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
L V
LV
Department of Microbiology, Boston University School of Medicine, 715 Albany Street, Boston, MA, 02118, USA.
Zalieckas
J M
JM
Fisher
S H
SH
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000166:cytology / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D051922:PII Nitrogen Regulatory Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012097:Repressor Proteins; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C085245:NrgA protein, Bacillus subtilis; C085246:NrgB protein, Bacillus subtilis; D051922:PII Nitrogen Regulatory Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C101458:ScgR protein, Bacillus subtilis; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases