A DNA Conformation-Dependent Genetic Switch in Halobacterium

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9604443 DasSarma This research tests the hypothesis that specific regions of the genome of Halobacterium halobium adopt supercoil density-dependent Z-DNA conformations that function as regulatory switches for the control of gene expression. Halobacterium halobium is an extremely halophilic Archaea in which the left-handed Z-DNA conformation is favored by a highly negative DNA supercoiling density, a high G+C content including alternating CG sequences, and a high cytoplasmic salt concentration (5M). A combination of mutagenic and biochemical analysis will determine if expression of the bop gene encoding the H. halobium purple membrane protein is controlled by B-to-Z DNA conformation-dependent switch at a potential Z-DNA sequence located 23 base pairs upstream from the bop gene transcription start site. Preliminary evidence indicates that bop gene transcription is induced under conditions of highly negative DNA supercoiling, and reduced when supercoiling is relieved by inhibition of DNA gyrase. Therefore, the DNA gyrase inhibitor novobiocin will be used to determine the role of DNA supercoiling in mediating DNA conformational changes and bop transcription, including the development of an in vitro transcription system for studying the effects of DNA supercoiling on transcription. %%% This research will evaluate the role of DNA conformation and conditions that affect DNA conformation in the regulation of gene expression in Halobacterium halobium, a Archaea bacteria that exists under conditions of extremely high salt concentration. ***