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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)

The promoter region of the agarase gene (dagA) of Streptomyces coelicolor A3(2) is complex; it consists of four distinct promoters with different -10 and -35 regions. We report the isolation of a form of RNA polymerase that mediates transcription in vitro from the dagAp4 promoter. The core components of this RNA polymerase are associated with a polypeptide of c. 66 kDa; holoenzyme reconstitution experiments show that the 66 kDa polypeptide functions as a sigma factor that directs transcription from the dagAp4 and Bacillus subtilis veg promoters in vitro. Alignment of the DNA sequences of these two promoters shows that they have bases in common in the -10 and -35 regions and that these sequences are similar to those observed for the major RNA polymerases of other bacteria. N-terminal amino acid sequence analysis of the 66 kDa polypeptide revealed it to be the product of the hrdB gene. Previous experiments showed that the predicted amino acid sequence of the hrdB gene product is very similar to the major sigma factors of other bacteria and suggested that disruption of the hrdB gene is lethal. These observations together lead to the conclusion that we have isolated the major RNA polymerase of Streptomyces coelicolor A3(2). We have developed an improved protocol for the renaturation of sigma factors that have been isolated by preparative sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). This method involves renaturing the polypeptide in the presence of the bacterial chaperonin GroEL. We expect this protocol to find general application for renaturation of other polypeptides that have been subjected to SDS-PAGE.
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PMID:Isolation and characterization of the major vegetative RNA polymerase of Streptomyces coelicolor A3(2); renaturation of a sigma subunit using GroEL. 135 Mar 15

Characterization of the heat shock response in Clostridium acetobutylicum has indicated that at least 15 proteins are induced by a temperature upshift from 30 to 42 degrees C. These so-called heat shock proteins include DnaK and GroEL, two highly conserved molecular chaperones. Several genes encoding heat shock proteins of C. acetobutylicum have been cloned and analysed. The dnaK operon includes the genes orfA (a heat shock gene with an unknown function), grpE, dnaK, and dnaJ; and the groE operon the genes groES and groEL. The hsp18 gene coding for a member of the small heat shock protein family constitutes a monocistronic operon. Interestingly, the heat shock response in this bacterium is regulated by a mechanism, which is obviously different from that found in Escherichia coli. So far, no evidence for a heat shock-specific sigma factor of the RNA polymerase in C. acetobutylicum has been found. In this bacterium, like in many Gram-positive and several Gram-negative bacteria, a conserved inverted repeat is located upstream of chaperone/chaperonin-encoding stress genes such as dnaK and groEL and may be implicated as a cis-acting regulatory site. The inverted repeat is not present in the promoter region of hsp18. Therefore, in C. acetobutylicum there are at least two classes of heat shock genes with respect to the type of regulation. Evidence has been found that a repressor is involved in the regulation of the heat shock response in C. acetobutylicum. However, this regulation seems to be independent of the inverted repeat motif, and the mechanism by which the inverted repeat motif mediates regulation remains to be elucidated.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of heat shock genes in Clostridium acetobutylicum. 757 72

This report is concerned with the structural characterization and genetic regulation of new bacterial groES and groEL chaperonin genes, and presents two novelties. The first is the discovery that the nitrogen fixing soybean root nodule bacterium, Bradyrhizobium japonicum, unlike all other prokaryotes investigated so far, possesses a multigene family consisting of five very similar, though not identical, groESL-like genes. The second novelty relates to the finding that these five homologues are expressed to different degrees and, in particular, that one family member (namely groESL3) is induced by a mechanism that does not involve the well-known heat shock response. By contrast, the groESL3 genes are co-regulated together with symbiotic nitrogen fixation genes, in that they are activated by the nitrogen fixation regulatory protein NifA at low oxygen conditions and transcribed from a -24/-12 promoter by the sigma 54 RNA polymerase. Two other members of the groESL gene family are apparently expressed constitutively at different levels, and yet another one is strongly induced by high temperature. As an attractive hypothesis it follows that B. japonicum may modulate its cellular contents of GroES- and GroEL-like chaperonins in response to specific environmental conditions and physiological needs.
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PMID:One member of a gro-ESL-like chaperonin multigene family in Bradyrhizobium japonicum is co-regulated with symbiotic nitrogen fixation genes. 810 85

We initiated a survey of the Streptococcus pneumoniae genome by DNA sequence sampling. More than 9,500 random DNA sequences of approximately 500 bases average length were determined. Partial sequences sufficient to identify approximately 95% of the aminoacyl tRNA synthetase genes and ribosomal protein (rps) genes were found by comparing the database of partial sequences to known sequences from other organisms. Many genes involved in DNA replication, repair, and mutagenesis are present in S. pneumoniae. Genes for the major subunits of RNA polymerase are also present, as are genes for two alternative sigma factors, rpoD and rpoN. Many genes necessary for amino acid or cofactor biosynthesis and aerobic energy metabolism in other bacteria appear to be absent from the S. pneumoniae genome. A number of genes involved in cell wall biosynthesis and septation were identified, including six homologs to different penicillin binding proteins. Interestingly, four genes involved in the addition of D-alanine to lipoteicoic acid in other gram positive bacteria were found, even though the lipoteicoic acid in S. pneumoniae has not been shown to contain D-alanine. The S. pneumoniae genome contains a number of chaperonin genes similar to those found in other bacteria, but apparently does not contain genes involved in the type III secretion commonly observed in gram negative pathogens. The G+C content of S. pneumoniae genomic DNA is approximately 43 mole percent and the size of the genome is approximately 2.0 Mb as determined by pulsed-field gel electrophoresis. Many of the genes identified by sequence sampling have been physically mapped to the 19 different SmaI fragments derived from the S. pneumoniae genome. The database of random genome sequence tags (GSTs) provides the starting material for determining the complete genome sequence, gene disruption analysis, and comparative genomics to identify novel targets for antibiotic development.
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PMID:DNA sequence sampling of the Streptococcus pneumoniae genome to identify novel targets for antibiotic development. 953 20

Highly purified Escherichia coli RNA polymerase contains a small subunit termed omega that has a molecular mass of 10 105 Da and is comprised of 91 amino acids. E. coli strains lacking omega (omega-less) are viable, but exhibit a slow-growth phenotype. Renaturation of RNA polymerase isolated from an omega-less mutant, in the presence of omega, resulted in maximum recovery of activity. The omega-less RNA polymerase from omega-less strains recruits the chaperonin, GroEL (unlike the wild-type enzyme), suggesting a structural deformity of the mutant enzyme. The GroEL-containing core RNA polymerase interacts efficiently with sigma70 to generate the fully functional holoenzyme. However, when GroEL was removed, the enzyme was irreversibly nonfunctional and was unable to bind to sigma70. The damaged enzyme regained activity after going through a cycle of denaturation and reconstitution in the presence of omega or GroEL. GroES was found to have an inhibitory effect on the core-sigma70 association unlike the omega subunit. The omega subunit may therefore be needed for stabilization of the structure of RNA polymerase.
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PMID:GroEL is involved in activation of Escherichia coli RNA polymerase devoid of the omega subunit in vivo. 1054 69

Atomic force microscopy (AFM) is capable of generating images within ranges of resolution that are of particular interest in biology. Although atomic resolution may not be possible with biological samples, a great deal of information can still be obtained from images that provide structures at a slightly lower level of resolution. The submolecular resolution images of bacteriorhodopsin and the chaperonin GroES, which revealed, respectively, individual loops and beta-turns, confirmed and complemented other structural investigations, while the molecular-level features in images of membrane-bound VacA, a cytotoxin from Helicobacter pylori, immediately suggested the possibility, subsequently proven, of channel-forming ability. A series of images with macromolecular resolution directly provided details on the mechanisms by which RNA polymerase nonspecifically translocates along DNA, and images with subcellular resolving power of erythrocytic cellular membranes showed, with unambiguous clarity, linear arrays of molecular complexes. In this review, we will describe some of the most biologically relevant findings that have been obtained with AFM within ranges of resolution from the submolecular to the molecular, and from the macromolecular to the subcellular. Furthermore, we will describe some of the sample conditions and imaging environments that are likely important to achieve a particular level of resolution.
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PMID:Atomic force microscopy in structural biology: from the subcellular to the submolecular. 1110 27

The vibrational analysis of elastic models suggests that the essential motions of large biomolecular assemblies can be captured efficiently at an intermediate scale without requiring knowledge of the atomic structure. While prior work has established a theoretical foundation for this analysis, we demonstrate here on experimental electron microscopy maps that vibrational modes indeed describe functionally relevant movements of macromolecular machines. The clamp closure in bacterial RNA polymerase, the ratcheting of 30S and 50S subunits of the ribosome, and the dynamic flexibility of chaperonin CCT are extracted directly from single electron microscopy structures at 15-27 A resolution. The striking agreement of the presented results with experimentally observed motions suggests that the motion of the large scale machinery in the cell is surprisingly independent of detailed atomic interactions and can be quite reasonably described as a motion of elastic bodies.
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PMID:Mega-Dalton biomolecular motion captured from electron microscopy reconstructions. 1255 16

Images of entire cells are preceding atomic structures of the separate molecular machines that they contain. The resulting gap in knowledge can be partly bridged by protein-protein interactions, bioinformatics, and electron microscopy. Here we use interactions of known three-dimensional structure to model a large set of yeast complexes, which we also screen by electron microscopy. For 54 of 102 complexes, we obtain at least partial models of interacting subunits. For 29, including the exosome, the chaperonin containing TCP-1, a 3'-messenger RNA degradation complex, and RNA polymerase II, the process suggests atomic details not easily seen by homology, involving the combination of two or more known structures. We also consider interactions between complexes (cross-talk) and use these to construct a structure-based network of molecular machines in the cell.
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PMID:Structure-based assembly of protein complexes in yeast. 1504 3

All archaeal genomes encode RNA polymerase (RNAP) subunits E and F that share a common ancestry with the eukaryotic RNAP subunits A43 and A14 (Pol I), Rpb7 and Rpb4 (Pol II), and C25 and C17 (Pol III). By gene replacement, we have isolated archaeal mutants of Thermococcus kodakarensis with the subunit F-encoding gene (rpoF) deleted, but we were unable to isolate mutants lacking the subunit E-encoding gene (rpoE). Wild-type T. kodakarensis grows at temperatures ranging from 60 degrees C to 100 degrees C, optimally at 85 degrees C, and the DeltarpoF cells grew at the same rate as wild type at 70 degrees C, but much slower and to lower cell densities at 85 degrees C. The abundance of a chaperonin subunit, CpkB, was much reduced in the DeltarpoF strain growing at 85 degrees C and increased expression of cpkB, rpoF or rpoE integrated at a remote site in the genome, using a nutritionally regulated promoter, improved the growth of DeltarpoF cells. RNAP preparations purified from DeltarpoF cells lacked subunit F and also subunit E and a transcription factor TFE that co-purifies with RNAP from wild-type cells, but in vitro, this mutant RNAP exhibited no discernible differences from wild-type RNAP in promoter-dependent transcription, abortive transcript synthesis, transcript elongation or termination.
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PMID:Archaeal RNA polymerase subunits E and F are not required for transcription in vitro, but a Thermococcus kodakarensis mutant lacking subunit F is temperature-sensitive. 1878 48

The four proteins CDK8, cyclin C, Med12, and Med13 can associate with Mediator and are presumed to form a stable "CDK8 subcomplex" in cells. We describe here the isolation and enzymatic activity of the 600-kDa CDK8 subcomplex purified directly from human cells and also via recombinant expression in insect cells. Biochemical analysis of the recombinant CDK8 subcomplex identifies predicted (TFIIH and RNA polymerase II C-terminal domain [Pol II CTD]) and novel (histone H3, Med13, and CDK8 itself) substrates for the CDK8 kinase. Notably, these novel substrates appear to be metazoan-specific. Such diverse targets imply strict regulation of CDK8 kinase activity. Along these lines, we observe that Mediator itself enables CDK8 kinase activity on chromatin, and we identify Med12--but not Med13--to be essential for activating the CDK8 kinase. Moreover, mass spectrometry analysis of the endogenous CDK8 subcomplex reveals several associated factors, including GCN1L1 and the TRiC chaperonin, that may help control its biological function. In support of this, electron microscopy analysis suggests TRiC sequesters the CDK8 subcomplex and kinase assays reveal the endogenous CDK8 subcomplex--unlike the recombinant submodule--is unable to phosphorylate the Pol II CTD.
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PMID:The human CDK8 subcomplex is a histone kinase that requires Med12 for activity and can function independently of mediator. 1904 73


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