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

Kim A. Susanna, Aleksandra M. Mironczuk, Wiep Klaas Smits, Leendert W. Hamoen, Oscar P. Kuipers*

*Corresponding author for this work

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The competence transcription factor ComK plays a central role in competence development in Bacillus subtilis by activating the 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 spacer 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 ComK's transcription activation and DNA-binding affinity on altered K-boxes with mutations either in the spacer 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 threefold reduction in transcription activation and DNA binding by ComK. Transcription activation, as well as DNA binding, was almost completely abolished when both AT-boxes contained a T-2-to-G mutation. This result was corroborated by in silico analyses demonstrating that a combination of mutations at the T-2 positions of both AT-boxes is not found among any ComK-activated K-boxes, indicating that at least one consensus T-2 position is required to maintain a functional K-box. The results suggest an important structural role for T-2 in ComK binding, probably by its specific position in the minor groove of the DNA.

Original languageEnglish
Pages (from-to)4718-4728
Number of pages11
JournalJournal of Bacteriology
Issue number13
Publication statusPublished - Jul-2007


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