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)

In Streptomyces coelicolor A3(2), two genes, tuf1 and tuf3, encode the apparent polypeptide chain elongation factors EF-Tu1 and EF-Tu3, respectively. While tuf1 appears to code for the major EF-Tu, the function of tuf3 is unknown. To assess the role of EF-Tu3, tuf3 was subjected to mutational and transcriptional analyses. Replacement of the 5'-half of tuf3 by an antibiotic resistance cassette had no detectable effect on phenotype, indicating that tuf3 is not essential for growth or differentiation. The transcription start site of tuf3 was located approximately 195 nt upstream of the translation start site. The sequence of the tuf3 promoter (Ptuf3) resembles the consensus for the major class of eubacterial promoters, and Ptuf3 was recognized preferentially by an RNA polymerase fraction enriched in sigma hrdB, the principal sigma factor of S. coelicolor. Nuclease S1 mapping failed to reveal tuf3 transcripts during growth of S. coelicolor in liquid culture, consistent with the apparent absence of EF-Tu3 in total protein extracts of the same strain. However, tuf3 transcription was observed in cultures of S. coelicolor M145 shortly after nutritional shiftdown (which resulted in the disappearance of tuf1 transcripts) and after addition of serine hydroxamate, both of which induce the stringent response. Transcription of tuf3 was also observed in transition-phase and stationary-phase cultures of S. coelicolor J1681, a strain deleted for bldA (which specifies a tRNA(Leu) for the rare leucine codon UUA). In all of these examples, transcription of tuf3 followed the production of ppGpp, consistent with the hypothesis that tuf3 is subject to positive stringent control.
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PMID:The tuf3 gene of Streptomyces coelicolor A3(2) encodes an inessential elongation factor Tu that is apparently subject to positive stringent control. 758 11

Dimers of P22 Arc repressor bind to half-sites of the 21 bp arc operator and interact cooperatively to stabilize a DNA-bound tetramer. Mutation of Ser35 (a residue in the dimer-dimer interface) to Arg or Leu disrupts cooperative binding. The mutant proteins have near wild-type stabilities, give operator footprints like wild-type, and prevent binding of RNA polymerase to the Pant promoter in vitro. These mutants are, however, largely inactive in vivo. Thus, although cooperativity is not structurally required for repression, it appears that the additional DNA-binding energy from dimer-dimer cooperativity is required for normal biological function. Altering the spacing between the DNA half-sites by even one base-pair eliminates dimer-dimer cooperativity, indicating that Arc dimers need to be oriented correctly by half-site binding to allow the interactions that stabilize the tetrameric complex.
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PMID:P22 Arc repressor: role of cooperativity in repression and binding to operators with altered half-site spacing. 760 85

Our long-term goal is to define the catalytic domains of the L protein subunit of the Sendai virus RNA polymerase. An aberrant polyadenylation phenotype in the vesicular stomatitis virus tsG16 L protein mutant has recently been identified as a phenylalanine to serine change at amino acid 1488 (Hunt and Hutchinson, Virology 193, 786-793, 1993). To test if functional domains are conserved in the L proteins of negative-strand RNA viruses, we attempted to create a similar polyadenylation defect in the Sendai virus L protein. Nine different amino acid substitutions at the analogous site in the Sendai L protein (cysteine at amino acid 1571) were constructed by site-directed mutagenesis of the gene. Each mutant L protein was synthesized and bound to the Sendai P protein to form the P-L polymerase complex. While none of these L mutants exhibited a change in polyadenylation, the single amino acid changes yielded a variety of activities in vitro. Mutants containing valine, leucine, or phenylalanine at amino acid 1571, amino acids found naturally in the L proteins of other paramyxoviruses, yielded polymerases that had biological activity equal to or better than the wild-type (WT) polymerase. Serine or threonine substitutions in the L protein at this position also resulted in polymerases with nearly WT synthetic activity. In contrast, a glycine substitution significantly decreased overall polymerase activity, whereas a tyrosine substitution gave decreased transcription, but virtually no DI genome replication in vitro. The tyrosine-substituted polymerase may be unable to carry out the packaging step of replication, since DI leader RNA synthesis was normal in this mutant. Mutant L proteins with basic arginine or histidine substitutions were inactive in all viral RNA synthesis in vitro, although the polymerase complexes could bind the nucleocapsid template.
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PMID:Alternative amino acids at a single site in the Sendai virus L protein produce multiple defects in RNA synthesis in vitro. 764 61

The gene on chromosome 10 at band p12 (AF10), involved in the t(10;11) translocation in acute myeloid leukemia, has been identified and shown to contain conserved zinc finger and leucine zipper domains. These regions are highly homologous to the equivalent regions on AF17, the gene involved in the t(11;17) translocations. A series of adult, childhood, and infant leukemias with either simple or complex versions of the t(10;11) has been examined by Southern analysis and shown to involve rearrangement to the HRX locus. Reverse transcriptase-polymerase chain reaction from either bone marrow or peripheral blood cells showed that HRX sequence was fused to AF10 sequence in all 8 cases and subsequent sequence analysis showed an in-frame fusion between the HRX and AF10 sequence. A consistent feature of these fusions was the juxtaposition of the leucine dimerization motif of AF10 onto the NH2-terminal region of HRX. The published data suggest that a similar conclusion can be drawn about the t(11;17) translocation, implying a critical role for this motif in the chimaeric HRX protein.
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PMID:The t(10;11) translocation in acute myeloid leukemia (M5) consistently fuses the leucine zipper motif of AF10 onto the HRX gene. 766 54

The cDNA of a small subunit (hRPB14.4) of RNA polymerase II (or B) from HeLa cells has been cloned. A 127 residue peptide sequence (calculated molecular weight of 14,478; isoelectric point of 3.7) was deduced and compared to that of the homologous subunit of Saccharomyces cerevisiae polymerase (ABC23, encoded by the RPB6/RPO26 gene). About 50% of the total residues were found to be conserved between yeast and man, with the C-terminal two-third being the most conserved (72% identity). A putative leucine-zipper comprising four properly spaced leucine residues, but not preceded by a basic domain, was identified near the C-terminal end of both proteins.
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PMID:A 14.4 KDa acidic subunit of human RNA polymerase II with a putative leucine-zipper. 780 19

Rodent BC1 RNA represents the first example of a neural cell-specific RNA polymerase III (Pol III) transcription product. By developing a rat brain in vitro system capable of supporting Pol III-directed transcription, we showed that the rat BC1 RNA intragenic promoter elements, comprising an A box element and a variant B box element, as well as its upstream region, containing octamer-binding consensus sequences and functional TATA and proximal sequence element sites, are necessary for transcription. The BC1 B box, lacking the invariant A residue found in the consensus B boxes of tRNAs, represents a functionally related and possibly distinct promoter element. The transcriptional activity of the BC1 B box element is greatly increased, in both a BC1 RNA and a chimeric tRNA(Leu) gene construct, when the BC1 5' flanking region is present and is appropriately spaced. Moreover, a tRNA consensus B-box sequence can efficiently replace the BC1 B box only if the BC1 upstream region is removed. These interactions, identified only in a homologous in vitro system, between upstream Pol II and intragenic Pol III promoters suggest a mechanism by which the tissue-specific BC1 RNA gene and possibly other Pol III-transcribed genes can be regulated.
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PMID:BC1 RNA: transcriptional analysis of a neural cell-specific RNA polymerase III transcript. 786 55

The ada gene of Escherichia coli K-12 encodes the 39-kDa Ada protein, which consists of two domains joined by a hinge region that is sensitive to proteolytic cleavage in vitro. The amino-terminal domain has a DNA methyltransferase activity that repairs the S-diastereoisomer of methylphosphotriesters while the carboxyl-terminal domain has a DNA methyltransferase activity that repairs O6-methylguanine and O4-methylthymine lesions. Transfer of a methyl group to Cys-69 by repair of a methylphosphotriester lesion converts Ada into a transcriptional activator of the ada and alkA genes. Activation of ada, but not alkA, requires elements contained within the carboxyl-terminal domain of Ada. In addition, physiologically relevant concentrations of the unmethylated form of Ada specifically inhibit methylated Ada-promoted ada transcription both in vitro and in vivo and it has been suggested that this phenomenon plays a pivotal role in the down-regulation of the adaptive response. A set of site-directed mutations were generated within the hinge region, changing the lysine residue at position 178 to leucine, valine, glycine, tyrosine, arginine, cysteine, proline, and serine. All eight mutant proteins have deficiencies in their ability to activate ada transcription in the presence or absence of a methylating agent but are proficient in alkA activation. AdaK178P (lysine 178 changed to proline) is completely defective for the transcriptional activation function of ada while it is completely proficient for transcriptional activation of alkA. In addition, AdaK178P possesses both classes of DNA repair activities both in vitro and in vivo. Transcriptional activation of ada does not occur if both the amino- and carboxyl-terminal domains are produced separately within the same cell. The mutation at position 178 might interfere with activation of ada transcription by changing a critical contact with RNA polymerase, by causing a conformational change of Ada, or by interfering with the communication of conformational information between the amino- and the carboxyl-terminal domains. These results indicate that the hinge region of Ada is important for ada but not alkA transcription and further support the notion that the mechanism(s) by which Ada activates ada transcription differs from that by which it activates transcription at alkA.
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PMID:Alteration of lysine 178 in the hinge region of the Escherichia coli ada protein interferes with activation of ada, but not alkA, transcription. 786 1

We report the identification of three new alpha-amanitin resistance mutations in the gene encoding the largest subunit of mouse RNA polymerase II (RPII215). These mutations are clustered in a region of the largest subunit that is important for transcription elongation. This same domain has been identified as the site of alpha-amanitin resistance mutations in both Drosophila and Caenarhabditis elegans. The sequences encompassing this cluster of mutations are highly conserved among RNA polymerase II genes from a number of species, including those that are naturally more resistant to alpha-amanitin suggesting that this region of the largest subunit is critical for a conserved catalytic function. The mutations reported here change leucine 745 to phenylalanine, arginine 749 to proline, or isoleucine 779 to phenylalanine. Together with the previously reported asparagine 792 to aspartate substitution these mutations define a potential alpha-amanitin binding pocket in a region of the mouse subunit that could be involved in translocation of polymerase during elongation.
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PMID:Clustered alpha-amanitin resistance mutations in mouse. 789 49

A novel human homeobox gene, HB9, was isolated from a cDNA library prepared from in vitro stimulated human tonsil B lymphocytes and from a human genomic library. The HB9 gene is composed of 3 exons spread over 6 kilobases of DNA. An open reading frame of 1206 nucleotides is in frame with a diverged homeodomain. The predicted HB9 protein has a molecular mass of 41 kilodaltons and is enriched for alanine, glycine, and leucine. The HB9 homeodomain is most similar to that of the Drosophila melanogaster homeobox gene proboscipedia. Northern blot analysis of poly(A) RNA purified from the human B cell line RPMI 8226 and from activated T cells revealed a major mRNA transcript of 2.2 kilobases. Similar analysis of poly(A) RNA from a variety of adult tissues demonstrated HB9 transcripts in pancreas, small intestine, and colon. Reverse transcriptase-polymerase chain reaction was used to examine HB9 RNA transcripts in hematopoietic cell lines. HB9 RNA transcripts were most prevalent in several human B cell lines and K562 cells. In addition, transcripts were detected in RNA prepared from tonsil B cells and in situ hybridization studies localized them in the germinal center region of adult tonsil. These findings suggest the involvement of HB9 in regulating gene transcription in lymphoid and pancreatic tissues.
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PMID:A novel human homeobox gene distantly related to proboscipedia is expressed in lymphoid and pancreatic tissues. 791 94

The bacteriophage P2 ogr gene product is a positive regulator of transcription from P2 late promoters. The ogr gene was originally defined by compensatory mutations that overcame the block to P2 growth imposed by a host mutation, rpoA109, in the gene encoding the alpha subunit of RNA polymerase. DNA sequence analysis has confirmed that this mutation affects the C-terminal region of the alpha subunit, changing a leucine residue at position 290 to a histidine (rpoAL290H). We have employed a reporter plasmid system to screen other, previously described, rpoA mutants for effects on activation of a P2 late promoter and have identified a second allele, rpoA155, that blocks P2 late transcription. This mutation lies just upstream of rpoAL290H, changing the leucine residue at position 289 to a phenylalanine (rpoAL289F). The effect of the rpoAL289F mutation is not suppressed by the rpoAL290H-compensatory P2 ogr mutation. P2 ogr mutants that overcome the block imposed by rpoAL289F were isolated and characterized. Our results are consistent with a direct interaction between Ogr and the alpha subunit of RNA polymerase and support a model in which transcription factor contact sites within the C terminus of alpha are discrete and tightly clustered.
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PMID:Mutations affecting two adjacent amino acid residues in the alpha subunit of RNA polymerase block transcriptional activation by the bacteriophage P2 Ogr protein. 800 64


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