Identification of a DNA binding region in GerE from Bacillus subtilis.
Proteins that have a structure similar to those of LuxR and FixJ comprise a large subfamily of transcriptional activator proteins. Most members of the LuxR-FixJ family contain a similar amino-terminal receiver domain linked by a small region to a carboxy-terminal domain that contains an amino acid sequence similar to the helix-turn-helix (HTH) motif found in other DNA-binding proteins. GerE from Bacillus subtilis is the smallest member of the LuxR-FixJ family. Its 74-amino-acid sequence is similar over its entire length to the DNA binding region of this protein family, including the HTH motif. Therefore, GerE provides a simple model for studies of the role of this HTH domain in DNA binding. Toward this aim, we sought to identify the amino acids within this motif that are important for the specificity of binding to DNA. We examined the effects of single base pair substitutions in the high-affinity GerE binding site on the sigK promoter and found that nucleotides at positions +2, +3, and +4 relative to the transcription start site on the sigK promoter are important for a high-affinity interaction with GerE. We next examined the effects of single alanine substitutions at two positions in the HTH region of GerE on binding to wild-type or mutant target sites. We found that the substitution of an alanine for the threonine at position 42 of GerE produced a protein that binds with equal affinity to two sites that differ by 1 bp, whereas wild-type GerE binds with different affinities to these two sites. These results provide evidence that the amino acyl residues in or near the putative HTH region of GerE and potentially other members of the LuxR-FixJ family determine the specificity of DNA binding.
Crater
D L
DL
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Moran
C P
CP
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019077:Helix-Turn-Helix Motifs; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor; D014157:Transcription Factors / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C084190:sigma K; C092468:spoIIR protein, Bacillus subtilis; C030069:spore-specific proteins, Bacillus