THE COMPETENCE TRANSCRIPTION FACTOR OF BACILLUS-SUBTILIS RECOGNIZES SHORT A T-RICH SEQUENCES ARRANGED IN A UNIQUE, FLEXIBLE PATTERN ALONG THE DNA HELIX/

The development of genetic competence in Bacillus subtilis is regulate d by a complex signal transduction cascade, which leads to the synthes is of the competence transcription factor (CTF). Previous studies sugg ested that CTF is encoded by comK. ComK is required for the transcript ion of comK itself, as well as of the late competence genes encoding t he DNA uptake machinery and of genes required for homologous recombina tion. Here, we used purified ComK to study its role in transcription a nd to determine the DNA recognition sequence for ComK. In vitro transc ription from the comG promoter, which depends on ComK in vivo, was obs erved on the addition of purified ComK together with Bacillus subtilis RNA polymerase, proving that ComK is CTF. To determine the DNA sequen ces involved in ComK recognition, footprinting analysis was performed with promoter fragments of the CTF-dependent genes: comC, comE, comF, comG, comK, and addAB. The ComK binding sites determined by DNase I pr otection experiments were unusually long, with average lengths of simi lar to 65 bp, and displayed only weak sequence similarities. Hydroxy-r adical footprinting, performed with the addAB promoter, revealed a uni que arrangement of four short A/T-rich sequences. Gel retardation expe riments indicated that four molecules of ComK bound the addAB promoter and the dyad symmetrical arrangement of the four A/T-rich sequences i mplied that ComK functions as a tetramer composed of two dimers each r ecognizing the motif AAAAN(5)TTTT. Comparable A/T-rich sequences were identified in all six DNase I footprints and could be used to predict ComK targets in the B. subtilis genome. On the basis of the variabilit y in distance between the ComK-dimer binding sites, ComK-regulated pro moters could be divided into three classes, demonstrating a remarkable flexibility in the binding of ComK. The pattern of hydroxy-radical pr otections suggested that ComK binds at one face of the DNA helix throu gh the minor groove. This inference was strengthened by the observatio n that minor groove binding drugs inhibited the binding of ComK.