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)

The time required for transcription of the lacZ gene in Escherichia coli was determined during exponential growth and under conditions, when the bacterium was exposed to partial isoleucine starvation. To do this, RNA was extracted from the cells at 10 s intervals following induction and quantified by Northern hybridization with probes complementary to either the beginning or the end of the lacZ mRNA. The time lag between inducer addition and the appearance of a hybridization signal at the 'late' probe represents the transit time for RNA polymerase on the lacZ gene, and this parameter and the known length of the transcribed sequence were used to calculate the lacZ mRNA chain growth-rate. The transcription elongation rate was c. 43 nucleotides s-1 during exponential growth and decreased abruptly to c. 20 nucleotides s-1 in a relA+ strain after the onset of isoleucine starvation, when massive concentrations of guanosine tetraphosphate (ppGpp) accumulated in the cells. The starvation condition did not affect initiation of transcription at the lac-promoter, but a substantial fraction of the initiated lacZ mRNA chains was never completed. For the rel+ strain the polarity was moderate, since c. 25% of the initiated lacZ mRNA' chains were continued into full-length mRNAs, but for the relA strain the polarity was so strong that no completed lacZ mRNA could be detected. The protein chain elongation rates decreased from 13 amino acids (aa) s-1 in the unperturbed growth phase to approximately 6 as s-1, when the cells starved for isoleucine. In combination, these results suggest that ppGpp plays a major role in maintaining the coupling between transcription and translation during the downshift by inhibiting mRNA chain elongation. The implications of this result for the control of stable RNA synthesis during the stringent response are discussed.
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PMID:Decreasing transcription elongation rate in Escherichia coli exposed to amino acid starvation. 140 59

The action of rifampicin on the RNA chain initiation catalysed by E. coli RNA polymerase over different templates has been studied. The steady-state formation of dinucleoside tetraphosphate under the condition of abortive initiation reaction was assayed. It was observed that rifampicin shows a spectrum of inhibitory effects on transcription initiation at different promoters. At two different promoters with a pyrimidine nucleotide at the 5'-initiation site, e.g. rrnB P2 having CTP and lacP2 having UTP, the effect of rifampicin on the abortive synthesis of the first phosphodiester bond was found to be total, even at low concentrations of the antibiotic. On the other hand, in most cases the effect of rifampicin on the abortive synthesis with a purine nucleotide at the 5'-initiation site was found to be only partial, with the exception of the T7A2 promoter, where rifampicin stimulates the abortive synthesis of pppGpC. It was also noticed that if there was a purine nucleotide at the second position of a dinucleotide which had already been synthesised by the enzyme, then further addition of the third nucleotide was not blocked in the presence of rifampicin. It appeared that a purine nucleotide at the initiation site or at the product terminus site of a translocated dinucleotide behaved similarly towards rifampicin. In the same way, if this position was occupied by a pyrimidine, rifampicin would inhibit further phosphodiester synthesis, even at a very low concentration. The stimulatory effect of rifampicin at the T7A2 promoter was presumably because here a ternary complex containing the promoter, enzyme and the abortive transcript pppGpC was initially stable, but dissociated upon addition of rifampicin, resulting in the rapid turn-over of the product.
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PMID:Differential inhibition of abortive transcription initiation at different promoters catalysed by E. coli RNA polymerase. Effect of rifampicin on purine or pyramidine-initiated phosphodiester synthesis. 162 42

The kinetics of formation of abortive initiation products during transcription of a synthetic template (encoding the transcript GAUGGC) by T7 RNA polymerase have been determined. This study revealed that while total RNA was formed in the reaction as expected, the levels of the dinucleoside tetraphosphate guanylyl-3',5'-adenosine-5'-triphosphate (pppGpA) and trinucleoside pentaphosphate guanylyl-3',5'-adenosine-3',5'-uridine-5'-triphosphate (pppGpApU) formed by premature termination of transcription reached a maximum after 10 min, and then decreased. Transcription of the same template, in the presence of either [gamma-32P]GTP and ATP, or GTP and [alpha-32P]ATP, gave the 32P-labeled dinucleotides *pppGpA and pppG*pA. Incorporation of each of these substrates into longer RNA transcripts in the same enzyme-template system was demonstrated. The incorporation was shown to require the presence of template in the reaction mixture. The requirement for base complementarity restricts the position of incorporation to that of initiating (5') nucleotide. Transcription of a second template, which encodes an RNA transcript having the partial sequence GpA at two internal positions, in the presence of each of the labeled dinucleoside tetraphosphates, failed to bring about the synthesis of significant yields of any longer radiolabeled transcripts. It is concluded that dinucleoside tetraphosphate (and perhaps trinucleoside pentaphosphate) can function as initiating nucleotides when complementary to the nucleotide sequence at promoter regions. However, a dinucleotide is not used as substrate for subsequent chain elongation in T7 RNA polymerase catalyzed transcription reactions.
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PMID:Abortive products as initiating nucleotides during transcription by T7 RNA polymerase. 171 17

The regulation of the synthesis of ribosomal RNA is a key problem for the understanding of bacterial growth. Many different regulatory mechanisms involving cis and trans acting components participate in a concerted way to achieve the very efficient, flexible and coordinated production of this class of molecules. We have studied three different sequence regions within a ribosomal RNA transcription unit which are believed to control different stages of ribosomal RNA expression. In the first part of the study the function of AT-rich sequences upstream of the -35 hexamer of rRNA promoter P1 in the activation of rRNA transcription was analyzed. We confirm that a sequence dependent bend upstream of P1 is responsible for the high promoter activity. Experiments employing linker scanning mutations demonstrated that the distance as well as the angular orientation of the bent DNA is crucial for the degree of activation. In addition, the effect of the trans activating protein Fis on the transcription initiation of promoter P1 was investigated. We can show, using the abortive initiation assay, that the predominant effect of Fis is due to an increase in the affinity of RNA polymerase for the promoter (binding constant KB) while the isomerisation rate (kf) from a closed to an open RNA polymerase promoter complex is not altered significantly. We also describe the characterization of sequence determinants important for stringent regulation and growth rate control. Evidence is provided that the discriminator motif GCGC is a necessary but not sufficient element for both types of control. Furthermore we show that not simply a particular DNA primary structure but the higher order conformation of the complete promoter region is recognized and triggers the two regulatory mechanisms, both of which are apparently mediated by the effector molecule guanosine tetraphosphate (ppGpp). Finally, we have carried out a systematic mutational analysis of the rrnB leader region preceding the structural gene for 16S RNA. We could demonstrate that highly conserved sequence elements within the rrnB leader, which were believed to be involved in transcription antitermination have post-transcriptional functions. We present evidence that these sequence elements direct the biogenesis of active ribosomal particles.
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PMID:Analysis of sequence elements important for the synthesis and control of ribosomal RNA in E coli. 176 16

We have fused the ribosomal RNA promoter P1 from the rrnB gene of Escherichia coli to lacZ and examined its guanosine tetraphosphate (ppGpp)-dependent expression at different growth rates. The rrnB P1-lacZ fusion was constructed on plasmid vectors and then recombined into the chromosome of a delta lac relA1 strain close to the normal location of the rrnB locus and in the normal orientation, coincident with the direction of replication. A series of spoT strains differing in the severity of their SpoT defect were analyzed with respect to their growth characteristics and ppGpp metabolism. The spoT203 allele was introduced into the rrnB P1-lacZ fusion containing strain and used to manipulate the ppGpp concentration independently of the growth medium. 1) The levels of ppGpp during exponential growth are decreased in rich media due to a decreased activity of (p)ppGpp synthetase II (PSII). 2) The specific activity of rrn P1-directed beta-galactosidase was increased in a parabolic fashion with increasing growth rate. A theoretical analysis showed that this was to be expected since enzyme expression from a stringently controlled promoter is affected by changes in the growth rate both via the control of the promoter, and indirectly via the control of bulk mRNA synthesis. 3) The observer specific enzyme activities were converted into rrnB P1 promoter activities, given as lacZ transcription relative to the total rate of transcription. The rrn P1 promoter activity decreased exponentially with increasing cytoplasmic concentration of ppGpp, independent of whether a given level of ppGpp was achieved by using different growth media or by using a spoT allele. These results support two hypotheses: (i) that RNA polymerase is partitioned by ppGpp into two fractions with different abilities to initiate transcription at rrn P1 promoters; and (ii) that during exponential growth ppGpp is derived from ppGpp synthetase II (PSII). Together these hypotheses predict that the growth rate control of rRNA synthesis reflects a control of PSII activity which is regulated by the composition of the growth medium.
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PMID:Guanosine tetraphosphate (ppGpp) dependence of the growth rate control of rrnB P1 promoter activity in Escherichia coli. 211

Terbium (III) upon complexation with guanosine 5'-triphosphate showed remarkable enhancement of fluorescence emission at 488 and 545 nm when excited at 295 nm. Analysis of the binding data yielded a value for the mean Kd between Tb(III) and GTP of 0.2 microM, with three binding sites for Tb(III) on GTP. 31P and 1H NMR measurements revealed that Tb(III) mainly binds the phosphate moiety of GTP. Fluorescence titration of the emission signals of the TbGTP complex with varying concentrations of Escherichia coli RNA polymerase resulted in a Kd value of 4 microM between the TbGTP and the enzyme. It was observed that TbGTP can be incorporated in the place of GTP during E. coli RNA polymerase catalyzed abortive synthesis of dinucleotide tetraphosphate at T7A2 promoter. Both the substrate TbGTP and the inhibitor of the initiation of transcription rifampicin bind to the beta-subunit of E. coli RNA polymerase. This allows the measurement of the fluorescence excited-state energy transfer from the donor TbGTP-RNA polymerase to the acceptor rifampicin. Both emission bands of Tb(III) overlap with the rifampicin absorption, and the distances at 50% efficiency of energy transfer were calculated to be 28 and 24 A for the 488- and 545-nm emission bands, respectively. The distance between the substrate binding site and the rifampicin binding site on the beta-subunit of E. coli RNA polymerase was measured to be around 30 A. This suggests that the nature of inhibition of transcription by rifampicin is essentially noncompetitive with the substrate.
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PMID:Resonance energy transfer study on the proximity relationship between the GTP binding site and the rifampicin binding site of Escherichia coli RNA polymerase. 240 99

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

Transcription in vitro of stringently controlled Escherichia coli genes by purified RNA polymerase holoenzyme is inhibited by guanosine tetraphosphate (ppGpp). In order to examine possible role of omega factor in this ppGpp sensitivity, RNA polymerases with or without the omega factor were reconstituted and tested for their ppGpp sensitivity using an in vitro mixed transcription system. RNA polymerase lacking the omega factor was found virtually insensitive to ppGpp but the addition of a purified omega factor restored the ppGpp sensitivity of this omega-free RNA polymerase. These results raise a possibility that the omega factor is a regulatory protein of RNA polymerase and is involved in the ppGpp-mediated alteration of the promoter selectivity.
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PMID:Promoter selectivity of Escherichia coli RNA polymerase: omega factor is responsible for the ppGpp sensitivity. 268 48

The effects of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), both produced by E. coli, were measured on the activities of several genes in a cell-free system. Gene activity is measured as gene-directed synthesis of biochemically competent protein or transfer RNA. Both ppGpp and pppGpp stimulated the activities of the ara, lac, and trp operons and inhibited the arg operon. Production of transfer-RNA(Tyr) was unaffected by moderate levels of either ppGpp or pppGpp and only slightly inhibited at higher levels of ppGpp. Since the cell-free reaction mixtures hydrolyze pppGpp to ppGpp, we performed similar studies with a hydrolysis-resistant analog of pppGpp, the beta-gamma methylenyl derivative (pcppGpp). In general, pcppGpp shows the same inhibitory potency as pppGpp for the arg operon, but lacks the stimulatory effects on the ara, lac, and trp operons. This result suggests that the stimulation of these gene activities is specific for ppGpp.Under similar conditions, pppGpp and ppGpp show a slight inhibitory effect on the messenger-directed synthesis of beta-galactosidase and no effect on the messenger-directed synthesis of MS2 viral-coat protein. These observations, together with the fact that in the same system these nucleotides affect coupled transcription and translation, lead us to surmise that the activities of pppGpp and ppGpp are exerted at the level of RNA polymerase activity.
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PMID:Effects of guanosine tetraphosphate, guanosine pentaphosphate, and beta-gamma methylenyl-guanosine pentaphosphate on gene expression of Escherichia coli in vitro. 435 31

We have determined the effects of the nusA gene protein and the regulatory nucleotide guanosine tetraphosphate (ppGpp) on pausing and termination of transcription in the leader region of the rrnB operon in vitro. The leader region of rrnB contains several types of potential regulatory sequences that act at the level of RNA chain elongation and may be involved in control of bacterial growth. We have mapped a termination site, tL, located 260 bases from rrnB promoter P1. Termination at tL is dependent on the nusA protein and is enhanced by ppGpp. The DNA sequence at tL shows striking homologies with trp t', a terminator also strongly affected in vitro by the nusA protein. These in vitro results suggest that rRNA transcription in vivo may be regulated in part through an attenuation mechanism that leads to termination of rrnB chains in the leader region. In addition to tL, elongation of transcription in the leader region is affected by several pause sites that are sensitive to the concentration of ppGpp and the presence of the nusA gene protein. The location and properties of these pause sites suggest that they may also play a role in regulation of rrnB transcription through a mechanism we have termed "turnstile" attenuation. One pause site, located 90 and 91 bases from P1, is unique in that it is not normally a site for transcriptional pausing, but is dependent on simultaneous binding of polymerase at rrnB promoter P2. This leads to blockage of P1 transcripts at high RNA polymerase densities and may provide an additional locus for regulation of rrn transcription.
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PMID:Pausing and attenuation of in vitro transcription in the rrnB operon of E. coli. 608 7


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