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Amino acid substitution studies reveal that substitution of alanine for the phenylalanine at position 6 and to the leucine at position 67 in the GerE transcription factor give rise to region 1 and region 2 promoter activation sites in Bacillus subtilis.

Results demonstrate that amino acyl residues in or near the putative helix-turn-helix motif of the DNA-binding protein GerE determine the specificity of DNA binding in Bacillus subtilis. Nucleotides at positions +2, +3, and +4 with reference to transcription start site on the sigk promoter are involved in the binding site.

The DNA-binding protein GerE acts as both a repressor and an activator of transcription of genes transcribed by σK-RNA polymerase (RNA-P) during the later stages of endospore formation in Bacillus subtilis. GerE represses transcription from the sigK promoter, and activates transcription from other promoters, including cotC and cotX. Two different regions of GerE (AR1 and AR2) are required for activation of cotC and cotX, respectively. We used a genetic screen to seek mutations that would define additional regions of GerE required for promoter activation. We found that a substitution of proline for leucine at position 12 of GerE (L12P) decreased cotC promoter activity but did not interfere with GerE-dependent repression of the sigK promoter or with activation of the cotX promoter in vivo. We also found that the L12P substitution had no effect on binding to cotC in vitro. However, the L12P-substituted GerE failed to stimulate cotC transcription in vitro, whereas it stimulated transcription from PcotX. The crystal structure of GerE suggests that L12 is not exposed on the surface of the molecule. Therefore, we propose that the L12P substitution reduces the flexibility of the N-terminal arm, preventing an interaction of AR1 with RNA-P that is essential for activation of the cotC promoter. [Copyright &y& Elsevier]

Examines the effects of single base pair substitutions in the high affinity GerE protein binding site on the sigK promoter in Bacillus subtilis. Production of a protein that binds with equal affinity to two sites that differ by one base pair; Identification of the nucleotide sequence that signals recognition and binding by GerE; Mutagenesis of the DNA binding region of GerE.