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)

Recognition sites of bovine mitochondrial serine tRNA specific for condons AGY [tRNA(Ser) (AGY)] by the cognate mitochondrial seryl-tRNA synthetase were studied using a range of tRNA(Ser)(AGY) variants which were obtained by the in vitro transcription of synthetic tRNA genes with T7 RNA polymerase. Base replacements in the anticodon and discriminator sites did not affect serine acceptance. However, deletion and/or replacement in the T-loop region completely deprived the variants of their charging activities. Point mutation experiments in this region also showed that the adenosine residue in the middle of the T-loop (position 58), which is involved in tertiary interaction between the T-loop and the truncated D-arm [de Bruijn and Klug, 1983] played a significant role in the recognition process by the synthetase.
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PMID:The T-loop region of animal mitochondrial tRNA(Ser)(AGY) is a main recognition site for homologous seryl-tRNA synthetase. 137 35

We have exploited the Escherichia coli lac operator/repressor system as a means to regulate the expression of a mammalian tRNA gene in vivo and in vitro. An oligonucleotide containing a lac operator (lacO) site was cloned immediately upstream of a human serine amber suppressor (Su+) tRNA gene. Insertion of a single lac repressor binding site at position -1 or -32 relative to the coding region had no effect on the amount of functional tRNA made in vivo, as measured by suppression of a nonsense mutation in the E. coli chloramphenicol acetyltransferase gene following cotransfection of mammalian cells. Inclusion of a plasmid expressing the lac repressor in the transfections resulted in 75 to 98% inhibition of suppression activity of lac operator-linked tRNA genes but had no effect on expression of the wild-type gene. Inhibition could be quantitatively relieved with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG). Similarly, transcription in vitro of lac operator-linked tRNA genes in HeLa cell extracts was repressed in the presence of lac repressor, and this inhibition was reversible with IPTG. These results demonstrate that the bacterial lac operator/repressor system can be used to reversibly control the expression of mammalian genes that are transcribed by RNA polymerase III.
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PMID:Regulated expression of a mammalian nonsense suppressor tRNA gene in vivo and in vitro using the lac operator/repressor system. 140 20

A cDNA clone that encodes oryzacystatin, a cysteine protease inhibitor from rice, was isolated and expressed in Escherichia coli BL-21 (DE3) using an expression plasmid under the control of a T7 RNA polymerase promoter. The construct pT7OC 9b encoded a fusion protein containing 11 amino acid residues of the NH2 terminus of the bacterial protein phi 10 and 79 residues of oryzacystatin lacking 23 NH2-terminal residues of the wild-type protein. Recombinant oryzacystatin (ROC) constituted approximately 10% of the total bacterial protein mass and was purified in a single step by anion-exchange chromatography. The inhibitory activity of ROC toward papain (Ki = 3 x 10(-8) M) was comparable with that of the naturally occurring protein isolated from rice. Caseinolytic activity in midgut homogenates from seven species of stored product insects was inhibited from 18 to 85% by ROC, whereas the same activity was inhibited from 14 to 69% by the serine proteinase inhibitor phenylmethylsulfonyl fluoride. Midguts of stored product insects apparently contain both cysteine proteinases and serine proteinases, but the relative amounts vary with the species. When fed to the red flour beetle, Tribolium castaneum, 10 wt% ROC in the diet suppressed growth approximately 35% relative to that of the control group of insects.
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PMID:Rice cystatin: bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein. 142 7

A unique form of nucleoplasmic and cytoplasmic protein glycosylation, O-linked GlcNAc, has previously been detected, using Gal transferase labeling techniques, on a myriad of proteins (for review see Hart, G. W., Haltiwanger, R. S., Holt, G. D., and Kelly, W. G. (1989a) Annu. Rev. Biochem. 58, 841-874), including many RNA polymerase II transcription factors (Jackson, S. P., and Tjian, R. (1988) Cell 55, 125-133). However, virtually nothing is known about the degree of glycosylation at individual sites, or, indeed, the actual sites of attachment of O-GlcNAc on transcription factors. In this paper we provide rigorous evidence for the occurrence and locations of O-GlcNAc on the c-fos transcription factor, serum response factor (SRF), expressed in an insect cell line. Fast atom bombardment mass spectrometry (FAB-MS) of proteolytic digests of SRF provides evidence for the presence of a single substoichiometric O-GlcNAc residue on each of four peptides isolated after sequential cyanogen bromide, tryptic, and proline specific enzyme digestion: these peptides are 306VSASVSP312, 274GTTSTIQTAP283, 313SAVSSADGTVLK324, and 374DSSTDLTQTSSSGTVTLP391. Using an array of techniques, including manual Edman degradation, aminopeptidase, and elastase digestion, together with FAB-MS, the major sites of O-GlcNAc attachment were shown to be serine residues within short tandem repeat regions. The highest level of glycosylation was found on the SSS tandem repeat of peptide (374-391) which is situated within the transcriptional activation domain of SRF. The other glycosylation sites observed in SRF are located in the region of the protein between the DNA binding domain and the transcriptional activation domain. Glycosylation of peptides (274-283) and (313-324) was found to occur on the serine in the TTST tandem repeat and on serine 316 in the SS repeat, respectively. The lowest level of glycosylation was recovered in peptide (306-312) which lacks tandem repeats. All the glycosylation sites identified in SRF are situated in a relatively short region of the primary sequence close to or within the transcriptional activation domain which is distant from the major sites of phosphorylation catalyzed by casein kinase II.
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PMID:Localization of O-GlcNAc modification on the serum response transcription factor. 151 32

A novel penicillin-binding protein (PBP 5*) with D,D-carboxypeptidase activity is synthesized by Bacillus subtilis, beginning at about stage III of sporulation. The complete gene (dacB) for this protein was cloned by immunoscreening of an expression vector library and then sequenced. The identity of dacB was verified not only by the size and cross-reactivity of its product but also by the presence of the nucleotide sequence that coded for the independently determined NH2 terminus of PBP 5*. Analysis of its complete amino acid sequence confirmed the hypothesis that this PBP is related to other active-site serine D,D-peptidases involved in bacterial cell wall metabolism. PBP 5* had the active-site domains common to all PBPs, as well as a cleavable amino-terminal signal peptide and a carboxy-terminal membrane anchor that are typical features of low-molecular-weight PBPs. Mature PBP 5* was 355 amino acids long, and its mass was calculated to be 40,057 daltons. What is unique about this PBP is that it is developmentally regulated. Analysis of the sequence provided support for the hypothesis that the sporulation specificity and mother cell-specific expression of dacB can be attributed to recognition of the gene by a sporulation-specific sigma factor. There was a good match of the putative promoter of dacB with the sequence recognized by sigma factor E (sigma E), the subunit of RNA polymerase that is responsible for early mother cell-specific gene expression during sporulation. Analysis of PBP 5* production by various spo mutants also suggested that dacB expression is on a sigma E-dependent pathway.
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PMID:Isolation and sequence analysis of dacB, which encodes a sporulation-specific penicillin-binding protein in Bacillus subtilis. 154 23

Three serine kinases which phosphorylate the CTD of RNA polymerase II have been identified in Aspergillus nidulans. The kinases (KI, KII, KIII) were identified using a synthetic peptide containing four copies of the CTD consensus heptamer repeat, and differ in chromatographic behavior, and apparent molecular mass (KI approximately 60kDa; KII approximately 82kDa; KIII approximately 43 kDa). KIII utilized, in addition to peptide, histone H1 as substrate, whereas casein was not phosphorylated by any of the three kinases. The kinases appear to be unrelated to the p34cdc2 kinase, as judged by Western blot analysis and the position of serine phosphorylation of the synthetic CTD peptide. KI was highly purified and renaturation experiments have shown that it consists of a single polypeptide of 57 kDa. KI also phosphorylated RNA polymerase II associated in a preinitiation complex.
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PMID:Protein kinases from Aspergillus nidulans that phosphorylate the carboxyl-terminal domain of the largest subunit of RNA polymerase II. 155 38

The phosphorylation, DNA-binding and dimerization properties of both forms of the RNA polymerase I transcription factor UBF were studied and compared. Tryptic peptide maps of in vivo 32P-labeled UBF contained four phospho-peptides. Two of these peptides are predicted to derive from the serine-rich, carboxyl-terminal of UBF. This region contains nine consensus phosphorylation sites for casein kinase II, and is one of the regions phosphorylated in vitro by casein kinase II. Analysis of the DNA-binding properties of recombinant forms of UBF1 and UBF2 by Southwestern blots revealed: (1) a role for the NH2-terminal 102 amino acid domain of UBF1/UBF2 in DNA-binding; (2) the importance of the bases from -106 to -101 of the rat ribosomal DNA promoter for the binding of UBF; and (3) functional differences between UBF1 and UBF2. Glutaraldehyde cross-linking and overlay assays using recombinant forms of UBF1 and UBF2 demonstrated that the molecules can form both homodimers and heterodimers. These assays also demonstrated that the NH2-terminal 102 amino acids of UBF plays a significant role in dimerization and that other domains contribute to dimerization. The dimerization properties of recombinant forms of UBF1 and UBF2 were different, suggesting that the HMG box 2 of UBF1, which is partially deleted in UBF2, also contributes to UBF dimerization.
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PMID:Analysis of the phosphorylation, DNA-binding and dimerization properties of the RNA polymerase I transcription factors UBF1 and UBF2. 156 Oct 86

We demonstrate that four different proteins from calf thymus are able to restore splicing in the same splicing-deficient extract using several different pre-mRNA substrates. These proteins are members of a conserved family of proteins recognized by a monoclonal antibody that binds to active sites of RNA polymerase II transcription. We purified this family of nuclear phosphoproteins to apparent homogeneity by two salt precipitations. The family, called SR proteins for their serine- and arginine-rich carboxy-terminal domains, consists of at least five different proteins with molecular masses of 20, 30, 40, 55, and 75 kD. Microsequencing revealed that they are related but not identical. In four of the family members a repeated protein sequence that encompasses an RNA recognition motif was observed. We discuss the potential role of this highly conserved, functionally related set of proteins in pre-mRNA splicing.
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PMID:SR proteins: a conserved family of pre-mRNA splicing factors. 157 77

UBF is a DNA binding protein which interacts with both the promoter and the enhancer of various vertebrate ribosomal RNA genes and functions as a transcription initiation factor for RNA polymerase I (pol I). We have purified murine UBF to apparent molecular homogeneity and demonstrate that its transactivating potential, but not its DNA binding activity, is modulated in response to cell growth. In vivo labelling experiments demonstrate that UBF is a phosphoprotein and that the phosphorylation state is different in growing and quiescent cells. We show that UBF is phosphorylated in vitro by a cellular protein kinase which by several criteria closely resembles casein kinase II (CKII). A major modification involves serine phosphoesterifications in the carboxy terminal hyperacidic tail of UBF. Deletions of this C-terminal domain severely decreases the UBF directed activation of transcription. The data suggest that phosphorylation of UBF by CKII may play an important role in growth dependent control of rRNA synthesis.
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PMID:The nucleolar transcription factor mUBF is phosphorylated by casein kinase II in the C-terminal hyperacidic tail which is essential for transactivation. 160 Sep 46

RNA-dependent RNA polymerases contain a highly conserved region of amino acids with a core segment composed of the amino acids YGDD which have been hypothesized to be at or near the catalytic active site of the molecule. Six mutations in this conserved YGDD region of the poliovirus RNA-dependent RNA polymerase were made by using oligonucleotide site-directed DNA mutagenesis of the poliovirus cDNA to substitute A, C, M, P, S, or V for the amino acid G. The mutant polymerase genes were expressed in Escherichia coli, and the purified RNA polymerases were tested for in vitro enzyme activity. Two of the mutant RNA polymerases (those in which the glycine residue was replaced with alanine or serine) exhibited in vitro enzymatic activity ranging from 5 to 20% of wild-type activity, while the remaining mutant RNA polymerases were inactive. Alterations in the in vitro reaction conditions by modification of temperature, metal ion concentration, or pH resulted in no significant differences in the activities of the mutant RNA polymerases relative to that of the wild-type enzyme. An antipeptide antibody directed against the wild-type core amino acid segment containing the YGDD region of the poliovirus polymerase reacted with the wild-type recombinant RNA polymerase and to a limited extent with the two enzymatically active mutant polymerases; the antipeptide antibody did not react with the mutant RNA polymerases which did not have in vitro enzyme activity. These results are discussed in the context of secondary-structure predictions for the core segment containing the conserved YGDD amino acids in the poliovirus RNA polymerase.
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PMID:Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif. 165 2


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