Purification and in vitro activities of the Bacillus subtilis TnrA transcription factor.

The Bacillus subtilis nitrogen regulatory protein TnrA was purified and its interaction with the nrgAB regulatory region examined. The TnrA protein activates transcription from the nrgAB promoter in vitro. DNase I footprinting and methylation protection experiments demonstrated that TnrA binds to an inverted repeat, upstream of the -35 region of the nrgAB promoter. Gel mobility retardation assays were used to determine the affinity of TnrA for its DNA-binding site. The equilibrium dissociation binding constant for the interaction of TnrA with the nrgAB promoter fragment was 7.7 nM under the conditions used here. Mutations in the TnrA consensus sequence that reduce nrgAB expression in vivo were found to reduce significantly the in vitro affinity for TnrA. An A+T rich region located upstream of the TnrA-binding site was found to be necessary for optimal transcriptional activation. A mutant protein, TnrA(HTH), was constructed in which the putative helix-turn-helix DNA-binding motif was altered by exchanging two arginine residues for alanine residues. The TnrA(HTH) protein was unable to activate the in vivo expression of nrgAB and had an in vitro affinity for the nrgAB promoter that was significantly lower than that of the wild-type protein.

Wray LV Jr ，Zalieckas JM ，Fisher SH 《JOURNAL OF MOLECULAR BIOLOGY》

Mutational analysis of the TnrA-binding sites in the Bacillus subtilis nrgAB and gabP promoter regions.

Wray LV Jr ，Zalieckas JM ，Ferson AE ，Fisher SH ... -  《-》

Cross-regulation of the Bacillus subtilis glnRA and tnrA genes provides evidence for DNA binding site discrimination by GlnR and TnrA.

Zalieckas JM ，Wray LV Jr ，Fisher SH 《-》

TnrA, a transcription factor required for global nitrogen regulation in Bacillus subtilis.

Wray LV Jr ，Ferson AE ，Rohrer K ，Fisher SH ... -  《-》

Analysis of a cell-cycle promoter bound by a response regulator.

Caulobacter crescentus CtrA protein, an OmpR-type response regulator, receives cell-cycle signals and binds the proposed consensus TTAA-N7-TTAA present inside the chromosome replication origin and cell-cycle transcription promoters. We synthesized a 42 bp cell-cycle promoter based on this consensus and elements of the fliL promoter. Over 100 promoter mutations were assayed for transcription directed by CtrA. Although both CtrA binding half-sites cooperate and the N7 spacing is critical for transcription, the upstream half-site is relatively flexible. The downstream CtrA half-site is less flexible and more important for cell-cycle regulation. A CtrA binding site and a -10 promoter element are sufficient for cell-cycle transcription, and both sequences cooperate and compensate for respective defects. Mutations in the CtrA binding site, but not in the -10 promoter sequence, perturb cell-cycle transcription. A single base-pair change switches a cell-cycle promoter into a strong conventional promoter. We propose rules for CtrA binding and promoter interactions implying how CtrA evolved into a versatile regulator of cell-cycle functions including flagellar biogenesis, cell division, DNA methylation, and chromosome replication.

Ouimet MC ，Marczynski GT 《JOURNAL OF MOLECULAR BIOLOGY》