Two regions of Bacillus subtilis transcription factor SpoIIID allow a monomer to bind DNA.
Nutrient limitation causes Bacillus subtilis to develop into two different cell types, a mother cell and a spore. SpoIIID is a key regulator of transcription in the mother cell and positively or negatively regulates more than 100 genes, in many cases by binding to the promoter region. SpoIIID was predicted to have a helix-turn-helix motif for sequence-specific DNA binding, and a 10-bp consensus sequence was recognized in binding sites, but some strong binding sites were observed to contain more than one match to the consensus sequence, suggesting that SpoIIID might bind as a dimer or cooperatively as monomers. Here we show that SpoIIID binds with high affinity as a monomer to a single copy of its recognition sequence. Using charge reversal substitutions of residues likely to be exposed on the surface of SpoIIID and assays for transcriptional activation in vivo and for DNA binding in vitro, we identify two regions essential for DNA binding, the putative recognition helix of the predicted helix-turn-helix motif and a basic region near the C terminus. SpoIIID is unusual among prokaryotic DNA-binding proteins with a single helix-turn-helix motif in its ability to bind DNA monomerically with high affinity. We propose that the C-terminal basic region of SpoIIID makes additional contacts with DNA, analogous to the N-terminal arm of eukaryotic homeodomain proteins and the "wings" of winged-helix proteins, but structurally distinct. SpoIIID is highly conserved only among bacteria that form endospores, including several important human pathogens. The need to conserve biosynthetic capacity during endospore formation might have favored the evolution of a small transcription factor capable of high-affinity binding to DNA as a monomer, and this unusual mode of DNA binding could provide a target for drug design.
Himes
Paul
P
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
McBryant
Steven J
SJ
Kroos
Lee
L
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D016384:Consensus Sequence; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D011485:Protein Binding; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C071977:SpoIIID protein, Bacillus; D014157:Transcription Factors