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

---

Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report the identification and partial characterization of the promoters for two chitinase genes from Streptomyces plicatus. Chitinases are a family of enzymes made by Streptomyces and other soil microbes to digest chitin, an abundant source of carbon and nitrogen in the soil. The promoter regions of two chitinases were defined by using transcriptional fusions to the xylE reporter gene. Transcription was shown to be glucose-sensitive and chitin-dependent. Each promoter contains a putative RNA polymerase binding site with a recognition sequence very similar to that observed in many eubacterial vegetatively expressed genes. In both promoters, a pair of 12-base-pair direct repeat sequences overlap the putative RNA polymerase binding sites. Further analysis of one of the promoters revealed that a single-base change within the direct repeat sequences resulted in glucose-resistant, chitin-independent expression in vivo. In addition, the promoter region that includes the direct repeat sequences was shown to interact with a sequence-specific DNA binding factor in vitro. Similar direct repeat sequences are present in other chitinase genes recently characterized, and we suggest that these repeats may be involved in repression and induction for this entire class of catabolite-controlled genes.
...
PMID:Direct repeat sequences are implicated in the regulation of two Streptomyces chitinase promoters that are subject to carbon catabolite control. 154 88

We have investigated whether the RNA polymerase III-driven transcription of eukaryotic tRNA genes can be regulated by the prokaryotic tetracycline operator-repressor system. The bacterial tet operator (tetO) was inserted at two different positions (-7 and -46) upstream of a tRNA(Glu) (amber) suppressor gene. Both constructs are transcribed in Saccharomyces cerevisiae and yield functional tRNAs as scored by suppression of an amber nonsense mutation in the met8-1 allele. Controlled expression of Tet repressor was achieved by fusing the bacterial tetR gene to the yeast gal1 promoter. This leads to expression of Tet repressor in yeast on galactose--but not on glucose--containing media. Regulation of the su-tRNA gene with the tetO fragment inserted at position -7 has been demonstrated. Under conditions which allow tetR expression, cells exhibit a met- phenotype. This methionine auxotrophy can be conditionally reverted to prototrophy by adding tetracycline. However, a su-tRNA gene with the tetO fragment inserted at position -46 cannot be repressed. Our results demonstrate clearly that the bacterial repressor protein binds to its operator in the yeast genome. Formation of this complex in the vicinity of the pol III transcription initiation site reduces the level of su-tRNA at least 50-fold as concluded from quantitative primer extension analyses. This indicates for the first time that class III gene expression can be regulated by a DNA binding protein with its target site in the 5'-flanking region and that a prokaryotic repressor can confer regulation of a suitably engineered tRNA gene.
...
PMID:RNA polymerase III catalysed transcription can be regulated in Saccharomyces cerevisiae by the bacterial tetracycline repressor-operator system. 156 52

Linker-insertion mutagenesis was used to isolate mutations in the Saccharomyces cerevisiae gene encoding the largest subunit of RNA polymerase II (RPO21, also called RPB1). The mutant rpo21 alleles carried on a plamid were introduced into a haploid yeast strain that conditionally expresses RPO21 from the inducible promoter pGAL10. Growth of this strain on medium containing glucose is sustained only if the plasmid-borne rpo21 allele encodes a functional protein. Of nineteen linker-insertion alleles tested, five (rpo21-4 to -8) were found that impose a temperature-sensitive (ts) lethal phenotype on yeast cells. Four of these five ts alleles encode mutant proteins in which the site of insertion lies near one of the regions of the largest subunit that have been conserved during evolution. Two of the ts mutants (rpo21-4 and rpo21-7) display pleiotropic phenotypes, including an auxotrophy for inositol and a decreased proliferation rate at the permissive temperature. The functional relationship between RPO21 and RPO26, the gene encoding the 17.9 kDa subunit shared by RNA polymerases I, II, and III was investigated by determining the ability of increased dosage of RPO26 to suppress the ts phenotype imposed by rpo21-4 to -8. Suppression of the ts defect was specific for the rpo21-4 allele and was accompanied by co-suppression of the inositol auxotrophy. These results suggest that mutations in the largest subunit of RNA polymerase II can have profound effects on the expression of specific subsets of genes, such as those involved in the metabolism of inositol. In the rpo21-4 mutant, these pleiotropic phenotypes can be attributed to a defective interaction between the largest subunit and the RPO26 subunit of RNA polymerase II.
...
PMID:Isolation and phenotypic analysis of conditional-lethal, linker-insertion mutations in the gene encoding the largest subunit of RNA polymerase II in Saccharomyces cerevisiae. 158 9

In Escherichia coli K-12, expression of zwf, the gene for glucose 6-phosphate dehydrogenase, is coordinated with the cellular growth rate and induced by superoxide-generating agents. To initiate the study of the molecular mechanisms regulating its expression, the gene was cloned and its DNA sequence was determined. The 5' ends of zwf mRNA isolated from cells growing in glucose and acetate minimal media were mapped. The map was complex in that transcripts mapped to -45, -52, and -62, with respect to the beginning of the coding sequence. Three analytical methods were used to search the DNA sequence for putative promoters. Only one sequence for a promoter recognized by the sigma 70 form of RNA polymerase was found by all three search routines that could be aligned with a mapped transcript, indicating that the other transcripts arise by processing of the mRNA. A computer-assisted search did not reveal a thermodynamically stable long-range mRNA secondary structure that is capable of sequestering the translation initiation region, which suggests that growth-rate-dependent regulation of glucose 6-phosphate dehydrogenase level may not be carried out by a mechanism similar to the one for the gene (gnd) for 6-phosphogluconate dehydrogenase. The DNA segment between the -10 hexamer and the start point of transcription resembles the discriminator sequence of stable RNA genes, which has been implicated in stringent control and growth-rate-dependent regulation.
...
PMID:Molecular characterization of the Escherichia coli K-12 zwf gene encoding glucose 6-phosphate dehydrogenase. 170 5

L-Aspartase was purified from Bacillus subtilis, its N-terminal amino acid sequence was determined to construct a probe for the aspartase gene, and the gene (termed ansB) was cloned and sequenced. A second gene (termed ansA) was found upstream of the ansB gene and coded for L-asparaginase. These two genes were in an operon designated the ans operon, which is 80% cotransformed with the previously mapped aspH1 mutation at 215 degrees. Primer extension analysis of in vivo ans mRNA revealed two transcription start sites, depending on the growth medium. In wild-type cells in log-phase growth in 2x YT medium (tryptone-yeast extract rich medium), the ans transcript began at -67 relative to the translation start site, while cells in log-phase growth or sporulating (t1 to t4) in 2x SG medium (glucose nutrient broth-based moderately rich medium) had an ans transcript which began at -73. The level of the -67 transcript was greatly increased in an aspH mutant grown in 2x YT medium; the -67 transcript also predominated when this mutant was grown in 2x SG medium, although the -73 transcript was also present. In vitro transcription of the ans operon by RNA polymerase from log-phase cells grown in 2x YT medium and log-phase or sporulating cells grown in 2x SG medium yielded only the -67 transcript. Depending on the growth medium, the levels of asparaginase and aspartase were from 2- to 40-fold higher in an aspH mutant than in wild-type cells, and evidence was obtained indicating that the gene defined by the aspH1 mutation codes for a trans-acting transcriptional regulatory factor. In wild-type cells grown in 2x SG medium, the levels of both aspartase and asparaginase decreased significantly by t0 of sporulation but then showed a small increase, which was mirrored by changes in the level of beta-galactosidase from an ansB-lacZ fusion. The increase in the activities of ans operon enzymes between t2 and t5 of sporulation was found primarily in the forespore, and the great majority of the increased was found in the mature spore. However, throughout sporulation the only ans transcript detected was the -73 form, and no sporulation-specific RNA polymerase tested yielded a -73 transcript in vitro.
...
PMID:Cloning, nucleotide sequence, and expression of the Bacillus subtilis ans operon, which codes for L-asparaginase and L-aspartase. 171 Oct 29

The Bacillus subtilis dciA operon, which encodes a dipeptide transport system, was induced rapidly by several conditions that caused the cells to enter stationary phase and initiate sporulation. The in vivo start point of transcription was mapped precisely and shown to correspond to a site of transcription initiation in vitro by the major vegetative form of RNA polymerase. Post-exponential expression was prevented by a mutation in the spo0A gene (whose product is a known regulator of early sporulation genes) but was restored in a spo0A abrB double mutant. This implicated AbrB, another known regulator, as a repressor of dciA. In fact, purified AbrB protein bound to a portion of the dciA promoter region, protecting it against DNase I digestion. Expression of dciA in growing cells was also repressed independently by glucose and by a mixture of amino acids; neither of these effects was mediated by AbrB.
...
PMID:Transcriptional regulation of a Bacillus subtilis dipeptide transport operon. 176 71

The gerD locus of Bacillus subtilis comprises a single gene whose function is essential for the germination of B. subtilis spores in media containing asparagine, glucose, and fructose. The expression of gerD has been characterized by using a chromosomal lacZ fusion to the gerD promoter. The promoter is switched on at the same time as the synthesis of glucose dehydrogenase, 2.5 h after sporulation has been initiated in the developing forespore. The gerD gene is not expressed in spoIIB or spoIIIA, -IIIB, -EIII, -FIII, or -IIIG mutants, but it is expressed in spoIIIC and -IIID and spoIVA mutant backgrounds. The in vivo transcriptional start point of the gene has been mapped by primer extension analysis, and sequences upstream from the start point show considerable homology with the promoter consensus sequences recognized by RNA polymerase containing the forespore-specific sigma factor sigma G (E sigma G). gerD is transcribed in vitro by E sigma G with a similar if not identical start point to that found in vivo, and expression of the gene can be rapidly induced in vegetative cells following the induction of sigma G synthesis. These results indicate that gerD is another member of the sigma G regulon, which includes a number of genes expressed only in the forespore compartment of sporulating cells of B. subtilis.
...
PMID:Analysis of transcriptional control of the gerD spore germination gene of Bacillus subtilis 168. 190 67

The transcription from the spoVG promoter of Bacillus subtilis is induced at the start of the stationary phase of growth and is dependent on the expression of the spoOA, spoOB, and spoOH genes. It is repressed in cells grown in the presence of excess glucose and glutamine and is under the negative control of the abrB gene. The spoOA and spoOB gene products function to suppress the negative control exerted by abrB. Transcription initiation requires the form of RNA polymerase holoenzyme that contains the spoOH gene product, sigma H. Optimal transcription also requires an upstream A-T-rich region termed the upstream activating sequence (UAS). The mechanism of UAS function was examined through mutational analysis of the spoVG promoter region. Deletion of the UAS or positioning the UAS one half turn or one full turn of the DNA helix upstream of its location in wild-type spoVG resulted in a severe reduction in promoter activity. Deletion of most of the UAS abolished the abrB-dependent repression of spoVG transcription. Higher activity was observed when the UAS was inserted 10 bp (one turn of the helix) upstream than when the sequence was repositioned either 5 or 13 bp upstream. Sequences upstream of the UAS were found not to be involved with the position-dependent function of the UAS. Positioning the UAS 42 or 116 bp upstream eliminated the stimulatory effect of the sequence on spoVG transcription. These data indicate that the UAS functions effectively when it is in close proximity to the -35 region. In vitro transcription analysis indicated that the deletion and insertion mutation affecting the UAS impair RNA polymerase-spoVG promoter interaction. Deletion of the UAS showed that the negative effect of exogenous glucose and glutamine is not dependent on the UAS but is exerted at a site within or near the -35 and -10 regions.
...
PMID:Analysis of the upstream activating sequence and site of carbon and nitrogen source repression in the promoter of an early-induced sporulation gene of Bacillus subtilis. 193 51

The SRP3-1 mutation is an allele-specific suppressor of temperature-sensitive mutations in the largest subunit (A190) of RNA polymerase I from Saccharomyces cerevisiae. Two mutations known to be suppressed by SRP3-1 are in the putative zinc-binding domain of A190. We have cloned the SRP3 gene by using its suppressor activity and determined its complete nucleotide sequence. We conclude from the following evidence that the SRP3 gene encodes the second-largest subunit (A135) of RNA polymerase I. First, the deduced amino acid sequence of the gene product contains several regions with high homology to the corresponding regions of the second-largest subunits of RNA polymerases of various origins, including those of RNA polymerase II and III from S. cerevisiae. Second, the deduced amino acid sequence contains known amino acid sequences of two tryptic peptides from the A135 subunit of RNA polymerase I purified from S. cerevisiae. Finally, a strain was constructed in which transcription of the SRP3 gene was controlled by the inducible GAL7 promoter. When this strain, which can grow on galactose but not on glucose, was shifted from galactose medium to glucose medium, a large decrease in the cellular concentration of A135 was observed by Western blot analysis. We have also identified the specific amino acid alteration responsible for suppression by SRP3-1 and found that it is located within the putative zinc-binding domain conserved among the second-largest subunits of eucaryotic RNA polymerases. From these results, it is suggested that this putative zinc-binding domain is in physical proximity to and interacts with the putative zinc-binding domain of the A190 subunit.
...
PMID:Suppressor analysis of temperature-sensitive mutations of the largest subunit of RNA polymerase I in Saccharomyces cerevisiae: a suppressor gene encodes the second-largest subunit of RNA polymerase I. 199 Feb 81

Human brain hexokinase (hexokinase I) was produced in Escherichia coli from a synthetic gene under control of the bacteriophage T7 promoter. The expressed coding region derives from a human cDNA clone thought to specify hexokinase I based on amino acid sequence identity between the predicted translation product and hexokinase I from rat brain. The open reading frame from this cDNA was fused to the promoter and 5' flanking region of T7 gene 10, and expressed in E. coli by induction of T7 RNA polymerase. Induced cells contained a hexokinase activity and an abundant protein of apparent molecular weight 100,000, neither of which was present in cells lacking T7 RNA polymerase. Enzyme purified to near homogeneity consisted of a 100,000 Da protein, the size predicted from the nucleotide sequence of the expressed cDNA. The purified enzyme had Michaelis constants of 32 microM and 0.3 mM for glucose and ATP, respectively, and bound to rat liver mitochondria in the presence of MgCl2. Enzymatic activity was inhibited by glucose-6-P and this inhibition was relieved by inorganic phosphate. Deinhibition by phosphate is a property specific to brain hexokinase.
...
PMID:Expression of human brain hexokinase in Escherichia coli: purification and characterization of the expressed enzyme. 204 17


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

---