Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have isolated a new class of mutations in rpoD, the gene encoding the sigma 70 subunit of Escherichia coli RNA polymerase, that alter the transcription initiation properties of RNA polymerase holoenzyme. The rpoD(Lac) mutations increase expression of the lac operon in the absence of CAP-cAMP, allowing a strain lacking adenyl cyclase to grow on lactose. Four of the six alleles isolated have three- to fivefold increases in the amount of lac mRNA and beta-galactosidase per cell. We show that these four mutations increase transcription initiation from the same promoter used by wild-type RNA polymerase. The mutations were mapped and sequenced. One mutation occurs in the codon for amino acid 389 of the sigma 70 polypeptide. The remaining five mutations are clustered, affecting residues 570, 571 and 575. These five mutations are within or near a proposed helix-turn-helix motif in the C terminus of sigma 70.
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PMID:Mutations in rpoD, the gene encoding the sigma 70 subunit of Escherichia coli RNA polymerase, that increase expression of the lac operon in the absence of CAP-cAMP. 284 53

Bacteriophage Mud1 (Ap, lac) was used to construct the cya-lac operon fusion in Escherichia coli chromosome. The maximal cya expression was observed by measuring the levels of beta-galactosidase in the strains lacking the cAMP. The inhibition of cya transcription was proportional either to the amount of cAMP added or to the copy number of cya gene in the cell. Functional cAMP receptor protein (CRP) was necessary for the effect. The cya gene expression was low in cAMP-independent crp mutant even in the absence of the cAMP.
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PMID:[Study of the expression of the cya gene in Escherichia coli K12 on a model of the hybrid cya-lac operon]. 285 91

The Escherichia coli lac promoter has been shown to contain an RNA polymerase binding site (P2) that overlaps with, and is shifted 22 base-pairs upstream from the normal lac promoter (P1). In this paper, we provide RNA polymerase protection data obtained in vitro that show that, in the absence of CAP-cAMP, in vitro P2 is the preferred polymerase binding site on the P+ template. In the presence of CAP-cAMP, polymerase binding to P2 is reduced and more polymerase is bound at P1. Two lac P1 "-35 region" mutations, L157 and 4, which increase the homology between this region and the consensus "-10 region" sequence, are both shown to have an increased affinity for polymerase binding at P2. CAP-cAMP is also able to decrease the amount of polymerase bound to P2 and to increase the amount bound to P1 on these mutant promoter fragments. P2 does not initiate transcription efficiently in vivo. Nuclease S1 mapping experiments detect only a low level of transcription from one of the P2 "up" mutations, but no beta-galactosidase synthesis is directed by this mutant. Mutations such as L157 and 4, which alter the P2-10 region, also alter lac P sensitivity to CAP-cAMP in vivo, suggesting that the P2 sequence plays a role in CAP-cAMP regulation of lac P. Possible roles for P2 in vivo are discussed.
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PMID:Properties of lac P2 in vivo and in vitro. An overlapping RNA polymerase binding site within the lactose promoter. 299 53

Kline et al. (1980) have reported that indole-3-acetic acid (IAA) and four other indole derivatives are able to substitute for cAMP in activating expression of the ara regulon of E. coli. We have examined this phenomenon in detail, utilizing fusions between the structural gene for beta-galactosidase and the promoters for the araBAD, araE, and araFG operons. We confirm that IAA potently stimulates transcription from the araBAD promoter. The effect is highly specific to araBAD, as IAA has no, or only slight, effects on the araE and araFG operons. However, contrary to the results of Kline et al., we find that the action of IAA does not require CAP. Thus, IAA fully stimulates the transcription of araBAD in a strain which bears a complete deletion of the crp gene.
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PMID:The catabolite gene activator protein (CAP) is not required for indole-3-acetic acid to activate transcription of the araBAD operon of Escherichia coli K-12. 299 82

Plasmids carrying the CYR1 gene of yeast Saccharomyces cerevisiae, which encodes adenylate cyclase, were introduced into the cya mutant strain of Escherichia coli. The transformants had a GTP-independent adenylate cyclase activity but did not produce cAMP. The E. coli transformant carrying the yeast RAS2 or RAS2val19 gene had no adenylate cyclase activity. Transformant cells carrying both CYR1 and RAS2 produced GTP-dependent adenylate cyclase and cAMP, and those carrying CYR1 and RAS2val19 produced GTP-independent adenylate cyclase and a large amount of cAMP. Production of cAMP in the transformant carrying CYR1 and either RAS2 or RAS2val19 was confirmed by staining colonies on maltose-MacConkey plates and by measuring induction of beta-galactosidase by isopropyl beta-D-thiogalactopyranoside. Mixing a crude extract from the E. coli transformant carrying CYR1 with a crude extract from cells carrying RAS2 reconstituted the GTP-dependent adenylate cyclase. Reconstitution of the GTP-dependent adenylate cyclase was observed by mixing the plasma membrane fraction of yeast CYR1 ras1 ras2 bcy1 mutant and a crude extract from the E. coli transformant carrying RAS2 or by mixing a crude extract from the E. coli transformant carrying CYR1 and the membrane fraction of yeast cyr1 RAS1 RAS2 BCY1 mutant. The data suggest that the yeast GTP-dependent adenylate cyclase consists of catalytic and regulatory subunits encoded by the CYR1 and RAS2 genes, respectively.
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PMID:Reconstitution of the GTP-dependent adenylate cyclase from products of the yeast CYR1 and RAS2 genes in Escherichia coli. 299 79

Mutations which permit cAMP binding protein (CRP) to act in the absence of cAMP have been isolated by in vitro mutagenesis of a plasmid containing the cloned crp gene. Adenylate cyclase deficient cells harbouring the mutant (crp*) plasmids exhibited a variety of fermentation profiles on MacConkey indicator plates containing various sugars. beta-galactosidase synthesis in cells carrying the crp* plasmids was activated most by the addition of cGMP as well as cAMP. The sites of mutations which are responsible for the cAMP independent phenotype were determined by in vitro recombination and DNA sequencing. The amino acid substitutions in the mutant proteins were found in two specific regions of the crp gene encoding residues 53-62 and 141-148 of CRP polypeptide. The first region may participate in cAMP binding, while the second appears to be the inter-domain region of the N-terminal cAMP-binding and C-terminal DNA-binding domains.
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PMID:Mutations that alter the allosteric nature of cAMP receptor protein of Escherichia coli. 300 51

Amplification of the cya gene of E. coli on the plasmid pBR325 leads to an increase of adenylate cyclase activity proportional to the gene dosage. In strains harboring hybrid plasmids with cya gene the intracellular level of cAMP and the rate of nucleotide secretion are also elevated. The adenylate cyclase activity in cells with truncated cya gene cloned on pBR322 remains sensitive to glucose inhibition. Amplification of the cya gene leads to considerable resistance of beta-galactosidase synthesis to transient repression by alpha-methylglucoside, but does not influence the permanent repression caused by glucose.
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PMID:[Amplification of the adenylate cyclase gene in Escherichia coli cells]. 300 39

cAMP receptor protein (CRP)-dependent operon expression in Escherichia coli requires the CRP X cAMP complex form of wild-type CRP. One class of crp mutants (crp*) activates CRP-dependent promoters in strains (cya) incapable of endogenous cAMP synthesis. Of fundamental interest is the difference in regulatory properties exhibited by crp* mutant strains, some of which exhibit glucose-mediated repression of beta-galactosidase synthesis, some of which do not. To gain a better understanding of the mechanisms of cAMP-independent promoter activation and repression we have: determined through cloning and DNA sequence analysis the primary structure of three CRP* forms of CRP; purified the mutant proteins; characterized the effect of these mutations on CRP secondary structure; and studied CRP*-activated lac promoter regulation in a purified in vitro transcription system. The results of this study provide strong evidence that mutations in crp alter the conformation of CRP and result in cAMP-independent activation of CRP-dependent promoters in vitro. In addition, a CRP allele-specific inhibition of CRP* activity by spermidine was observed in vitro that parallels crp* strain-specific sensitivity to glucose-mediated repression of CRP-dependent enzyme synthesis in vivo. This observation provides evidence that catabolite repression in cells lacking cAMP may be mediated through a mechanism that inhibits CRP* activity.
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PMID:Structure-function analysis of three cAMP-independent forms of the cAMP receptor protein. 302 48

The regulatory and promoter regions of the gene for acid phosphatase of optimum pH 2.5 (appA) of Escherichia coli has been characterized by constructing appA-lacZ protein fusions. Monitoring of beta-galactosidase activity showed that the cloned fragment harbored a region that was responsible for a negative control of transcription mediated by the cAMP-cAMP receptor (CAP) complex. The nucleotide sequence of the segment was determined. A region containing a putative CAP binding site, overlapping the -10 region of the promoter was found. Deletion analysis around the promoter region, using appA-lacZ protein fusions substantiated the hypothesis for a role of the 'consensus' sequence in the negative control mediated by cyclic AMP. In addition, the coding sequence revealed the likely existence of a signal peptide similar to the one found for other exported proteins, upstream from the phosphatase protein sequence.
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PMID:The structure of the promoter and amino terminal region of the pH 2.5 acid phosphatase structural gene (appA) of E. coli: a negative control of transcription mediated by cyclic AMP. 303 1

Antibodies raised to a mixture of the 46 and 48 kDa rat CNS 2',3'-cyclic nucleotide 3-phosphodiesterases (CNPs) recognized apparently identical proteins in peripheral nervous system (PNS), thymus, and circulating blood lymphocytes. These antibodies were used to identify, in a rat brain phage lambda gt11 expression library, cDNA clones encoding beta-galactosidase-CNP fusion proteins, some of which showed CNP activity. In RNA blots, the subcloned CNP cDNA inserts hybridized to mRNAs of approximately 2400 and approximately 2800 nucleotides (nts), and to a approximately 2500 nt mRNA from thymus. Several nonexpressing CNP cDNAs were identified by plaque hybridization, and the mRNA transcribed in vitro from one of these cDNAs (pCNP7) encoded a complete 46 kDa CNP polypeptide. Examination of the deduced amino acid sequence revealed an apparent homology to cAMP binding sites in several other proteins. A 373 bp segment from the 5' end of this pCNP7 hybridized only to the 2800 nt nervous system mRNAs, thus revealing that not all CNP mRNAs share the same 5'-ends. Genomic DNA blots probed with CNP cDNAs suggest that there is a single gene which can be alternatively spliced to produce the various mRNA transcripts in the nervous and lymphoid tissues.
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PMID:Molecular cloning of a 2',3'-cyclic nucleotide 3'-phosphodiesterase: mRNAs with different 5' ends encode the same set of proteins in nervous and lymphoid tissues. 304 Sep 24


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