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)

Histone-DNA templates for bacteriophage T7 RNA polymerase were assembled from a plasmid containing a promoter and a terminator for T7 RNA polymerase, intact (H3.H4)2 tetramers, and either untreated or chemically acetylated H2A.H2B dimers. The nucleosomal particles containing acetylated H2A.H2B dimers protect 145 base pairs of DNA against micrococcal nuclease digestion and prevent the reaction with psoralen of 80 to 145 DNA base pairs. The inhibition of transcriptional initiation caused by the association of DNA with intact core histone octamers decreases significantly when the histone octamers contain acetylated H2A.H2B dimers. These results suggest a role for H2A.H2B dimers in the control of transcription, which might be mediated through acetylation and deacetylation of their lysine residues.
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PMID:Acetylation of histone H2A.H2B dimers facilitates transcription. 775 16

The DNA-binding protein MetR belongs to the LysR family of transcriptional activators and is required for expression of the metE and metH promoters in Escherichia coli. However, it is not known if this activation is mediated by a direct interaction of MetR with RNA polymerase. In a search for RNA polymerase mutants defective in MetR-mediated activation of the metE gene, we isolated a mutation in the alpha subunit of RNA polymerase that decreases metE expression independently of the MetR protein. The mutation does not affect expression from the metH promoter, suggesting that the alpha subunit of RNA polymerase interacts differently at these two promoters. The mutation was mapped to codon 261 of the rpoA gene, resulting in a change from a glutamic acid residue to a lysine residue. Growth of the mutant is severely impaired in minimal medium even when supplemented with methionine and related amino acids, indicating a pleiotropic effect on gene expression. This rpoA mutation may identify either a site of contact with an as yet unidentified activator protein for metE expression or a site of involvement by the alpha subunit in sequence-specific recognition of the metE promoter.
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PMID:A mutation in the rpoA gene encoding the alpha subunit of RNA polymerase that affects metE-metR transcription in Escherichia coli. 783 82

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

Although the TATA-binding protein (TBP) is highly conserved throughout the eukaryotic kingdom, human TBP cannot functionally replace yeast TBP for cell viability. To investigate the basis of this species specificity, we examine the in vivo transcriptional activity of human TBP at different classes of yeast promoters. Consistent with previous results, analysis of yeast/human hybrid TBPs indicates that growth defects are not correlated with the ability to promote TATA-dependent polymerase II (Pol II) transcription or to respond to acidic activator proteins. Human TBP partially complements the growth defects of a yeast TBP mutant with altered TATA element-binding specificity, suggesting that it carries out sufficient Pol II function to support viability. However, human TBP does not complement the defects of yeast TBP mutants that are specifically defective in transcription by RNA polymerase III. Three independently isolated derivatives of human TBP that permit yeast cell growth replace arginine 231 with lysine; the corresponding amino acid in yeast TBP (lysine 133) has been implicated in RNA polymerase III transcription. Transcriptional analysis indicates that human TBP functions poorly at promoters recognized by RNA polymerases I and III and at RNA Pol II promoters lacking a conventional TATA element. These observations suggest that species specificity of TBP primarily reflects evolutionarily diverged interactions with TBP-associated factors (TAFs) that are necessary for recruitment to promoters lacking TATA elements.
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PMID:Conserved and nonconserved functions of the yeast and human TATA-binding proteins. 792 34

Dermal fibroblasts from a Chinese Ehlers-Danlos syndrome type VII patient synthesized approximately equal amounts of normal pro-alpha 2(I) chains of type I procollagen and abnormal ones with electrophoretic mobility of pN alpha 2(I) chains, in which the amino-propeptide (N-propeptide) was retained. Reverse-transcriptase PCR analysis of the proband's RNA showed outsplicing of the 54 base exon 6 in half of the pro-alpha 2(I) mRNAs. Exon 6 encodes 18 amino acids of the N-telopeptide which contains the procollagen N-proteinase cleavage site and a cross-link precursor lysine. Loss of these sequences would result in failure to cleave the amino-propeptide of pro-alpha 2(I) and the accumulation of pN-alpha 2(I) chains. Nucleotide sequencing analyses of the proband's COL1A2 gene showed the presence of a T to C transition at position +2 of intron 6 in one allele and the proband is heterozygous for the defect. This mutation which destroyed the consensus GT dinucleotide at the 5' splice donor site of the intron is responsible for the loss of exon 6 by exon skipping. Electron microscopic analysis of the patient's dermis showed the presence of abnormal collagen I fibrils of irregular diameter and circularity. This mutation in COL1A2 in an EDS VII patient is the first reported case in the Chinese population and is identical to one reported for another EDS-VII (Libyan) patient. The occurrence of an identical mutation in two probands of different ethnic origin is direct evidence that the mutant genotype is the cause of the EDS VII phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Further evidence that the failure to cleave the aminopropeptide of type I procollagen is the cause of Ehlers-Danlos syndrome type VII. 808 89

Late transcription of bacteriophage Mu initiates at four promoters, P(lys), PI, PP and Pmom, and requires the Mu C protein and the host RNA polymerase. Promoter-containing DNA fragments extending approximately 200 bp upstream and downstream of the 5' starts of the lys, I and P transcripts were cloned into a multicopy lacZ-expression plasmid. Promoter activity, assayed by beta-galactosidase expression, was determined under two different conditions: (1) with C provided from a compatible plasmid in the absence of other Mu factors and (2) with C provided from an induced Mu prophage. beta-galactosidase activities were greatest for P(lys), intermediate for PI, and lowest for PP. Similar analysis of plasmids containing nested sets of deletions removing 5' or 3' sequences of P(lys) demonstrated that a 68-bp region was sufficient for full activity. Point mutations were generated within the 68-bp region by mutagenic oligonucleotide-directed PCR (Mod-PCR). Properties of the lys promoter mutants indicated that, in addition to the -10 region, a 19-bp region from -52 to -34 containing the C footprint is required for C-dependent promoter activity.
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PMID:Mutational analysis of a C-dependent late promoter of bacteriophage Mu. 829 68

The carboxy-terminal domain (CTD) of RNA polymerase II consists of multiple repeats of the unique heptad sequence -(Ser-Pro-Thr-Ser-Pro-Ser-Tyr)- which may interact with DNA through the intercalation of adjacent tyrosine aromatic rings. We have examined details of the interaction of this motif with calf thymus DNA through analysis of peptide analogues that contain (1) an amino-terminal tyrosine which mimics the presence of an adjacent heptad repeat and (2) positively-charged lysine residues which facilitate the initial contact between peptide and DNA. Results of fluorescence experiments, NMR titrations, and viscometric analyses indicate that these peptides bind to the DNA helix through a non-classical intercalation mode involving partial aromatic stacking of the tyrosine rings with the Watson-Crick base pairs.
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PMID:Aromatic stacking and bending of the DNA helix by the individual repeat units of the carboxy-terminal domain of RNA polymerase II. 829 83

Dipicolinic acid (DPA) is a small polar molecule that accumulates to high concentrations in bacterial endospores, and is thought to play a role in spore heat resistance, or the maintenance of heat resistance. Previous work has shown that mutations in the spoVF locus of Bacillus subtilis prevent the formation of DPA, and give rise to heat-sensitive spores. Addition of exogenous DPA during spore development led to the restoration of heat resistance. This suggested that the spoVF locus encoded dipicolinic acid synthetase, the enzyme thought to catalyse the single reaction needed to synthesise DPA from dihydroxydipicolinic acid, an intermediate in the lysine biosynthetic pathway. We have now cloned and sequenced the spoVF locus of Bacillus subtilis and show that it comprises two coordinately regulated genes, now designated dpaA and dpaB. Expression of fragments of the dpa operon in Escherichia coli has shown that the two gene products together specify DPA synthetase activity. The promoter of the dpa operon, which lies just upstream of the first gene, has been identified by primer extension analysis. Sequences in this region show strong sequence similarity to several promoters recognized by the sigma K form of RNA polymerase. Transcription from this promoter was detected four hours after the onset of sporulation, at about the same time that sigma K activity is known to appear. Furthermore, transcription was abolished by mutations in a series of genes that are known to be required for the synthesis of active sigma K. These results are in accordance with previous work indicating that DPA synthetase activity was present only during the late stages of sporulation and specifically in the mother cell compartment. Transcription was enhanced by a gerE mutation, indicating that, like the previously described cotA gene, spoVF is negatively regulated by GerE. The mother-cell-specific synthesis of an enzyme responsible for a compound that accumulates to high concentrations in the prespore raises interesting questions about intercellular transport mechanisms.
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PMID:Cloning, DNA sequence, functional analysis and transcriptional regulation of the genes encoding dipicolinic acid synthetase required for sporulation in Bacillus subtilis. 834 20

The compatible plasmids pKGP1-1 and pCM-X# will confer chloramphenicol resistance to Escherichia coli harboring the two plasmids if the T7 RNA polymerase produced from pKGP1-1 can recognize the T7 promoter carried on pCM-X# and transcribe the CAT gene that is cloned behind the promoter [Ikeda et al. (1992) Biochemistry 31, 9073-9080]. When E. coli harbor pKGP1-1 and a pCM-X# plasmid that carries a point mutation in the T7 promoter that destroys promoter activity (an inactive pCM-X#), the T7 RNA polymerase will not utilize the T7 promoter point mutant, will not produce CAT, and will not induce chloramphenicol resistance. The selection of mutants of T7 RNA polymerase that exhibit altered promoter recognition was pursued by randomly mutagenizing pKGP1-1 with aqueous hydroxylamine, cotransforming E. coli with the mutagenized pKGP1-1 and a mixture of seven different inactive pCM-X# plasmids, and isolating and characterizing the RNA polymerase that was present in those colonies that exhibited chloramphenicol resistance. It was established that E. coli harboring the mutant plasmid pKGP-HA1mut4 and an inactive pCM-X# are chloramphenicol-resistant and that the mutation responsible for the expression of CAT from the inactive pCM-X# plasmid is a G to A transition at nucleotide 664 of T7 gene 1 that converts glutamic acid (222) to lysine. Apparently this mutation expands the range of T7 promoter sequences that can be utilized by the enzyme. The mutant T7 RNA polymerase, GP1(Lys222), utilizes all seven inactive T7 promoter point mutants more efficiently than wild-type T7 RNA polymerase both in vivo and in vitro. Furthermore, the correlation of in vivo and in vitro promoter utilization suggests that the restoration of chloramphenicol resistance in the cotransformed E. coli results from the ability of GP1(Lys222) to initiate transcription from T7 promoter point mutants that are normally inactive.
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PMID:Selection and characterization of a mutant T7 RNA polymerase that recognizes an expanded range of T7 promoter-like sequences. 836 83

The last enzymatic step for L-cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL, EC 4.2.99.8) which synthesizes L-cysteine from O-acetylserine and "sulfide." We have isolated and characterized a full-length cDNA (1432 bp) from a lambda gt11 library of spinach leaf encoding the complete precursor of the chloroplast isoform. The 1149-nucleotide open reading frame coding for O-acetylserine(thiol)lyase was in the direction opposite that of the lambda gt11 beta-galactosidase gene. The derived amino acid sequence indicates that the protein precursor consists of 383 amino acid residues including a N-terminal presequence peptide of 52 residues. The amino acid sequence of mature spinach chloroplast O-acetylserine(thiol)lyase shows 40 and 57% homology with its bacterial counterparts. Sequence comparison with several pyridoxal 5'-phosphate-containing proteins reveals the presence of a lysine residue assumed to be involved in cofactor binding. A synthetic cDNA was constructed, coding for the entire 331-amino-acid mature O-acetylserine(thiol)lyase and for an initiating methionine. A high level of expression of the active mature chloroplast isoform was achieved in an Escherichia coli strain carrying the T7 RNA polymerase system (F. W. Studier, A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff, 1990, in Methods in Enzymology, D. V. Goeddel, Ed., Vol. 185, pp. 60-89, Academic Press, San Diego, CA). Addition of pyridoxine to the bacterial growth medium enhanced the enzyme activity due to the recombinant protein. The extent of production is 25-fold higher than in chloroplast from spinach leaves and the recombinant protein presents the relative molecular mass and immunological properties of the natural enzyme from spinach leaf chloroplast. This work, together with our previous biochemical studies, are in accordance with a prokaryotic type enzyme for L-cysteine biosynthesis in higher plant chloroplasts. Southern blot analysis indicated that O-acetylserine(thiol)lyase is encoded by multiple genes in the spinach leaf genomic DNA.
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PMID:O-acetylserine(thiol)lyase from spinach (Spinacia oleracea L.) leaf: cDNA cloning, characterization, and overexpression in Escherichia coli of the chloroplast isoform. 842 55


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