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)

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

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

An RNA polymerase chain reaction strategy was used to amplify and clone a cDNA segment encoding for the complete constant part of the axolotl IgY heavy (C upsilon) chain. C upsilon is 433 amino acids long and organized into four domains (C upsilon 1-C upsilon 4); each has the typical internal disulfide bond and invariant tryptophane residues. Axolotl C upsilon is most closely related to Xenopus C upsilon (40% identical amino acid residues) and C upsilon 1 shares 46.4% amino acid residues among these species. The presence of additional cysteines in C upsilon 1 and C upsilon 2 domains is consistent with an additional intradomain S-S bond similar to that suggested for Xenopus C upsilon and C chi, and for the avian C upsilon and the human C epsilon. C upsilon 4 ends with the Gly-Lys dipeptide characteristic of secreted mammalian C gamma 3, human C epsilon 4, and avian and anuran C upsilon 4, and contains the consensus [G/GT(AA)] nucleotide splice signal sequence for joining C upsilon 4 to the transmembrane region. These results are consistent with the hypothesis of an ancestral structural relationship between amphibian, avian upsilon chains, and mammalian epsilon chains. However, these molecules have different biological properties: axolotl IgY is secretory Ig, anuran and avian IgY behave like mammalian IgG, and mammalian IgE is implicated in anaphylactic reactions.
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PMID:Phylogeny of immunoglobulin heavy chain isotypes: structure of the constant region of Ambystoma mexicanum upsilon chain deduced from cDNA sequence. 834 18

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

Escherichia coli RNA polymerase is composed of four different subunits, 2 alpha, beta, beta' and sigma. Among these subunits, the role of beta' is poorly understood. The rpoC10 mutation affecting beta' has been isolated as a suppressor mutation of the temperature-sensitive nusA11 mutant. DNA sequence analysis revealed that the rpoC10 mutant is a substitution of Lys for Glu-402. This increased positive charge appears to compensate for the increased negative charge present in the nusA11 protein (Asp for Gly-181). In vivo measurements of reporter gene expression have revealed that rpoC10 restores rho-dependent termination but fails to restore rho-independent termination in nusA11. Moreover, the rpoC10 mutation, in combination with any nusA mutation, inhibited lambda Q-mediated antitermination without affecting N antitermination and severely restricted lambda phage development. The inhibition of Q function and lambda growth could be compensated for by overproducing Q. These results suggest that the RNA polymerase beta' subunit plays a crucial role in factor-dependent transcription termination and antitermination.
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PMID:Pleiotropic effects of the rpoC10 mutation affecting the RNA polymerase beta' subunit of Escherichia coli on factor-dependent transcription termination and antitermination. 841 81

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

A study was carried out on the influence of point mutations of the functional amino acid residues on the secondary and ternary structure of bacteriophage T7 RNA polymerase as well as on the activity of the enzyme. A change in residue 631 is accompanied by significant changes in secondary structure (alpha-->beta transition). The substitution Lys-172 Leu changes both the secondary and ternary structures whereas the deletion of residues 172-173 does not lead to such changes. Changes in residues 631-632 do not affect the ability of the enzyme to bind the promoter and/or the synthesis of a full-length transcript but disturbs phosphodiester bond formation.
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PMID:[Study of the form of bacteriophage T7 RNA polymerase containing point substitutions in the region of functionally important amino acid residues]. 848 76

The gene encoding protein p10, a structural protein of African swine fever (ASF) virus, has been mapped, sequenced and expressed in E. coli. Protein p10 was purified from dissociated virus by reverse-phase HPLC, and its NH2-terminal end identified by automated Edman degradation. To map the gene encoding protein p10, a mixture of 20-mer oligonucleotides based upon a part of the amino acid sequence was hybridized to cloned ASF virus restriction fragments. This allowed the localization of the gene in fragment Eco RI K of the ASF virus genome. The nucleotide sequence obtained from this region revealed an open reading frame encoding 78 amino acids, with a high content of Ser and Lys residues. Several of the Ser residues are found in Ser-rich regions, which are also found in some nucleic acid-binding proteins. The gene coding for protein p10 has been inserted in an expression vector which contains the promoter for T7 RNA polymerase. The recombinant plasmid was used to produce the ASF virus protein in E. coli. The bacterially produced p10 protein shows a strong DNA binding activity with similar affinity for both double-stranded and single-stranded DNA.
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PMID:Structure and expression in E. coli of the gene coding for protein p10 of African swine fever virus. 850 90


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