Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The coat protein of Bacillus subtilis spores comprises about 10% of the total dry weight of spores and 25% of the total spore protein. One protein with a molecular weight of 13,000 to 15,000 comprises a major portion of the spore coat. This mature spore coat protein has histidine at its NH2 terminus and is relatively rich in hydrophobic amino acids. Netropsin, and antibiotic which binds to A-T-rich regions of DNA and inhibits sporulation, but not growth, decreased the synthesis of this spore coat protein by 75%. A precursor spore coat protein with a molecular weight of 25,000 is made initially at t1 of sporulation and is converted to the mature spore coat protein with a molecular weight of 13,500 at t2 - t3. These data indicate that the spore coat protein gene is expressed very early in sporulation prior to the modifications of RNA polymerase which have been noted.
 ...
PMID:Spore coat protein of Bacillus subtilis. Structure and precursor synthesis. 9 46

The behavior of nucelotides with thioketo-substituted pyrimidine bases (4-thiouracil, 2-thiouracil and 2-thiocytosine) or amino-analogue purine bases (2-aminopurine and 2,6-diaminopurine) in transcription and translation was investigated. The experimental results obtained led to the following conclusions. 1. The stereochemical basis of substrate selection in transcription is the geometry of Watson-Crick base pairs A-U (or A-T) and G-C between substrate and template bases. 2. The topology of the active site of Escherichia coli RNA polymerase is precisely adopted to the geometry of Watson-Crick base pairs. 3. The enzyme active site discriminates between A-U (A-T) and G-C base pairs. An essential feature in this discrimination is the 6-NH2 group of the A-U (A-T) base pair and the 2-keto group of cytosine in the G-C base pair. 4. The codon properties of a nucleic acid base in messenger RNA can be predicted on the basis of its specificity in polynucleotide interactions. There seems to be no evidence for the participation of protein topological sites in the control of the specificity of codon-anticodon interactions in translation.
 ...
PMID:The stereochemical basis of template function. 31

Protease-free bovine pancreatic deoxyribonuclease (DNase) (1.6 X 10(-4) mmol) was thiolated on the NH2 groups with N-acetyl-DL-homocysteine thiolactone (2.4 X 10(-2) mmol) at pH 10.5 with imidazole (2.4 X 10(-2) mmol) as the catalyst in the presence of 4,4'-dithiodipyridine (4.2 X 10(-2) mmol). The product obtained after 16 h at 4 degrees C, 2-acetamido-4-(4'-dithiopyridyl)butyryl-DNase, isolated by gel filtration, contained an average of 0.87 +/- 0.13 mol of mixed disulfide per mol of DNase. Ribonuclease (RNase) was thiolated in a similar manner, but under N2 in the absence of 4,4'-dithiodipyridine. The protein N-acetylhomocysteinyl-RNase contained on the average 0.94 +/- 0.11 mol of sulfhydryl groups per mol of RNase. The coupling of RNase ot DNase was accomplished by thiol-disulfide interchange at pH 6.2 and 25 degrees C for 90 min. The hybrid enzyme (yield 25--33%, based upon the DNase derivative used) was freed from unreacted DNase, RNase, and homodimers by gel filtration, affinity chromatography, and salting-out chromatography. The purified enzyme contained one molecule each of DNase and RNase and hydrolyzed thymus deoxyribonucleic acid (DNA) and yeast or transfer ribonucleic acid (RNA) with 75 and 40% of the efficiencies, respectively, of the parent enzymes. The RNA strand of the hybrid substrate, phage f1 DNA-[3H]RNA, prepared from phage DNA with RNA polymerase, was hydrolyzed rapidly by the hybrid enzyme but was not hydrolyzed by RNase alone. A conjugate of the two enzymes offers the possibility in vivo of delivering two enzymes that differ in size, charge, and biological function to the same site at the same time.
 ...
PMID:Preparation of the bifunctional enzyme ribonuclease-deoxyribonuclease by cross-linkage. 48 31

We examined the expression of choB, encoding cholesterol oxidase of Brevibacterium sterolicum ATCC 21387, in Escherichia coli JM105 and Streptomyces lividans TK23 using various deletion DNA fragments within the 5'-flanking region. The enzyme activity could be detected intracellularly in E. coli only when the 5'-flanking region was reduced to less than 256-bp and choB was transcribed by the lac promoter. A large amount of the enzyme were produced as inactive inclusion bodies when ChoB protein was fused with the NH2-terminal portion of LacZ protein. In contrast, choB with more than 256-bp of the 5'-flanking region was efficiently expressed in S. lividans TK23, and about 85 times as much of the active enzyme (170 U/ml) was secreted into the culture filtrate as with B. sterolicum in flask culture. These results suggest that the promoter of choB exist within 256-bp of the 5'-flanking region and can be efficiently recognized by the RNA polymerase of S. lividans. The characteristics of the enzyme purified from the culture filtrate of the S. lividans transformant and that of B. sterolicum were identical although the NH2-terminal amino acid sequence of the enzyme from the S. lividans transformant was 6 amino acids shorter than that from B. sterolicum.
 ...
PMID:Hyperexpression and analysis of choB encoding cholesterol oxidase of Brevibacterium sterolicum in Escherichia coli and Streptomyces lividans. 136 73

DNA-dependent RNA polymerase (EC 2.7.7.6) was purified from Pseudomonas putida. The enzyme had the typical composition of beta',beta,alpha, and sigma subunits of eubacterial RNA polymerases. The molecular masses of the subunits were 156,000 Da, 151,000 Da, 87,000 Da, and 42,000 Da, respectively, as measured by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The NH2-terminal amino acid residues of the alpha subunit had a marked homology with those of the alpha subunit of Escherichia coli RNA polymerase. The enzyme activity was dependent on ribonucleoside triphosphates, Mg2+, and a DNA template, and was inhibited in vitro by rifampicin. The enzyme activity was maximal in the presence of 10 mM MgCl2. In an in vitro transcription assay using the tac promoter-controlled DNA as a template, the RNA polymerase of P. putida initiated transcription at the same site as that of E. coli.
 ...
PMID:Purification and characterization of a DNA-dependent RNA polymerase from Pseudomonas putida. 136 75

Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. R. capsulatus nifA1 and nifA2 encode identical NIFA proteins that activate transcription of nifHDK and other nif genes. In this study, we report that nifA1-lacZ and nifA2-lacZ fusions are repressed in the presence of NH3 and activated to similar levels under nitrogen-deficient conditions. This nitrogen-controlled activation was dependent on R. capsulatus ntrC (which encodes a transcriptional activator) but not rpoN (which encodes an RNA polymerase sigma factor). We have used primer extension analyses of nifA1, nifA2 and nifH and deletion analyses of nifA1 and nifA2 upstream regions to define likely promoters and cis upstream activation sequences required for nitrogen control of these genes. Primer extension mapping confirmed that ntrC but not rpoN is required for nifA1 and nifA2 activation, and that nifA1 and nifA2 do not possess typical RPON-activated promoters.
 ...
PMID:Analysis of the promoters and upstream sequences of nifA1 and nifA2 in Rhodobacter capsulatus; activation requires ntrC but not rpoN. 137 28

H-protein, a component of the glycine cleavage system with lipoic acid as a prosthetic group, was expressed in Escherichia coli using a T7 RNA polymerase plasmid expression system. After induction with 25 microM isopropyl-beta-D-thiogalactopyranoside, bacteria harboring the recombinant plasmid expressed mature bovine H-protein as a soluble form at a level of about 10% of the total bacterial protein. Little of the H-protein was lipoylated in E. coli cultured without added lipoate, but when the cells were cultured in medium supplemented with 30 microM lipoate, about 10% of the recombinant protein expressed was the correctly lipoylated active form, 10% was an inactive aberrantly modified form, presumably with an octanoyl group, and the remaining 80% was the unlipoylated apoform. Each of the three forms was purified to homogeneity and shown to have the same NH2-terminal amino acid sequence as that of native bovine H-protein. The specific activity of the lipoylated form of H-protein expressed was consistent with that of H-protein purified from bovine liver. The purified recombinant apo-H-protein was lipoylated and consequently activated in vitro with lipoyl-AMP as a lipoyl donor by lipoyltransferase purified 150-fold from bovine liver mitochondria. The lipoylation was dependent on lipoyl-AMP, apo-H-protein, and lipoyltransferase. The partially purified lipoyltransferase had no lipoate-activating activity. These results provide the first evidence that in mammals two consecutive reactions are required for the attachment of lipoic acid to the acceptor protein: the activation of lipoic acid to lipoyl-AMP catalyzed by lipoate-activating enzyme and the transfer of the lipoyl group to an N epsilon-amino group of a lysine residue to apoprotein by lipoyl-AMP:N epsilon-lysine lipoyltransferase.
 ...
PMID:Expression of mature bovine H-protein of the glycine cleavage system in Escherichia coli and in vitro lipoylation of the apoform. 140 Mar 16

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.
 ...
PMID:Rice cystatin: bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein. 142 7

Two duplexes (20-mers) were constructed containing either a single cis-[Pt(NH3)2[d(GpG)]] or cis-[Pt(NH3)2[d(ApG)]] intrastrand cross-link, the major DNA adducts of the antitumor drug cis-diamminedichloroplatinum(II). These synthetic duplexes were multimerized and the resultant polymers used as templates in single-step addition reactions of condensation of a single nucleoside triphosphate substrate to a dinucleotide primer (abortive elongation reaction) catalyzed by prokaryotic or eukaryotic RNA polymerases. Primer-substrate combinations were selected so as to direct trinucleotide product formation within the platinated bases of the templates. Transcription experiments established that cis-DDP-DNA adducts formed at d(ApG) or d(GpG) sites are not an absolute block to formation of a single phosphodiester bond by either Escherichia coli RNA polymerase or wheat germ RNA polymerase II. Furthermore, the kinetic data indicate that single-step addition reactions are much more impeded at the platinated d(GpG) than at the platinated d(ApG) site and that the mechanisms of inhibition of RNA polymerase activity are different at the two platinated sites. In particular, binding affinity between E. coli RNA polymerase and the d(GpG)-containing platinated template is lowered, as the apparent Km of enzyme for the platinated polymer is increased by a factor of 4-5. In contrast, binding affinity between the RNA polymerase and the d(ApG)-containing template is not affected by modification of the d(ApG) site by cis-diamminedichloroplatinum(II). Similar experiments were carried out with synthetic templates containing the adducts at the d(GpG) sites, in which one of the two platinated dG residues is paired with a dT residue.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:RNA polymerases react differently at d(ApG) and d(GpG) adducts in DNA modified by cis-diamminedichloroplatinum(II). 153 34

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.
 ...
PMID:Isolation and sequence analysis of dacB, which encodes a sporulation-specific penicillin-binding protein in Bacillus subtilis. 154 23


1 2 3 4 5 6 7 8 9 10 Next >>