PhoP~P and RNA polymerase σA holoenzyme are sufficient for transcription of Pho regulon promoters in Bacillus subtilis: PhoP~P activator sites within the coding region stimulate transcription in vitro

Ying, Qi; Hulett, F. Marion

doi:10.1046/j.1365-2958.1998.00882.x

Cited by 134 publications

(118 citation statements)

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“…The B. subtilis pst operon is induced in response to phosphate starvation from a single sigma A-type promoter upstream of pstS in a PhoPR-dependent manner (Qi & Hulett, 1998). This is confirmed by reporter gene experiments in which the first (pstS) and last (pstBB) genes in the operon showed virtually identical activity profiles (Fig.…”

Section: Discussionsupporting

confidence: 57%

Post-transcriptional regulation of the Bacillus subtilis pst operon encoding a phosphate-specific ABC transporter

et al. 2004

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During phosphate starvation, Bacillus subtilis regulates genes in the PhoP regulon to reduce the cell's requirement for this essential substrate and to facilitate the recovery of inorganic phosphate from organic sources such as teichoic and nucleic acids. Among the proteins that are highly induced under these conditions is PstS, the phosphate-binding lipoprotein component of a high-affinity ABC-type phosphate transporter. PstS is encoded by the first gene in the pst operon, the other four members of which encode the integral membrane and cytoplasmic components of the transporter. The transcription of the pst operon was analysed using a combination of methods, including transcriptional reporter gene technology, Northern blotting and DNA arrays. It is shown that the primary transcript of the pst operon is processed differentially to maintain higher concentrations of PstS relative to other components of the transporter. The comparative studies have revealed limitations in the use of reporter gene technology for analysing the transcription of operons in which the messenger RNA transcript is differentially processed.

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Section: Discussionsupporting

confidence: 57%

Post-transcriptional regulation of the Bacillus subtilis pst operon encoding a phosphate-specific ABC transporter

et al. 2004

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“…His-tagged B. subtilis RNAP was isolated as described by Qi and Hulett (19). E. coli RNAP, prepared as described elsewhere (12), was provided FIG.…”

Section: Methodsmentioning

confidence: 99%

Kinetic Analysis of tRNA-Directed Transcription Antitermination of the Bacillus subtilis glyQS Gene In Vitro

Grundy

Henkin

2004

J Bacteriol

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Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription antitermination mechanism. Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA. tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix. tRNA Gly -dependent antitermination of the Bacillus subtilis glyQS leader was previously demonstrated in a purified in vitro assay system. This assay system was used to investigate the kinetics of transcription through the glyQS leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and antitermination. Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on antitermination efficiency was analyzed. We found that addition of tRNA Gly can promote antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition.The rate of transcription elongation plays a crucial role in many genetic regulatory systems that operate at the level of premature termination of transcription. Pausing of RNA polymerase (RNAP) allows interaction of the transcription elongation complex (TEC) with cis-acting elements within the nascent transcript or with trans-acting factors that modulate the structure of the RNA or the properties of the TEC. The rate of transcription elongation in vivo has been estimated at 40 to 50 nucleotides (nt)/s (17, 23). The decision between termination and antitermination is therefore made within a very short timeframe. This is especially important in systems in which the nascent transcript folds into alternate terminator and antiterminator structures (13). In these cases, termination can be prevented by sequestration of the 5Ј portion of the terminator helix by pairing with upstream sequences to form the antiterminator element. It is likely that formation or stabilization of the antiterminator element must occur before synthesis of the 3Ј portion of the terminator helix; once the terminator helix is established in the appropriate position within the TEC, the residues participating in this helix are unlikely to be available for formation of the antiterminator element. Pausing of the TEC prior to synthesis of the complete terminator helix may therefore be required to allow an opportunity to monitor the relevant regulatory signal. A role for pausing has been established or postulated in a number of termination control systems, including the Escherichia coli trp operon attenuation system (14), L4-mediated attenuation in the E. coli S10 operon (22), the Bacillus subtilis TRAP (24) and PyrR (25) systems, and the E. coli ...

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“…The His tags were cleaved by factor Xa treatment, whereas the GST tag was removed by thrombin cleavage (29). For preparation of the B. subtilis His 6 -tagged RNA polymerase ( His6 RNAP) core enzyme (E), B. subtilis strain MH5636 (38) was grown in 12 1-liter portions of LB medium. Cells were collected at the end of the exponential phase of growth.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were collected at the end of the exponential phase of growth. Cell lysis and RNAP core enzyme purification were performed as previously described (38).…”

Section: Methodsmentioning

confidence: 99%

Bacillus subtilisPhosphorylated PhoP: Direct Activation of the Eσ^A- and Repression of the Eσ^E-ResponsivephoB-P_S+VPromoters during Pho Response

et al. 2005

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The phoB gene of Bacillus subtilis encodes an alkaline phosphatase (PhoB, formerly alkaline phosphatase III) that is expressed from separate promoters during phosphate deprivation in a PhoP-PhoR-dependent manner and at stage two of sporulation under phosphate-sufficient conditions independent of PhoP-PhoR. Isogenic strains containing either the complete phoB promoter or individual phoB promoter fusions were used to assess expression from each promoter under both induction conditions. The phoB promoter responsible for expression during sporulation, phoB-P S , was expressed in a wild-type strain during phosphate deprivation, but induction occurred >3 h later than induction of Pho regulon genes and the levels were approximately 50-fold lower than that observed for the PhoPR-dependent promoter, phoB-P V . E E was necessary and sufficient for P S expression in vitro. P S expression in a phoPR mutant strain was delayed 2 to 3 h compared to the expression in a wild-type strain, suggesting that expression or activation of E is delayed in a phoPR mutant under phosphate-deficient conditions, an observation consistent with a role for PhoPR in spore development under these conditions. Phosphorylated PhoP (PhoPϳP) repressed P S in vitro via direct binding to the promoter, the first example of an E E -responsive promoter that is repressed by PhoPϳP. Whereas either PhoP or PhoPϳP in the presence of E A was sufficient to stimulate transcription from the phoB-P V promoter in vitro, roughly 10-and 17-fold-higher concentrations of PhoP than of PhoPϳP were required for P V promoter activation and maximal promoter activity, respectively. The promoter for a second gene in the Pho regulon, ykoL, was also activated by elevated concentrations of unphosphorylated PhoP in vitro. However, because no Pho regulon gene expression was observed in vivo during P i -replete growth and PhoP concentrations increased only threefold in vivo during phoPR autoinduction, a role for unphosphorylated PhoP in Pho regulon activation in vivo is not likely.

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PhoP~P and RNA polymerase σ^A holoenzyme are sufficient for transcription of Pho regulon promoters in Bacillus subtilis: PhoP~P activator sites within the coding region stimulate transcription in vitro

Cited by 134 publications

References 39 publications

Post-transcriptional regulation of the Bacillus subtilis pst operon encoding a phosphate-specific ABC transporter

Post-transcriptional regulation of the Bacillus subtilis pst operon encoding a phosphate-specific ABC transporter

Kinetic Analysis of tRNA-Directed Transcription Antitermination of the Bacillus subtilis glyQS Gene In Vitro

Bacillus subtilisPhosphorylated PhoP: Direct Activation of the Eσ^A- and Repression of the Eσ^E-ResponsivephoB-P_S+VPromoters during Pho Response

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PhoP~P and RNA polymerase σA holoenzyme are sufficient for transcription of Pho regulon promoters in Bacillus subtilis: PhoP~P activator sites within the coding region stimulate transcription in vitro

Cited by 134 publications

References 39 publications

Post-transcriptional regulation of the Bacillus subtilis pst operon encoding a phosphate-specific ABC transporter

Post-transcriptional regulation of the Bacillus subtilis pst operon encoding a phosphate-specific ABC transporter

Kinetic Analysis of tRNA-Directed Transcription Antitermination of the Bacillus subtilis glyQS Gene In Vitro

Bacillus subtilisPhosphorylated PhoP: Direct Activation of the EσA- and Repression of the EσE-ResponsivephoB-PS+VPromoters during Pho Response

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PhoP~P and RNA polymerase σ^A holoenzyme are sufficient for transcription of Pho regulon promoters in Bacillus subtilis: PhoP~P activator sites within the coding region stimulate transcription in vitro

Bacillus subtilisPhosphorylated PhoP: Direct Activation of the Eσ^A- and Repression of the Eσ^E-ResponsivephoB-P_S+VPromoters during Pho Response