Transciptional Activation by Rhizobium meliloti DCTD

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HOOVER 96-30454 The long term goal of this project is to understand be mechanisms involved in transcriptional activation with (54 RNA polymerase holoenzyme (E(54) ~n bacteria Although much is known at a phenomenological level, many details concerning the mechanisms involved in this process need to be elucidated. E(54 binds to the promoter to form a closed complex that is unable to initiate transcription. The activator, which binds upstream of the promoter, must make productive contact with E(54 through DNA looping and hydrolyze ATP in order to activate transcription. Interactions between the activator and E(54 are transient, and nothing is known about specific contact sites between E(54 and any c~ dependent activator. Identifying these contact sites is crucial for dissecting the mechanisms of transcriptional activation with E(54, and the goal of this project is to identify these contact sites. The (54 -dependent activator DctD) (C4 dicarboxylic acid transport protein D) from Rhizobium meliloti will be used for these studies. DctD can be crosslinked to (54 and the ,B subunits of RNA polymerase, suggesting that DctD may engage these subunits of E(54 to activate transcription. A collection of DctD) mutants that fail to activate transcription bas been generated, and some of these mutants are likely to be defective in interactions with E(54. A search will be made for alleles of ntrA (encodes (54) and rpoB (encodes the B subunit of RNA polymerase) that suppress dctD mutants. Gene products of these suppressors u ill be purified and examined for their ability to function with the DctD mutants in an In vitro transcription assay, as well as their ability to interact with DctD mutants in the crosslinking assay. A second research goal is to isolate (54 mutants that are defective in protein-protein interactions with DctD:). Such (54 mutants are expected to allow E(54 to bind to the promoter to form a closed complex, but will not allow the isomerization of these closed complexes ~ open complexes that are tr anscriptionally productive. A genetic screen based on the ability of E(54 to repress transcription from the phage P22 ant gene has been developed, and will be used to isolate (54 mutants with these properties. The (54 mutants isolated from this screen will be examined for their ability to recognize promoter sequences, bind core RNA polymerase, and interact with DctD in the crosslinking assay The results of these experiments will help define the contact sites between DctD and E(54. Examination of how (54 -dependent activators function is crucial, as it will contribute to our understanding of how many important metabolic processes are controlled in bacteria. The occurrence of (54 -dependent genes in bacteria is widespread, and the products of these genes are involved in such diverse processes as nitrogen fixation and metabolism, degradation of environmental pollutants, and regulation of virulence factors in plant and human pathogens. Understanding how bacteria control such metabolic processes is essential for the control and exploitation of these organisms in agriculture, environmental protection, and medicine.