title: 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.
abstract: 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.
authors.affiliation: Department of Genetics, University of Groningen, NL-9751 NN Haren, The Netherlands.
authors.forename: Kim A
authors.initials: KA
authors.lastname: Susanna
authors.forename: Aleksandra M
authors.initials: AM
authors.lastname: Mironczuk
authors.forename: Wiep Klaas
authors.initials: WK
authors.lastname: Smits
authors.forename: Leendert W
authors.initials: LW
authors.lastname: Hamoen
authors.forename: Oscar P
authors.initials: OP
authors.lastname: Kuipers
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D009154:Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D013936:Thymidine / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D014157:Transcription Factors; C087449:comK protein, Bacillus subtilis; D001616:beta-Galactosidase; D013936:Thymidine