MUTATIONAL ANALYSIS OF THE TNRA-BINDING SITES IN THE BACILLUS-SUBTILIS NRGAB AND GABP PROMOTER REGIONS

Transcription of the Bacillus subtilis nrgAB promoter is activated dur ing nitrogen-limited growth by the TnrA protein. A common inverted rep eat, TGTNAN(7)TNACA (TnrA site), is centered 49 to 51 bp upstream of t he transcriptional start sites for the TnrA-regulated nrgAB, gabP P2, and nas promoters. Oligonucleotide-directed mutagenesis of the nrgAB p romoter region showed that conserved nucleotides within the TnrA site, the A+T-rich region between the two TnrA half-sites, and an upstream A tract are all required for high-level activation of nrgAB expression . Mutations that alter the relative distance between the two half-site s of the nrgAB TnrA site abolish nitrogen regulation of nrgAB expressi on. Spacer mutations that change the relative distance between the Tnr A site and -35 region of the nrgAB promoter reveal that activation of nrgAB expression occurs only when the TnrA site is located 49 to 51 bp upstream of the transcriptional start site. Mutational analysis of th e conserved nucleotides in the gabP P2 TnrA site shelved that this seq uence is also required for nitrogen-regulated gabP P2 expression. The TnrA protein, expressed in an overproducing Escherichia coli strain, h ad a 625-fold-higher affinity for the wild-type nrgAB promoter DNA tha n for a mutated nrgAB promoter DNA fragment that is unable to activate nrgAB expression in vivo. These results indicate that the proposed Tn rA site functions as the binding site for the TnrA protein. TnrA was f ound to activate nrgAB expression during late exponential growth in nu trient sporulation medium containing glucose, suggesting that cells be come nitrogen limited during growth in this medium.