Transcriptional Activation with Sigma54-Holoenzyme

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ABSTRACTMCB 9974558PI: Timothy HooverSinorhizobium meliloti C4-dicarboxylic acid protein D (DctD) will be used as a model to study transcriptional activation with sigma54-RNA polymerase holoenzyme. DctD binds to enhancer sites located upstream of the dctA promoter and then contacts sigma54-holoenzyme bound at the promoter through DNA looping to activate transcription. At high concentrations, DctD can activate transcription in the absence of the enhancer by contacting sigma54-holoenzyme directly from solution. Some mutant forms of DctD cannot activate transcription in the absence of the enhancer, and these mutant proteins may have reduced affinities for sigma54-holoenzyme. In the first specific aim, interactions between enhancer-dependent DctD mutants and sigma54-holoenzyme will be examined by isothermal titration calorimetry and surface plasmon resonance to determine if the enhancer-dependent DctD mutants have reduced affinities for sigma54-holoenzyme. In addition, a search will be made for mutant forms of sigma54-holoenzyme that suppress enhancer-dependent DctD mutants. Information gained from these studies will help identify sites that are involved in protein-protein interactions. In the second specific aim, mutant forms of sigma54 that are defective in steps following binding of sigma54-holoenzyme to the promoter will be isolated and characterized in vitro. These studies will help elucidate the role of sigma54 in transcriptional activation. Finally, Helicobacter pylori FlgR (flagellar regulatory protein) will be purified and characterized in vitro. FlgR is an activator of sigma54-holoenzyme but it lacks the DNA-binding domain found in other activators. FlgR presumably either contacts sigma54-holoenzyme directly from solution at physiological concentrations to catalyze open complex formation, or it contains another subunit that is responsible for DNA binding. Both possibilities are unusual for activators of sigma54-holoenzyme and experiments will be done to distinguish between the two.Many bacteria contain sigma54-RNA polymerase holoenzyme, and this form of holoenzyme is responsible for the expression of genes whose products are involved in a variety of important microbial processes, including nitrogen assimilation, nitrogen fixation, toluene degradation, hydrogen metabolism, flagellar biosynthesis, and pilin formation. Many of these microbial processes have significant environmental, industrial, agricultural or medical implications. Understanding how sigma54-dependent genes are regulated is critical for exploiting and controlling the bacteria that are responsible for these microbial activities.