Two regions of GerE required for promoter activation in Bacillus subtilis.
GerE from Bacillus subtilis is the smallest member of the LuxR-FixJ family of transcription activators. Its 74-amino-acid sequence is similar over its entire length to the DNA binding domain of this protein family, including a putative helix-turn-helix (HTH) motif. In this report, we sought to define regions of GerE involved in promoter activation. We examined the effects of single alanine substitutions at 19 positions that were predicted by the crystal structure of GerE to be located on its surface. A single substitution of alanine for the phenylalanine at position 6 of GerE (F6A) resulted in decreased transcription in vivo and in vitro from the GerE-dependent cotC promoter. However, the F6A substitution had little effect on transcription from the GerE-dependent cotX promoter. In contrast, a single alanine substitution for the leucine at position 67 (L67A) reduced transcription from the cotX promoter, but not from the cotC promoter. The results of DNase I protection assays and in vitro transcription reactions lead us to suggest that the F6A and L67A substitutions define two regions of GerE, activation region 1 (AR1) and AR2, that are required for activation of the cotC and cotX promoters, respectively. A comparison of our results with those from studies of MalT and BvgA indicated that other members of the LuxR-FixJ family may use more than one surface to interact with RNA polymerase during promoter activation.
Crater
Dinene L
DL
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Moran
Charles P
CP
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor; D014157:Transcription Factors / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C081620:cotX protein, Bacillus subtilis; C092468:spoIIR protein, Bacillus subtilis; C030069:spore-specific proteins, Bacillus; D000409:Alanine