Transcriptional regulation of the ilv-leu operon of Bacillus subtilis

Abstract

We used primer extension and mutational analysis to identify a promoter upstream of iIvB, the first gene in the ilv-keu operon of Bacilus subtilis. Between the promoter and ilvB, there is a 482-bp leader region which contains a sequence that resembles a factor-independent transcription terminator. In in vitro transcription experiments, 90%o of transcripts initiated at the ilvB promoter ended at a site near this terminator. Primer extension analysis of RNA synthesized in vivo showed that the steady-state level of mRNA upstream of the terminator was twofold higher from cells limited for leucine than it was from cells grown with excess leucine. mRNA downstream of the terminator was 14-fold higher in cells limited for leucine than in cells grown with excess leucine. Measurement of mRNA degradation rates showed that the half-life of ilv-leu mRNA was the same when the cells were grown with or without leucine. These data demonstrate that the ilv-Iu operon is regulated by transcription attenuation.In Bacillus subtilis, 7 of the 10 genes required for synthesis of leucine, isoleucine, and valine form a single operon called the ilv-leu operon (15). The operon was cloned, and a putative promoter, a leader region, and the genes ilvB, ilvN, and ilvC were sequenced (14). By using transcriptional fusions of the ilv-leu regulatory region to the lacZ gene of Escherichia coli, Vandeyar et al. found that ilv-leu promoter expression was 10-fold higher in cells starved for leucine than in cells grown with excess leucine. Starvation for isoleucine and valine had no such effect (14). In this article, we identify the ilv-leu operon promoter and show that, between the promoter and the first structural gene, there is a 482-bp leader region with a sequence resembling a factorindependent transcription termination site. We demonstrate that most transcription terminates at or near this site in vitro and also in vivo when cells are grown in a medium containing excess leucine. MATERIALS AND METHODSBacterial strains and growth conditions. B. subtilis CU4609 (trpC2 leuB16 ilvN::Tn917) was used for leucine limitation experiments. B. subtilis CU1065 (trpC2) was a recipient strain for promoter insertion mutagenesis. E. coli XL1 Blue [recA4 lac endAI gyrA96 thi hsdRl7supE44 relAI (F'proAB lacIq lacZAM15 TnlO)] (Stratagene) was used for plasmid construction. Complex medium was LB broth containing 50 jig of ampicillin per ml when required. Minimal medium contained 0.5% glucose, 1 ,ug of biotin per ml, 20 ,ug of tryptophan per ml, 35 ,ug of isoleucine per ml, 70 ,ug of valine per ml, and a salts mixture to produce final concentrations of, per liter, 2 g of (NH4)2SO4, 13.8 g of K2HPO4 3H20, 6 g of KH2PO4, 0.2 mg of MnCl2 .4H20, 1 g of sodium citrate, and 0.2 g of MgSO4. Cultures were incubated at 37°C and aerated by shaking in baffled flasks.Plasmid construction. A 791-bp polymerase chain reaction product which contained sequences 240 bp upstream and 544

Regions of the Bacillus subtilis ilv-leu operon involved in regulation by leucine

“…Figure 6B shows that with RNA from the strain containing construct ACwt as template, primer B produced a cDNA product that was about 310 nucleotides (nt) long, indicating that transcription started at the + 1 nucleotide identified previously (Fig. 6B, lane 1, arrow A) (4). Extension of primer Z produced a cDNA about 200 nt long, corresponding to an endpoint just downstream of the proposed stem and loop of the terminator (Fig.…”

“…The effects of the azAL115, azlA152, and azlA153 mutations on ilv-leu transcription were determined by comparing ,B-galactosidase activities in the wild-type and mutant strains under conditions of leucine limitation and leucine excess (4). Cell cultures were limited for leucine by substituting N-acetyl leucine for leucine.…”

“…Transcription of this operon is repressed by leucine but not by isoleucine or valine (21). In work published previously we showed that transcription is initiated 482 bp upstream of the start codon for ilvB, the first gene in the operon (4).…”

“…Ninety percent of transcripts initiated at the promoter in vitro were terminated at this site. Analysis of mRNA levels in vivo suggested that repression by leucine occurs by attenuation of transcription within the 482-bp leader region and that the transcription terminator within the ilv-leu leader region is involved in the mechanism of leucine control (4).…”

Qualitative Reasoning for Reaction Networks with Partial Kinetic Information

“…Therefore the presence of CcpA slows down reaction r 4 [1,16] but it does not block it on average in a steady state. The promoter PIlv−Leu is also down-regulated by Leu in terms of a T-box [1,4], which is captured by the negative control of the reaction r 5 by Leu. Protein TnrA forms a further inhibitor whose impact on the PIlv−Leu promoter is represented by the binding side BS TnrA through the reaction r 7 .…”