Elucidation of the regulation of Escherichia coli fis gene promoter by interactions between a module of RNA polymerase molecules and DNA architectural proteins

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Initiation of bacterial gene transcription is subject to complex regulation by concerted actions of DNA architectural transcription factors, protein cofactors of transcription, small effector molecules and the DNA topology. In addition, the promoter regions of certain pleiotropic genes demonstrate arrays of multiple RNA polymerase (RNAP) binding sites. The expression of the major chromatin protein FIS is regulated by several DNA architectural transcription factors, including CRP, IMF, H-NS and FIS itself. Previously we identified a module of RNAP binding sites in the regulatory region of Escherichia colt fis operon. These RNAP binding sites demonstrate strong promoter activities when cloned separately on plasmid constructs, but their function in the chromosomal context remains controversial - it is unclear whether the identified polymerase binding sites serve as true promoters, or have a purely regulatory function. Especially intriguing is the role of a overlapping module of fisP2 and a divergent promoter (divP/fisP2 module) identified upstream of the fisP1 site. We observed that an up mutation of the d/VP -10 hexamer increases the d/V transcription both in in vivo and in vitro. We also found that this mutation not only affects the binding of polymerase at the divP/fisP2 module but also at the fisP1 site, suggesting that upon occupation of these two sites the RNAP molecules interact with each other. In keeping with this notion, by using atomic force microscopy we observed that simultaneous binding of divP/fisP2 module and fisP1 sites stabilizes a unique complex containing at least two polymerase molecules presumably forming a RNAP dimer. Thus, our preliminary data strongly suggest a novel mechanism of transcriptional control involving direct interactions between RNAP molecules. In this project we intend to use the model system of divP/fisP2 and fisP1 promoters to investigate this novel regulation mechanism. We are keen to elucidate the relationships between the polymerase dimer formation at the fis promoter and the regulatory effects on fis transcription exerted by cAMP-CRP, IHF, H-NS and FIS itself. We believe this study will provide new insights into the diversity of transcriptional control mechanisms.