Mechanism of gene regulation by CcpA bound to cre sites located upstream, downstream and overlapping the regulated promoter

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The global transcription factor CcpA mediates activation or repression of more than 100 genes in Bacillus subtilis by binding to cre sequences. Cre can be upstream, downstream, even as far as in the translated region of the regulated gene, or overlapping the regulated promoter. We propose in vivo crosslinking of proteins to CcpA-Strep bound to promoters representing these different locations of cre elements, followed by their identification by mass spectrometry. Using bacterial two-hybrid analysis and surface plasmon resonance we will identify the directly with CcpA interacting proteins and quantify their interaction with CcpA thermodynamically and kinetically. We will also clarify which cofactors of CcpA are involved in these interactions. Indirectly with CcpA interacting proteins will be expressed at various levels under tet control to elucidate their participation in CcpA mediated regulation. Furthermore, we will identify proteins bound to CcpA at glucose independently regulated promoters. The crystal structures of the most interesting protein-CcpA complexes will be determined. We expect to elucidate novel mechanistic details of how CcpA accomplishes regulation of transcription from such different sites with respect to the regulated promoters. The expected results will be relevant for bacterial regulation of transcription in general.