unresolved in ML trees inferred by the quartet puzzling algorithm. A rooting of the RNA polymerase-subunit tree at the mycoplasma level seen in both the MP trees and the ML trees reconstructed with suboptimal amino acid substitution models is not supported by the EF-based phylogenies which robustly affiliate mycoplasmas with low-G+C gram-positives and, most probably, reflects a "long branch attraction" artifact.
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PMID:Phylogenetic depth of the bacterial genera Aquifex and Thermotoga inferred from analysis of ribosomal protein, elongation factor, and RNA polymerase subunit sequences. 1079 28

The carboxyl-terminal domain of RNA polymerase II, which is rich in phosphorylation sites, contains 17--52 tandem repeats with the consensus sequence of the heptapeptide, YSPTSPS. The repeat unit of the heptapeptide has two SPXX motifs showing potential beta-turns, SPTS and SPSY. NMR studies were performed in water at pH 4.0 for two cyclic peptides containing one and two repeat units, cyclo-[C(1)R(2)D(3)Y(4)S(5)P(6)T(7)S(8)P(9)S(10)Y(11)S(12)R(13)D(14)C(15)] (peptide 1) and cyclo-[C(1)R(2)D(3)Y(4)S(5)P(6)T(7)S(8)P(9)S(10)Y(11)S(12)P(13)T(14)S(15)P(16)N(17)Y(18)S(19)R(20)D(21)C(22)] (peptide 2), which are cyclized with a disulfide bridge of two Cys residues at the N- and C-termini. SP in 1 and 2 are predominantly in trans form. The following NMR parameters were detected: (1) lower temperature coefficients of amide proton chemical shifts of T7 and S8 in 1, and Tx (T7 or T14), Sx (S8 or S15), Tz (T14 or T7) and Sz (S15 or S8) in 2, (2) significantly large deviation of H(alpha) chemical shifts from its random coil value (Delta H(alpha)) of Pro preceding the Thr (P6 in 1, and Px and Pz in 2), (3) relatively large (3)J(HNH alpha) coupling constants (>8.7 Hz) of T7 in 1 and Tx and Tz in 2, and (4) NOE (d(NN) (i, i+1)) connectivities between the amide protons of T7-S8 and S10-Y11 in 1, and Tx-Sx, S10-Y11, Tz-Sz, and N17-Y18 in 2, although two Pro-Thr-Ser segments in 2 (each of these are annotated by 'x' and 'z') in the first and second repeat units were not distinguishable. Comparison of the NMR parameters between the cyclic peptides and the corresponding linear peptides indicates that cyclization promotes structural stabilization in water. The present NMR data were consistent with the presence of a beta-turn at both SPTS and SPSY: S(5)P(6)T(7)S(8) and S(8)P(9)S(10)Y(11) in 1, and SPxTxSx, SPzTzSz, SP(9)S(10)Y(11), SP(16)N(17)Y(18) in 2. However, the structure of the SPTS segment is more stable than that of the SPSY segment. Conformations consistent with NMR parameters including NOE distances were obtained through molecular dynamics and energy minimization methods. These calculations yielded two stable conformers for the SPTS segment. One of the two corresponds to a type I beta-turn.
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PMID:Structure of the YSPTSPS repeat containing two SPXX motifs in the CTD of RNA polymerase II: NMR studies of cyclic model peptides reveal that the SPTS turn is more stable than SPSY in water. 1145 41

Two conservative gene clusters, the S10 ribosomal protein region and one (of the two) set of rRNA genes, were split in a genome crossover rearrangement event in Mycoplasma gallisepticum. As a result of the rearrangement the major part of the S10 ribosomal protein cluster is located upstream of genes for 23S-5S rRNA (rrn23-5), but the genes infA-rpl36-rps13-rpoA-rpl17 are located immediately downstream of the isolated gene for 16S rRNA (rrn16). A new ribosomal protein cluster infA-rpl36-rps13-rpoA-rpl17-rps16-trmD-rpl19 was formed. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that this ribosomal protein cluster is an operon.
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PMID:Mycoplasma gallisepticum rpoA gene cluster. 1195 50

In bacteria, conditions that uncouple translation from transcription activate intragenic terminators located within cistrons. We analyzed the function of NusA in intragenic termination, making use of two tandem terminators located within the hisG cistron, GTTE1 and GTTE2. GTTE2 is a canonical Rho site, capable to terminate with Rho alone in vitro. By contrast, GTTE1 is a suboptimal terminator, featuring a boxA element and requiring a functional NusB to terminate efficiently in vivo. We found that a functional NusA is necessary for efficient termination events to occur at both GTTE1 and 2. To enhance termination at GTTE1 in conditions in which the transcript is free of ribosomes, NusA acts at the same step as NusB and NusE/S10. In the presence of concomitant translation, termination at GTTE1 is dependent on the relative position of the translation stop codon and boxA. If translation stops upstream of boxA, NusA acts at the same step as NusB enhance termination. Ribosomes terminating translation at boxA influence termination at GTTE1. Interactions of NusA and/or NusB with ribosomal components, including NusE/S10, might facilitate termination. Differently from what observed at GTTE1, the NusA-stimulated pausing seems to be sufficient for the occurrence of complete termination events at GTTE2. A functional NusA is also necessary to prevent premature termination of normally translated transcripts. Our data support the hypothesis that NusA may program a fraction of the RNA polymerase to terminate transcription upon interactions with specific sites on the nascent mRNA and either other Nuses or ribosomes.
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PMID:NusA modulates intragenic termination by different pathways. 1271 96

The 109 amino acid residue Nun protein expressed from prophage HK022 excludes superinfecting phage lambda by arresting transcription on the lambda chromosome near the lambdanut sites. In vitro, the Nun N terminus binds to nascent lambdanutRNA, whereas the C terminus interacts with RNA polymerase and DNA template. Escherichia coli host factors, NusA, NusB, NusE (S10), and NusG, stimulate Nun-arrest. NusA binds the Nun C terminus and enhances formation of the Nun-nutRNA complex. Because of these in vitro activities of NusA, and since a nusA mutation (nusAE136K) blocked Nun in vivo, we assumed that NusA was required for Nun activity. However, using a nusAts strain, we find that NusA is required for termination at nutR but not at nutL. Furthermore, nusAE136K is dominant to nusA(+) for Nun-arrest, both in vitro and in vivo. NusAE136K shows increased affinity for Nun and, unlike NusA(+), can readily be recovered in a ternary complex with Nun and nutRNA. We propose NusAE136K suppresses Nun-arrest when it is a component of the transcription elongation complex, perhaps, in part, by blocking interactions between the Nun C terminus and RNA polymerase and DNA. We also find that in contrast to Nun-arrest, antitermination by lambda N requires NusA.
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PMID:Role of E.coli NusA in phage HK022 Nun-mediated transcription termination. 1663 Nov 97


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