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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)

During carbon-starvation-induced entry into stationary phase, Escherichia coli cells exhibit a variety of physiological and morphological changes that ensure survival during periods of prolonged starvation. Induction of 30-50 proteins of mostly unknown function has been shown under these conditions. In an attempt to identify C-starvation-regulated genes we isolated and characterized chromosomal C-starvation-induced csi::lacZ fusions using the lambda placMu system. One operon fusion (csi2::lacZ) has been studied in detail. csi2::lacZ was induced during transition from exponential to stationary phase and was negatively regulated by cAMP. It was mapped at 59 min on the E. coli chromosome and conferred a pleiotropic phenotype. As demonstrated by two-dimensional gel electrophoresis, cells carrying csi2::lacZ did not synthesize at least 16 proteins present in an isogenic csi2+ strain. Cells containing csi2::lacZ or csi2::Tn10 did not produce glycogen, did not develop thermotolerance and H2O2 resistance, and did not induce a stationary-phase-specific acidic phosphatase (AppA) as well as another csi fusion (csi5::lacZ). Moreover, they died off much more rapidly than wild-type cells during prolonged starvation. We conclude that csi2::lacZ defines a regulatory gene of central importanc e for stationary phase E. coli cells. These results and the cloning of the wild-type gene corresponding to csi2 demonstrated that the csi2 locus is allelic with the previously identified regulatory genes katF and appR. The katF sequence indicated that its gene product is a novel sigma factor supposed to regulate expression of catalase HPII and exonuclease III (Mulvey and Loewen, 1989). We suggest that this novel sigma subunit of RNA polymerase defined by csi2/katF/appR is a central early regulator of a large starvation/stationary phase regulon in E. coli and propose 'rpoS' ('sigma S') as appropriate designations.
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PMID:Identification of a central regulator of stationary-phase gene expression in Escherichia coli. 184 9

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 responds to combined nitrogen deprivation by forming specialized nitrogen-fixing cells at regular intervals along the filament. Genetic and biochemical studies have indicated that regulation of gene expression during differentiation occurs at the transcriptional level. As part of a characterization of RNA polymerase during differentiation, the gene encoding the 52-kDa principal sigma factor of the Anabaena sp. strain PCC 7120 vegetative-cell RNA polymerase was isolated by using an oligonucleotide probe based on the sequence of the N-terminal seven amino acids of the purified protein. sigA codes for a 390-amino-acid polypeptide that has a predicted molecular weight of 45,641. The amino acid sequence of the polypeptide encoded by sigA contains four regions corresponding to conserved domains of the principal RNA polymerase sigma factors of Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43). Thus, although the subunit composition of cyanobacterial RNA polymerase core differs from that of other eubacteria (G. J. Schneider and R. Haselkorn, J. Bacteriol. 170:4136-4140, 1988), the principal sigma factor of at least one cyanobacterium is typically eubacterial. In contrast to sigma 70 and sigma 43 operon organization, sigA is monocistronic and encodes two transcripts of 1.7 and 2.2 kb. The abundance of the 1.7-kb transcript remains constant under both nitrogen-replete and nitrogen-limiting conditions, whereas the 2.2-kb transcript is induced following the removal of combined nitrogen. Continued or enhanced transcription of sigA under nitrogen starvation conditions is consistent with the observation that the principal RNA polymerase in differentiating cells contains SigA.
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PMID:Isolation and characterization of the gene encoding the principal sigma factor of the vegetative cell RNA polymerase from the cyanobacterium Anabaena sp. strain PCC 7120. 190 66

Genes of higher eucaryotic cells are considered to show only a limited response to nutritional stress. Here we show, however, that omission of a single essential amino acid from the medium caused a marked rise in the mRNA levels of c-myc, c-jun, junB and c-fos oncogenes and ornithine decarboxylase (ODC) in CHO cells. There was no general accumulation of mRNAs in amino acid-starved cells, since the gamma-actin, beta-tubulin, protein kinase C, RNA polymerase II, and glyceraldehyde-3-phosphate dehydrogenase mRNAs and the total poly(A)+ mRNA were not increased. The levels of c-myc, ODC, and c-jun mRNAs were elevated more by amino acid starvation than by inhibition of protein synthesis with cycloheximide, which is known to increase the levels of these mRNAs. Importantly, however, cycloheximide present during amino acid starvation reduced the rise in the levels of the mRNAs down to the level obtained with cycloheximide alone. This implies that protein synthesis is required for the accumulation of c-myc, ODC, and c-jun mRNAs in amino acid-deprived cells. The junB and c-fos mRNAs, instead, were increased to the same extent or less by amino acid starvation than by cycloheximide treatment. The accumulation of the c-myc mRNA in amino acid-starved cells was due to both stabilization of the mRNA and increase of its transcription. The rise in the c-jun mRNA level seemed to be caused merely by stabilization of the mRNA. Further, despite the inhibition of general protein synthesis, amino acid starvation led to an increase in the synthesis of c-myc polypeptide. The results suggest that mammalian cells have a specific mechanism for registering shortages of amino acids in order to make adjustments compatible with cellular growth.
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PMID:Deprivation of a single amino acid induces protein synthesis-dependent increases in c-jun, c-myc, and ornithine decarboxylase mRNAs in Chinese hamster ovary cells. 212 33

During amino acid starvation the synthesis of rRNA and tRNA is specifically inhibited (stringently controlled) in wild type Escherichia coli but not in relaxed strains carrying the relA mutation. We have found that the in vivo transcription of a hybrid rrnB rRNA operon, in which the normal promoter region has been replaced by the lambda PL promoter, is under stringent control even though this promoter lacks the "stringent discriminator" sequence postulated to be required for stringent control. Furthermore, we have found that transcription of the rrnB operon from a phage T7 promoter, as well as T7 genes in general, by phage T7 RNA polymerase is also subject to stringent control in vivo. These results are consistent with the idea that stringent control acts in a relatively nonspecific manner to inhibit some step(s) in transcription that are often rate-limiting for very active transcription. The relative simplicity of transcription by phage T7 RNA polymerase should offer a good system to study the molecular mechanisms of stringent control.
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PMID:Stringent control in Escherichia coli applies also to transcription by T7 RNA polymerase. 243 27

Weak stringent or relaxed responses were induced in Escherichia coli (relA+), using mild amino acid starvation or treatment with chloramphenicol at low concentrations, respectively, such that the growth rate was barely reduced. In this manner, the intracellular concentration of the nucleotide guanosine tetraphosphate, ppGpp, could be varied in any desired range between 0 and 1000 pmol of ppGpp per OD460 unit of culture mass. At the same time, the rate of synthesis of stable RNA (rs; rRNA and tRNA) was measured, relative to the total instantaneous rate of RNA synthesis (rt). The correlation between the cytoplasmic concentration of ppGpp and stable RNA gene activity (rs/rt) was the same as that observed previously with relA+ and relA strains growing exponentially at different rates in different media. This suggests that the distinction between growth control and stringent control of stable RNA synthesis is arbitrary, and that both kinds of control reflect the same ppGpp-dependent phenomenon. By increasing the stable RNA gene dosage, using high copy number plasmids carrying an rrn gene, we have tested the idea that ppGpp partitions the bacterial RNA polymerase into two forms with different probabilities to initiate at stable RNA and mRNA promoters. The relaxed response was not significantly altered, but the extent of the stringent response was reduced by the presence of extra rrn genes. The results agree with quantitative predictions derived from the RNA polymerase partitioning hypothesis.
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PMID:Stringent and growth control of rRNA synthesis in Escherichia coli are both mediated by ppGpp. 244 28

Previous studies on two Escherichia coli rpoB mutants, carrying single amino acid substitutions at approximate amino acid positions 736 and 906 in the beta subunit, showed that these alterations in the RNA polymerase resulted in an apparent reduced response to valine-induced amino acid starvation in vivo and prevented ppGpp-mediated inhibition of transcriptional initiation at stable RNA promoters in vitro. These observations suggested that the mutations had altered either the ppGpp binding site or the promoter selectivity of the enzyme. The in vivo analysis presented here indicates that these mutants encode an RNA polymerase that responds normally to changes in the level of ppGpp; their apparent relaxedness is due to a reduced accumulation of ppGpp during isoleucine starvation. Thus, there is no indication that the mutations have altered ppGpp binding sites. These observations and the difference between in vitro and in vivo results can be explained by the assumption that the mutations produce an extended ppGpp-dependent pausing of RNA polymerase during the transcription of unstable RNA. Comparison of the vivo and in vitro effects of ppGpp on rrn transcription further suggests that these reflect different phenomena, although in both cases ppGpp inhibits rrn transcription.
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PMID:Studies in vivo on Escherichia coli RNA polymerase mutants altered in the stringent response. 246 Jul 32

The Escherichia coli spoT gene encodes a guanosine-3',5'-bispyrophosphate (ppGpp) 3'-pyrophosphohydrolase known to be responsible for cellular (ppGpp) degradation. The DNA sequence of the spoT region is presented. The spoT gene is deduced to be 702 codons long, with a probable UUG initiation codon, and a deduced mass of 79,342 daltons. Two spoT mutations (spoT202 and spoT203) have been localized to an open reading frame by complementation of function as well as by genetic marker rescue. The ability to overexpress the spoT gene is limited, but enough ppGppase activity can be made to reverse ppGpp accumulation during the stringent response to amino acid starvation. The spoT gene is located within a larger spo operon and is flanked by two smaller genes. The first gene in the operon encodes omega, a protein that copurifies with RNA polymerase (Gentry, D. R., and Burgess, R. R. (1986) Gene (Amst.) 48, 33-40). The spoT gene is the second gene in the operon; it is followed by a third open reading frame deduced to encode a protein with a mass of 25,343 daltons. Insertion of a kanamycin resistance gene in the omega gene reduces spoT gene expression as judged by lowered ppGppase activity, relA-dependent reduction of growth rate, and abolition of spoT mutant complementation activity. These effects are reversed by expression of the spoT gene, but not the omega gene, in trans. Transcription of the spo operon occurs in a clockwise direction on the E. coli chromosome and is probably directed by at least two promoters.
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PMID:Characterization of the spoT gene of Escherichia coli. 254 50

Replication of oriC-dependent minichromosomes was found to be transiently stimulated when protein synthesis was inhibited by the addition of chloramphenicol. Initiation of replication was also induced by amino acid starvation of relA mutant strains and a nutritional upshift. The results are explained on the basis that these treatments rendered RNA polymerase more available for participation in the initiation process. As a consequence, the oriC duplex may be transcriptionally activated to an open form, a necessary prerequisite for DNA polymerization.
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PMID:Inhibition of protein synthesis transiently stimulates initiation of minichromosome replication in Escherichia coli. 266 26

Transcription of the 26-kilobase (kb) dihydrofolate reductase (dhfr) gene in CHO cells is initiated at two sites: a major site (approximately 85% of the dhfr mRNA) at -63 relative to the translation start and a minor site (approximately 15%) at -107. Transcription also occurs from the opposite DNA strand in the dhfr 5' region, with a probable initiation site at approximately -195 relative to the dhfr translation start. A 4-kb polyadenylated RNA that is derived from the opposite-strand transcription increases threefold in abundance after serum starvation of CHO cells for 24 h. dhfr mRNA levels do not change during this time. The first dhfr exon lies within a 1-kb genomic region marked by exceptionally high G + C content and lack of DNA methylation. This region also includes a 214-base-pair (bp) exon for the opposite-strand transcript and five of the six DNase I-hypersensitive sites identified at the dhfr locus. Analysis of the DNA sequences of hamster, human (M. Chen, T. Shimada, A. D. Moulton, A. Cline, R. K. Humphries, J. Maizel, and A. W. Nienhuis, J. Biol. Chem. 259:3933-3943, 1984), and mouse (M. McGrogan, C. C. Simonsen, D. T. Smouse, P. J. Farnham, and R. T. Schimke, J. Biol. Chem. 260:2307-2314, 1985) dhfr genes reveals the presence of a 29-bp unit that is conserved 45 to 49 bp upstream of major and minor dhfr transcription start sites. This unit follows the consensus: GRGGCGGTGGCCTNNNNTGTCRCAARTRGGTR. The 5' part of the 29-bp unit contains a GC box that agrees with the GGGCGG consensus-binding site for the RNA polymerase II transcription factor Sp1 (D. Gidoni, W. A. Dynan, and R. Tjian, Nature (London) 312:409-413, 1984). Each of the three mammalian dhfr genes has several G-rich GC boxes proximal to the major dhfr transcription start site and several GC boxes of the opposite orientation (C rich) in a distal region about 500 bp upstream.
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PMID:Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene. 302 46

The alternative pathway of DNA replication in rnh mutants of Escherichia coli can be continuously initiated in the presence of chloramphenicol, giving rise to constitutive stable DNA replication (cSDR). We conducted a physiological analysis of cSDR in rnh-224 mutants in the presence or absence of the normal DNA replication system. The following results were obtained. cSDR allowed the cells to grow in the absence of the normal replication system at a 30 to 40% reduced growth rate and with an approximately twofold-decreased DNA content. cSDR initiation was random with respect to time in the cell cycle as well as choice of origins. cSDR initiation continued to increase exponentially for more than one doubling time when protein synthesis was inhibited by chloramphenicol. cSDR initiation was inhibited during amino acid starvation in stringent (relA+) but not in relaxed (relA1) strains, indicating its sensitivity to ppGpp. cSDR initiation was rifampin sensitive, demonstrating that RNA polymerase was involved. cSDR functioned in dnaA+ rnh-224 strains parallel to the normal oriC+ dnaA+-dependent chromosome replication system.
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PMID:Mode of initiation of constitutive stable DNA replication in RNase H-defective mutants of Escherichia coli K-12. 303 62


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