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)

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

Gal4p regulates expression of genes necessary for galactose catabolism in Saccharomyces cerevisiae. We have previously shown that phosphorylation of Gal4p requires both its DNA binding and transcriptional-activation functions and have suggested that phosphorylation occurs as a consequence of interaction with general transcription factors. In this study, we show that phosphorylation occurs rapidly on a limited fraction of overexpressed Gal4p present in a sodium dodecyl sulfate-extractable subcellular fraction while a significant fraction remains stably unphosphorylated. Taken together with our previous observations, we conclude that Gal4p is phosphorylated only if it becomes localized to the nucleus and is capable of both DNA binding and transcriptional activation. We demonstrate that Gal4p is multiply phosphorylated at both the C and N termini, and we identify the precise locations of three sites of phosphorylation at serines 691, 696, and 699. Of these sites, only serine 699 must be phosphorylated for galactose-inducible transcription to occur. Mutation of S-699 to alanine significantly impairs GAL induction by galactose in GAL80+ cells but does not affect transcriptional activation by Gal4p in gal80- cells. In gal80- cells, Gal4p phosphorylation, including that of serine 699, is stimulated by the presence of both galactose and glucose, indicating that phosphorylation at this site is not specifically activated by galactose. Serine 699 phosphorylation requires Gal4p's DNA binding function and is influenced by the function of the RNA polymerase II holoenzyme component Gal11p. These results suggest that a phosphorylation on Gal4p, likely resulting from interaction with the holoenzyme, modulates the induction process by regulating interaction between Gal4p and Gal80p.
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PMID:Phosphorylation of Ga14p at a single C-terminal residue is necessary for galactose-inducible transcription. 875 47

The human La antigen is an RNA-binding protein that facilitates transcriptional termination and reinitiation by RNA polymerase III. Native La protein fractionates into transcriptionally active and inactive forms that are unphosphorylated and phosphorylated at serine 366, respectively, as determined by enzymatic and mass spectrometric analyses. Serine 366 comprises a casein kinase II phosphorylation site that resides within a conserved region in the La proteins from several species. RNA synthesis from isolated transcription complexes is inhibited by casein kinase II-mediated phosphorylation of La serine 366 and is reversible by dephosphorylation. This work demonstrates a novel mechanism of transcriptional control at the level of recycling of stable transcription complexes.
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PMID:Phosphorylation of the human La antigen on serine 366 can regulate recycling of RNA polymerase III transcription complexes. 905 10

The c-Abl tyrosine kinase has been shown to interact with the COOH-terminal repeated domain (CTD) of mammalian RNA polymerase II and can phosphorylate the tyrosine residues in the CTD. Interestingly, the Drosophila or the yeast CTD were not efficiently phosphorylated by the mammalian c-Abl. This species-specificity was found to be determined by the extreme COOH-terminal CTD sequences that are not conserved through evolution. In vitro, COOH-terminal-truncated CTD could neither bind to, nor be phosphorylated by, c-Abl. In vivo, coexpression of a full length CTD prevents c-Abl from inducing the tyrosine phosphorylation of endogenous RNA polymerase II, and such inhibitory effect was not observed with the coexpression of COOH-terminal-truncated CTD. Serine/threonine phosphorylation of the CTD has been linked to the regulation of transcription elongation. Transcription from the human immunodeficiency virus type 1 (HIV-1) promoter requires CTD-phosphorylation, which is stimulated by the viral Tat protein through the recruitment of cellular Ser/Thr CTD kinases. In transient cotransfection experiments, the c-Abl kinase was found to activate the HIV promoter in the absence of Tat. The activation of the HIV promoter required the nuclear localization of c-Abl and could be correlated with increased tyrosine phosphorylation of RNA polymerase II. These observations suggest that tyrosine phosphorylation of the CTD may be functionally equivalent to its serine/threonine phosphorylation in stimulating transcription elongation.
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PMID:Nuclear c-Abl is a COOH-terminal repeated domain (CTD)-tyrosine (CTD)-tyrosine kinase-specific for the mammalian RNA polymerase II: possible role in transcription elongation. 1039

Serine at position 624 of PA subunit of the Influenza A virus RNA polymerase is the active site of a serine protease domain. To examine the role of this protease activity in the viral infection cycle, we compared the growth and the pathogenesis of influenza A/WSN/33 (WSN) and the virus encoding a PA with a S624A mutation (S624A virus), which were generated by the plasmid-based rescue system. The growth of S624A virus was less extensive than that of WSN in cells. The LD50 of S624A virus and WSN for intranasal infection in Balb/C mice was 4.0 x 10(4) and 9.3 x 10(3) PFU, respectively. That for intracranial infection was 460 and 200 PFU, respectively. These data indicated that Ser624, the active site of the serine protease activity of PA, is not essential for viral growth and pathogenesis, but is required for the maximal viral growth.
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PMID:Ser624 of the PA subunit of influenza A virus is not essential for viral growth in cells and mice, but required for the maximal viral growth. 1450 82

The largest subunit of RNA polymerase II contains a unique C-terminal domain important for coupling of transcription and mRNA processing. This domain consists of a repeated heptameric sequence (YSPTSPS) phosphorylated at serines 2 and 5. Serine 5 is phosphorylated during initiation and recruits capping enzyme. Serine 2 is phosphorylated during elongation by the Ctk1 kinase, a protein similar to mammalian Cdk9/P-TEFb. Chromatin immunoprecipitation was used to map positions of transcription elongation and mRNA processing factors in strains lacking Ctk1. Ctk1 is not required for association of elongation factors with transcribing polymerase. However, in ctk1Delta strains, the recruitment of polyadenylation factors to 3' regions of genes is disrupted and changes in 3' ends are seen. Therefore, Serine 2 phosphorylation by Ctk1 recruits factors for cotranscriptional 3' end processing in vivo.
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PMID:Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3' end processing. 1473 95

Fibrobacteres, Chlorobi, and Bacteroidetes (FCB group) comprise three main bacterial phyla recognized on the basis of 16S rRNA trees. Presently, there are no distinctive biochemical or molecular characteristics known that can distinguish these bacteria from other bacterial phyla. The relationship of these bacteria to other phyla is also not known. This review describes many signatures, consisting of defined and conserved inserts in widely distributed proteins, that provide distinctive molecular markers for these groups of bacteria. These signatures serve to clarify the evolutionary relationship between members of the FCB group, and to other bacterial phyla. A 4 aa insert in DNA Gyrase B (GyrB) and a 45 aa insert in the SecA proteins are uniquely shared by various Bacteroidetes species. The insert in GyrB is present in all Bacteroidetes species (>100) covering different orders and families, indicating that it is a distinctive characteristic of the group. Three signatures consisting of an 18 aa insert in ATPase alpha-subunit, an 8-9 aa insert in the FtsK protein and a 1 aa insert in the UvrB protein are commonly shared only by the Bacteroidetes and Chlorobi homologs providing evidence that these two groups are specifically related to each other. Two additional inserts in the RNA polymerase beta'-subunit (5-7 aa) and Serine hydroxymethyl-transferase (14-16 aa), which are commonly present in various Bacteroidetes, Chlorobi, and Fibrobacteres homologs, but not any other bacteria, provide evidence that these groups shared a common ancestor exclusive of all other bacteria. The FCB groups of bacteria are indicated to have diverged from this common ancestor in the following order: Fibrobacteres --> Chlorobi --> Bacteriodetes. The inferences from signature sequences are strongly supported by phylogenetic analyses. These observations suggest that the FCB groups of bacteria should be placed in a single phylum rather than three distinct phyla. Signature sequences in a number of other proteins provide evidence that the FCB group of bacteria diverged at a similar time as the Chlamydiae group, and that the Spirochetes and Aquificales groups are its closest relatives.
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PMID:The phylogeny and signature sequences characteristics of Fibrobacteres, Chlorobi, and Bacteroidetes. 1523 83

rRNA transcription is a fundamental requirement for all cellular growth processes and is activated by the phosphorylation of the upstream binding factor (UBF) in response to growth stimulation. Even though it is well known that phosphorylation of UBF is required for its activation and is a key step in activation of rRNA transcription, as yet, there has been no direct mapping of the UBF phosphorylation sites. The results of the present studies employed sophisticated nano-flow HPLC-microelectrospray-ionization tandem mass spectrometry (nHPLC-muESI-MS/MS) coupled with immobilized metal affinity chromatography (IMAC) and computer database searching algorithms to identify 10 phosphorylation sites on UBF at serines 273, 336, 364, 389, 412, 433, 484, 546, 584, and 638. We then carried out functional analysis of two of these sites, serines 389 and 584. Serine-alanine substitution mutations of 389 (S389A) abrogated rRNA transcription in vitro and in vivo, whereas mutation of serine 584 (S584A) reduced transcription in vivo but not in vitro. In contrast, serine-glutamate mutation of 389 (S389E) restored transcriptional activity. Moreover, S389A abolished UBF-SL1 interaction in vitro, while S389E partially restored UBF-SL1 interaction. Taken together, the results of these studies suggest that growth factor stimulation induces an increase in rRNA transcriptional activity via phosphorylation of UBF at serine 389 in part by facilitating a rate-limiting step in the recruitment of RNA polymerase I: i.e., recruitment of SL1. Moreover, studies provide critical new data regarding multiple additional UBF phosphorylation sites that will require further characterization by the field.
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PMID:Mass spectrometric identification of phosphorylation sites of rRNA transcription factor upstream binding factor. 1718 30

The Microtubule-Associated Serine/Threonine Kinase family (MAST1-4, and MAST-like) is characterised by the presence of a serine/threonine kinase domain and a postsynaptic density protein-95/discs large/zona occludens-1 domain (PDZ). This latter domain gives the MAST family the capacity to scaffold its own kinase activity. In the present study we have profiled the mRNA for each member of the MAST family transcripts across various tissues, with particular focus on rodent brain. Reverse-transcriptase polymerase chain reaction (RT-PCR) has shown equivalent patterns of expression for MAST1 and 2 in multiple tissues. Both MAST3 and 4 show more distinct expression in several tissues, and MAST-like appears to be predominantly expressed in heart and testis. In situ hybridisation reveals overlapping expression of MAST1 and 2 in specific brain regions. In contrast, MAST3 shows selective expression in the striatum and cerebral cortex. MAST4 also exhibits distinct expression in oligodendrocytes of white matter containing brain regions. In keeping with previous results, this family member also shows increased expression in the hippocampus following seizure-like activity. Our analysis of MAST family expression provides support for the role of these kinases in a broad range of neural functions.
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PMID:Expression of the MAST family of serine/threonine kinases. 1820 61

Mycobacterium tuberculosis CRP(Mt), encoded by Rv3676 (crp), is a CRP-like transcription factor that binds with the serC-Rv0885 intergenic region. In the present study, we evaluated CRP(Mt) 's regulation of serC and Rv0885 in M. tuberculosis and M. bovis BCG, using site-specific mutagenesis, promoter fusions and reverse-transcriptase PCR (RT-PCR). The CRP(Mt) binding site was required for full expression of serC and Rv0885, and expression of both genes was reduced in M. tuberculosis and M. bovis BCG crp mutants. These data show that CRP(Mt) binding directly activates both serC and Rv0885 expression. M. tuberculosis serC restored the ability of an Escherichia coli serC mutant to grow in serine-dropout medium, demonstrating that M. tuberculosis serC encodes a phosphoserine aminotransferase. Serine supplementation, or overexpression of serC, accelerated the growth of M. tuberculosis and M. bovis BCG crp mutants in mycomedium, but not within macrophages. These results establish a role for CRP(Mt) in the regulation of amino acid biosynthesis, and show that reduced serine production contributes to the slow-growth phenotype of M. tuberculosis and M. bovis BCG crp mutants in vitro. Restoration of serine biosynthesis by serC expression will facilitate identification of additional CRP(Mt)-regulated factors required by M. tuberculosis during macrophage and host infection.
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PMID:Dysregulation of serine biosynthesis contributes to the growth defect of a Mycobacterium tuberculosis crp mutant. 2190 33


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