Action-at-a-distance: Competing structures and functions in the regulation of transcription in supercoiled DNA

This dissertation uses the DNA-supercoiling dependent ilvP{dollar}sb{lcub}rm G{rcub}{dollar} promoter of the ilvGMEDA operon of Escherichia Coli as a model system to study the role of DNA topology on transcriptional initiation. Integration host factor (IHF) binds to an upstream activating sequence (UAS1) of this promoter. IHF enhances transcriptional initiation at the ilvP{dollar}sb{lcub}rm G{rcub}{dollar} promoter by increasing the rate of open complex formation on a supercoiled template without interacting with RNA polymerase.; Using biochemical structural probing techniques in conjunction with transcriptional analysis in vitro with plasmid topoisomers of defined superhelical densities, it is demonstrated that the formation of a superhelically-induced duplex destabilized (SIDD) structure in the DNA helix of the UAS1 is required for IHF activation. IHF binding in this region inhibits the formation of this DNA structure. Thus, the superhelicity involved in the formation of this structure is redistributed to structural transitions at other DNA sites. One of these sites is located in the {dollar}-{dollar}10 region of the downstream ilvP{dollar}sb{lcub}rm G{rcub}{dollar} promoter where duplex destabilization facilitates open complex formation to activate transcription initiation.; It is demonstrated that the Z-DNA forming sequence, (CG){dollar}sb{lcub}13{rcub}{dollar}AATT(CG){dollar}sb{lcub}22{rcub}{dollar}, can act to both inhibit and activate transcription at-a-distance. Experiments with the ilvP{dollar}sb{lcub}rm G{rcub}{dollar} and gyrA promoters demonstrate that the effect of this Z-DNA transition on transcription is promoter-specific. This specificity is determined by the intrinsic response of the promoter to the level of superhelicity. When this Z-DNA froming sequence was inserted in the superhelical domain in which both the UAS1 and ilvP{dollar}sb{lcub}rm G{rcub}{dollar} promoter are present the basal level of transcription was repressed. Furthermore, the formation of the SIDD structure in the UAS1 was offset towards more negative superhelical densities which was directly correlated with an equivalent offset of IHF-mediated activation. Thus, DNA secondary structures influence superhelically-dependent processes at-a-distance by modulating the local DNA topology. The deliberate establishment of competition between DNA secondary structures to alter the intrinsic superhelical response of a biological process, shown here with transcriptional regulation, demonstrates a novel method for dissecting the consequences of DNA sequence from structure on a wide array of superhelically-dependent biological processes.