Defective antitermination of rRNA transcription and derepression of rRNA and tRNA synthesis in the nusB5 mutant of Escherichia coli.

Sharrock, Robert A.; Gourse, Richard L.; Nomura, Masayasu

doi:10.1073/pnas.82.16.5275

Cited by 64 publications

(73 citation statements)

References 32 publications

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“…Unlike the G+C-rich sequence, however, these other determinants and the spacer sequence are not as strongly conserved among tRNA promoters (27). From gene dosage experiments, it appears that tRNA operons are under the control of the same negative feedback system that regulates rRNA promoters (16,17,28,39). Since their promoter sequences differ from those of the rRNAs, it could be that tRNAs are regulated slightly differently than rRNAs, consistent with the observation that there are small but reproducible differences between rRNA and tRNA promoters in the level of repression observed in the gene dosage experiments cited above.…”

Section: Discussionsupporting

confidence: 73%

Identification of promoter mutants defective in growth-rate-dependent regulation of rRNA transcription in Escherichia coli

et al. 1989

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We measured the activities of 50 operon fusions from a collection of mutant and wild-type rrnB P1 (rrnBlp in the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoters under different nutritional conditions in order to analyze the DNA sequence determinants of growth rate-dependent regulation of rRNA transcription in Escherichia coli. Mutants which deviated from the wild-type -10 or -35 hexamers or from the wild-type 16-base-pair spacer length between the hexamers were unregulated, regardless of whether the mutations brought the promoters closer to the E. coli promoter consensus sequence and increased activity or whether the changes took the promoters further away from the consensus and reduced activity. These data suggest that rRNA promoters have evolved to maintain their regulatory abilities rather than to maximize promoter strength. Some double substitutions outside the consensus hexamers were almost completely unregulated, while single substitutions at several positions outside the -10 and -35 consensus hexamers exerted smaller but significant effects on regulation. These studies suggest roles for specific promoter sequences and/or structures in interactions with regulatory molecules and suggest experimental tests for models of rRNA regulation.Ribosome synthesis rates in Escherichia coli are a direct function of rRNA transcription rates under most growth conditions (26,34). In order to meet the cell's requirements for protein synthesis, cells transcribe rRNA and tRNA at approximately the square of the growth rate, a phenomenon termed growth rate-dependent regulation (29).The mechanism responsible for growth rate control remains unclear. Previously, it was shown that a negative feedback system is responsible for rRNA and tRNA regulation (15-17, 28, 39) and that the system responds to the level of ribosomes that are capable of translation (10, 49). The magnitude of the signal made in response to the cell's translational capacity varies with the nutritional state of the culture. The identity of the signal is not known, although guanosine tetraphosphate (ppGpp) has been implicated as playing some role because of the virtually perfect inverse correlation between stable RNA synthesis and ppGpp concentration (36).The target of the signal, whatever its identity, has been shown in at least three of the seven rRNA operons to be P1, the more upstream of the two rRNA promoters (rrnB and rrnE [15]; rrnA [37] there is a region called the upstream activation sequence (UAS), which is required for maximal promoter activity (4,15,23).Mutations have been targeted to specific sites in the promoters rrnB P1 and tyrT, and activities resulting from the fusion of the mutant promoters to "reporter" genes have been measured under different growth conditions (15,46). Such experiments have implicated DNA sequences required for growth rate-dependent control in the region between -20 and -50 (15) and in the region just downstream of the -10 hexamer (46). The mutations examined in both studies contained ...

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Section: Discussionsupporting

confidence: 73%

Identification of promoter mutants defective in growth-rate-dependent regulation of rRNA transcription in Escherichia coli

et al. 1989

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“…3 may suggest that transcription initiation is increased in the topA null mutant not overproducing RNase H (compare MA249, MA251, and MA251/pSK760). As shown before (26), it can also be seen that rRNA transcription initiation is increased in the nusB5 mutant (Fig. 3, compare K37 and K450).…”

Section: Resultssupporting

confidence: 54%

“…In order to show that our approach can indeed be used to reveal problems at the level of transcription elongation, we included a pair of isogenic strains, K37 and K450. The latter carries the nusB5 mutation and was shown previously to be defective in antitermination of rRNA transcription (26). One consequence of such a defect is an increase in both rRNA transcription initiation and in the rate of 16 S rRNA synthesis (26).…”

Section: Resultsmentioning

confidence: 99%

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RNase H Overproduction Corrects a Defect at the Level of Transcription Elongation during rRNA Synthesis in the Absence of DNA Topoisomerase I in Escherichia coli

Hraiky

Raymond

Drolet

2000

Journal of Biological Chemistry

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It has been suggested that the major function of DNA topoisomerase I in Escherichia coli is to suppress the formation of R-loops, which could inhibit growth. Although the currently available data suggest that the inhibitory effect of R-loops is exerted at the level of gene expression, this has never been demonstrated. In the present report, we show that rRNA synthesis is significantly impaired at the level of transcription elongation in a bacterial strain lacking DNA topoisomerase I. We found that this inhibition is due to transcriptional blocks. RNase H overproduction is also shown to considerably reduce the extent of such transcriptional blocks during rRNA synthesis. Moreover, one of these transcriptional blockage sites is located within a region where extensive R-loop formation was previously shown to occur on a plasmid DNA in the absence of DNA topoisomerase I. Together, these results allow us to propose that an important function of DNA topoisomerase I is to inhibit the formation of R-loops, which may otherwise translate into roadblocks for RNA polymerases. Our results also highlight the potential regulatory role of DNA supercoiling at the level of transcription elongation.Escherichia coli DNA topoisomerase I, a member of the type IA family of topoisomerases, specifically relaxes negatively supercoiled DNA (1, 2). This specificity is explained by the fact that this enzyme binds to the junction of single-stranded and double-stranded DNA regions. DNA opening, and hence the generation of single-stranded DNA regions, is promoted by negative but not positive supercoiling. Hot spots for relaxation by DNA topoisomerase I are provided during transcription elongation in the frame of the twin-domain model (3). Indeed, very high levels of negative supercoiling can be generated behind the moving RNA polymerase during transcription elongation (4, 5). An R-loop, in which the template strand is paired with the nascent RNA, leaving the nontemplate strand unpaired, also provides a hot spot for relaxation by this enzyme (6).The accumulated evidence over the last few years has allowed us to conclude that a major function of DNA topoisomerase I in E. coli is to inhibit R-loop formation during transcription elongation. Indeed, the growth problem of topA (encoding DNA topoisomerase I) null mutants was shown to be partially corrected by overproducing RNase H, an enzyme that degrades the RNA moiety of an R-loop (7). A correlation was also established between the level of DNA gyrase activity, the enzyme that introduces negative supercoiling within the chromosomal DNA, and the amount of RNase H required to stimulate the growth of topA null mutants (7) and to inhibit R-loop formation during transcription (8). The finding that several topA null mutants carry compensatory gyr mutations (in gyrA or gyrB) that reduce DNA gyrase activity and correct their growth defect (9, 10) was therefore explained by the supercoiling activity of DNA gyrase, which promotes R-loop formation (7). On the contrary, DNA topoisomerase I activity inhibits R-lo...

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“…3), or by spectinomycin or chloramphenicol treatment of wild-type cells grown in minimal or complex medium ( In wild-type cells, the rRNA synthesis rate is finely tuned to the cell's nutritional environment, yet remains remarkably constant following most genetic manipulations that might be expected to perturb it. For example, when the rRNA gene dose was altered by adding rRNA operons on plasmids or by inactivating chromosomal rRNA operons (35)(36)(37), when rRNA transcription initiation was altered by deletion of the fis gene or by mutation of the gene coding for the RNAP ␣-subunit (6, 7), or when rRNA transcription elongation was compromised by mutation of genes coding for Nus factors (38), rRNA core promoter activity changed to keep the overall rRNA synthesis rate appropriate for the growth rate.…”

Section: Ntp-sensing Plays a Role In Homeostaticmentioning

confidence: 99%

NTP-sensing by rRNA promoters in Escherichia coli is direct

Schneider

Gaál

Gourse

2002

Proc. Natl. Acad. Sci. U.S.A.

105

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We showed previously that rrn P1 promoters require unusually high concentrations of the initiating nucleoside triphosphates (ATP or GTP, depending on the promoter) for maximal transcription in vitro. We proposed that this requirement for high initiating NTP concentrations contributes to control of the rrn P1 promoters from the seven Escherichia coli rRNA operons. However, the previous studies did not prove that variation in NTP concentration affects rrn P1 promoter activity directly in vivo. Here, we create conditions in vivo in which ATP and GTP concentrations are altered in opposite directions relative to one another, and we show that transcription from rrn P1 promoters that initiate with either ATP or GTP follows the concentration of the initiating NTP for that promoter. These results demonstrate that the effect of initiating NTP concentration on rrn P1 promoter activity in vivo is direct. As predicted by a model in which homeostatic control of rRNA transcription results, at least in part, from sensing of NTP concentrations by rrn P1 promoters, we show that inhibition of protein synthesis results in an increase in ATP concentration and a corresponding increase in transcription from rrnB P1. We conclude that translation is a major consumer of purine NTPs, and that NTP-sensing by rrn P1 promoters serves as a direct regulatory link between translation and ribosome synthesis.B ecause overexpression of ribosomes would be energetically costly, whereas underexpression would prevent the cell from taking full advantage of its nutritional environment, ribosome synthesis is regulated with the demand for protein synthesis. rRNA transcription is the rate-limiting step in ribosome synthesis in Escherichia coli and is controlled by several regulatory mechanisms acting at the level of transcription initiation (1, 2). In addition, an antitermination system ensures efficient rRNA transcription elongation (3).Each of the seven rRNA (rrn) operons in E. coli has two promoters, P1 and P2. The P1 promoters are responsible for the majority of rRNA transcription at moderate to fast growth rates and have been characterized extensively. Much of the intrinsic strength of the rrn P1 promoters results from AϩT-rich sequences (UP elements) upstream of the core promoters that recruit RNA polymerase (RNAP) to the promoter through specific interactions with the RNAP ␣-subunit (4-6). At least two trans-acting proteins affect rRNA transcription. Fis activates transcription from each of the 7 rrn P1 promoters by binding to sites upstream of Ϫ60 relative to the transcription start site, ϩ1 (4, 7), whereas H-NS contributes to repression of rrn P1 promoters during stationary phase (8).Although UP elements and Fis sites are required for maximal strength, rrn P1 promoters lacking these sequences (core promoters) are still regulated in response to the cell's nutritional environment (9, 10). Consistent with this finding, cells lacking the fis gene regulate transcription from rrn P1 promoters similarly to wild-type strains, because feedback systems comp...

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Defective antitermination of rRNA transcription and derepression of rRNA and tRNA synthesis in the nusB5 mutant of Escherichia coli.

Cited by 64 publications

References 32 publications

Identification of promoter mutants defective in growth-rate-dependent regulation of rRNA transcription in Escherichia coli

Identification of promoter mutants defective in growth-rate-dependent regulation of rRNA transcription in Escherichia coli

RNase H Overproduction Corrects a Defect at the Level of Transcription Elongation during rRNA Synthesis in the Absence of DNA Topoisomerase I in Escherichia coli

NTP-sensing by rRNA promoters in Escherichia coli is direct

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