title: Mechanism of transcription activation at the comG promoter by the competence transcription factor ComK of Bacillus subtilis.
abstract: The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK. ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machinery and of genes required for homologous recombination. In vivo and in vitro experiments have shown that ComK is responsible for transcription activation at the comG promoter. In this study, we investigated the mechanism of this transcription activation. The intrinsic binding characteristics of RNA polymerase with and without ComK at the comG promoter were determined, demonstrating that ComK stabilizes the binding of RNA polymerase to the comG promoter. This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNA resulted in an almost complete abolishment of stabilization of RNA polymerase binding. Furthermore, a strong requirement for the presence of an extra AT box in addition to the common ComK-binding site was shown. In vitro transcription with B. subtilis RNA polymerase reconstituted with wild-type alpha-subunits and with C-terminal deletion mutants of the alpha-subunits was performed, demonstrating that these deletions do not abolish transcription activation by ComK. This indicates that ComK is not a type I activator. We also show that ComK is not required for open complex formation. A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase.
authors.affiliation: Department of Genetics, University of Groningen, NL-9751 NN Haren, The Netherlands.
authors.forename: K A
authors.initials: KA
authors.lastname: Susanna
authors.forename: A F
authors.initials: AF
authors.lastname: van der Werff
authors.forename: C D
authors.initials: CD
authors.lastname: den Hengst
authors.forename: B
authors.initials: B
authors.lastname: Calles
authors.forename: M
authors.initials: M
authors.lastname: Salas
authors.forename: G
authors.initials: G
authors.lastname: Venema
authors.forename: L W
authors.initials: LW
authors.lastname: Hamoen
authors.forename: O P
authors.initials: OP
authors.lastname: Kuipers
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation
chemical_list: D001426:Bacterial Proteins; D014157:Transcription Factors; C087449:comK protein, Bacillus subtilis; D012321:DNA-Directed RNA Polymerases