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J. Bacteriol. doi:10.1128/JB.00281-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A single, specific thymine mutation in the ComK-binding site severely decreases binding and transcription activation by the competence transcription factor ComK of Bacillus subtilis

Department of Genetics, University of Groningen, NL-9751 NN Haren, the Netherlands

^* To whom correspondence should be addressed. Email: O.P.Kuipers{at}rug.nl.

	Abstract

The competence transcription factor ComK plays a central role in competence development in Bacillus subtilis by activating transcription of the K-regulon. ComK-activated genes are characterized by the presence of a specific sequence to which ComK binds, a K-box, in their upstream DNA region. Each K-box consists of two AT-boxes, with the consensus sequence AAAA-(N)₅-TTTT, which are separated by a flexible spacing resulting in either two, three or four helical turns between the starting nucleotides of the repeating AT-box units. In this study, the effects of potential determinants of ComK-regulation in K-boxes were investigated by testing transcription activation and DNA-binding affinity on altered K-boxes, which were either mutated in the spacing between the AT-boxes or in the consensus sequence of the AT-boxes.

The most striking result demonstrates the importance of the second thymine base in the AT-boxes. Mutation of this T into a guanine resulted in a 3-fold reduction in transcription activation and DNA-binding by ComK. Transcription activation, as well as DNA-binding, were almost completely abolished when both AT-boxes contained a T₂ to G mutation. This result is corroborated by in silico analyses, demonstrating that a combination of mutations at the T₂ positions of both AT-boxes is not found among any ComK-activated K-boxes, indicating that at least one consensus T₂-position is required to maintain a functional K-box. The results suggest an important structural role of T₂ in ComK-binding, probably by its specific position in the minor groove of the DNA.