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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)

RNA polymerase molecules pause at a single site during in vitro transcription of the tryptophan (trp) operon leader region. Pausing was observed when DNA templates derived from Escherichia coli. Salmonella typhimurium, and Klebsiella aerogenes were used. Fingerprint analyses showed that the major RNA species produced by the transcriptional pause is 91 nucleotides long. A minor RNA species 90 nucleotides long was also detected. Single-round transcription experiments were used to study the kinetics of pausing. Time course, pulse-chase, and delayed-labeling experiments suggest that every RNA polymerase molecule transcribing the trp leader region pauses. A suboptimal ribonucleoside triphosphate concentrations, the half-life of paused-leader RNA was approximately 3 min at 22 degrees C and 0.7 min at 37 degrees C. At near-optimal ribonucleoside triphosphate concentrations, the half-time of the paused species dropped to about 0.3 min at 22 degrees C. The appearance and half-life of the paused species were unaffected by salt concentration, rho factor, guanosine 3'-5'-bis(diphosphate), or point mutations in the trp attenuator region. It is postulated that transcriptional pausing may play a role in maintaining the synchronization of transcription and translation that is vital in the control of transcription termination at the trp operon attenuator.
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PMID:Pausing of RNA polymerase during in vitro transcription of the tryptophan operon leader region. 616 81

A DNA oligomer 15 nucleotides long was used to probe the involvement of RNA secondary structure in the control of transcription termination at the attenuator of the tryptophan (trp) operon of Escherichia coli. This 15-mer is perfectly complementary to a segment of trp RNA that is thought to play a role in regulation of attenuation. When added to an in vitro transcription reaction mixture containing wild-type E. coli or Salmonella typhimurium trp operon templates, the complementary 15-mer caused a 4-fold increase in read-through transcription. By contrast, the 15-mer did not affect attenuation when a mutant E. coli template was used that does not allow formation of a crucial RNA secondary structure. Control experiments established that oligomers that were not complementary to E. coli trp leader RNA did not affect attenuation and that the 15-mer did not reduce termination when the transcript lacked a complementary region. Other experiments established that the 15-mer did not increase read-through transcription by allowing RNA polymerase molecules that might have already stopped at the attenuator to resume transcription. These findings provide direct support for the view that alternate base-paired structures control transcription termination at the trp attenuator.
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PMID:Transcription termination at the tryptophan operon attenuator is decreased in vitro by an oligomer complementary to a segment of the leader transcript. 617 92

Transcription of the tryptophan (trp) operon of Escherichia coli and other bacterial species is regulated by the formation of alternative secondary structures in the leader segment of the transcript. During in vitro transcription of the trp leader region RNA polymerase pauses at base pair 92 after synthesis of an RNA hairpin secondary structure. We studied the dependence of pausing on hairpin stability by examining mutant trp templates containing base pair substitutions in the region corresponding to the hairpin secondary structure. Base changes that lower the stability of the hairpin were found to reduce both the frequency and half-life of RNA polymerase pausing while base changes that do not affect hairpin stability had little effect on pausing. Pausing was enhanced by the nusA protein; this enhancement was greatly reduced on mutant templates specifying less stable hairpins. The frequency of pausing on some mutant templates was correlated with the extent of read-through transcription beyond the trp attenuator, suggesting a possible role for pausing in the coupling of transcription and translation during transcription of the leader region of the operon.
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PMID:Stability of an RNA secondary structure affects in vitro transcription pausing in the trp operon leader region. 620 69

In vivo, transcription of tryptophan (trp) operon mRNA appears to terminate at a site (trp t) 36 nucleotides after the last structural gene, and efficient function at this site requires the protein factor rho. However, distal nucleotide sequences also seem to play a role in modulating termination at trp t. We report here our in vitro studies of DNA fragments carrying portions of the trp termination region. Transcription of these DNA fragments in a purified system demonstrates that RNA polymerase actually recognizes two different termination sites. Termination at the previously characterized site, trp t, is only 25% efficient, and it is unaffected by the presence of rho factor in vitro. However, addition of rho to the transcription reaction mixture reveals that termination also occurs within a region that we have designated trp t', located about 250 bases past trp t. These two sites behave independently in vitro, whether in the tandem configuration or cloned separately, and their structural features and functional characteristics are quite different. This contrasts with the observation that termination of transcription at the end of the trp operon in vivo appears to require a rho-mediated interaction between trp t and trp t'. The possible involvement of other factors and the significance of multiple termination sites is discussed.
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PMID:Tandem termination sites in the tryptophan operon of Escherichia coli. 626 23

The DNA sequence of a cluster of twenty-one tRNA genes distal to a rRNA gene set in B. subtilis was determined. None of the tRNA genes are repeated in the sequence. The only classes of tRNAs that are not represented are those for cysteine, glutamine, tryptophan, and tyrosine. Three of the tRNA genes in this cluster do not have the 3'-CCA sequence encoded in the gene. There is no RNA polymerase terminator sequence in the region between the 5S gene and the first tRNA gene or within the tRNA gene cluster. A terminator sequence was found directly after the last tRNA gene. This rRNA and tRNA gene cluster probably represents one transcriptional unit. However, there may be an RNA polymerase promoter site within this sequence, which raises some interesting questions concerning the regulation of transcription for these tRNA genes.
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PMID:Sequence analysis of a cluster of twenty-one tRNA genes in Bacillus subtilis. 631 May 12

10(-4) cleavage of alpha-amanitin after the procedure of Wieland & Fahrmeir (1) but without prior protective methylation of the 6'-hydroxyl of the tryptophan residue affords the alpha-amanitin aldehyde in 45% yield. The aldehyde was found to exhibit Ki = 3.0 and 12 microM for Drosophila melanogaster and wheat germ RNA polymerase II, respectively. This value is approximately 100-fold greater than for the parent alpha-amanitin. Treatment of the alpha-amanitin aldehyde with 2,4-dinitrophenylhydrazine in CH3OH, CH3CN, or dimethylsulfoxide yielded three products. Two of these did not contain the 2,4-dinitrophenyl moiety, showed Ki = 3.3 and 0.26 microM for wheat germ RNA polymerase II (alpha-amanitin, Ki = 0.09 microM), and accounted for 30-60% and 3% of the input alpha-amanitin aldehyde, respectively. The alpha-amanitin-2,4 dinitrophenylhydrazone was recovered in less than 10% yield regardless of reaction condition and showed a Ki = 0.26 microM on wheat germ RNA polymerase II. This hydrazone establishes that the amatoxin molecule can be modified in the dihydroxyisoleucine residue without disruption of binding to the RNA polymerase.
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PMID:2,4-Dinitrophenylhydrazone formation at the aldehyde derived by periodate cleavage of alpha-amanitin. 631 83

The role of alternative RNA secondary structures in regulating transcription termination at the attenuator of the tryptophan (trp) operon of Serratia marcescens was examined in vitro by transcribing mutant DNA templates having deletions of different segments of the trp leader region. Deletions that removed sequences corresponding to successive segments of postulated RNA secondary structures either increased or decreased transcription termination at the attenuator. The results obtained are consistent with the hypothesis that transcription termination results from RNA polymerase recognition of a particular RNA secondary structure, the terminator. This structure forms only in the absence of an alternative, preceding, RNA secondary structure, the antiterminator.
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PMID:Transcription termination in vitro at the tryptophan operon attenuator is controlled by secondary structures in the leader transcript. 634 Jan 18

Using the in vitro mixed transcription system (Kajitani, M. and Ishihama, A. (1983) Nucleic Acids Res. 11, 671-686), we determined the two parameters of the promoter strength, i.e., the rate of open complex formation between RNA polymerase and promoter, and the saturation level of the open complex formation at equilibrium, for the promoters of ribosomal RNA (rrnE), ribosomal protein S1 (rpsA) and recA protein (recA) operons from Escherichia coli. Taken together with the previous determinations for lactose (lac(UV5)), tryptophan (trp) and ribosomal protein L10 (rp1J) operons, these studies revealed that the relative promoter strengths with respect to the kinetic parameter are 200, 70, 50, 40, 30, 20 and 2% of the reference promoter lacP(UV5) for recAp, rp1Jp, rpsAp3, trpP, rpsAp1, rrnEp1 and rrnEp2, respectively, under our standard reaction conditions (50 mM NaCl and 37 degrees C); and those with respect to the thermodynamic parameter are 70, 35, 20, 10, 10, 10 and 5% the level of lacP(UV5) for rrnEp2, trpP, rpsAp3, rp1Jp, rpsAp1, rrnEp1 and recAp, respectively. The order of the promoter strength, however, changes with variation of the salt concentration or reaction temperature.
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PMID:Determination of the promoter strength in the mixed transcription system. II. Promoters of ribosomal RNA, ribosomal protein S1 and recA protein operons from Escherichia coli. 634 67

The formation of alternative secondary structures in the transcript of the tryptophan (trp) operon leader region regulates expression of the trp operons of Escherichia coli and other bacterial species. During in vitro transcription RNA polymerase pauses near base pair 90 after the first hairpin secondary structure in E. coli trp leader mRNA is formed. The E. coli L-factor enhances transcription pausing at this site (Farnham, P. J., Greenblatt, J., and Platt, T. (1982) Cell 29, 945-951); presumably it does so by facilitating recognition of the RNA hairpin by polymerase. We show that addition of a DNA oligomer complementary to the proximal segment of the RNA hairpin relieves transcription pausing in vitro both in the presence and absence of L-factor. The oligomer apparently interferes with formation of the RNA hairpin which we believe is recognized by polymerase as the pause signal. The oligomer also relieves pausing in L-factor-induced paused complexes, suggesting that the oligomer can disrupt a preformed secondary structure in the transcript.
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PMID:A complementary DNA oligomer releases a transcription pause complex. 634 40

Using the in vitro mixed transcription system (Kajitani, M., and Ishihama, A. (1983) Nucleic Acids Res. 11, 671-686; Kajitani, M., and Ishihama, A. (1983) Nucleic Acids Res., 11, 3873-3889) we examined the effect of guanosine 3'-diphosphate, 5'-diphosphate (ppGpp), the chemical mediator of stringent control, on transcription of various Escherichia coli DNA fragments, each carrying a single specific promoter. We found that ppGpp inhibits transcription of stringently controlled genes, rrnE, rpsA, and rplJ, coding for ribosomal RNA, ribosomal protein S1 and L10, respectively, but not that of trp (tryptophan) and lacUV5 (lactose) genes. Among the multiple promoters of the rrnE and rpsA operons, the upstream promoters, rrnEp1 and rpsAp1, are subject to repression by ppGpp but the downstream promoters, rrnEp2 and rpsAp3, are insensitive. Taking these facts and the intrinsic strength of the respective promoters together, we suggest that the multiple promoters within the single and same operons play different physiological roles and are regulated by independent mechanisms. The inhibition by ppGpp takes place even after formation of open complexes, suggesting that the RNA polymerase bound to the sensitive promoters is accessible for interaction with ppGpp leading to rapid decay of the open complexes. During this study, we noticed that some promoters including recAp are activated in the presence of ppGpp, raising a possibility that ppGpp has dual effects on the promoter function.
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PMID:Promoter selectivity of Escherichia coli RNA polymerase. Differential stringent control of the multiple promoters from ribosomal RNA and protein operons. 636 18


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