Integration Of Macromolecular Synthesis

Child Health And Human Development

Investigators

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Abstract

We study the coordination of global patterns of bacterial gene expression by nutrient availability with focus on the roles of 3'-pyrophosphorylated analogs of GDP and GTP, collectively abbreviated as (p)ppGpp. Limiting growth for any of a variety of nutrients leads to changes (p)ppGpp levels that provoke regulatory adjustments at levels of transcription, translation, and metabolism. We have previously collaborated to work out the structure of the catalytically active portion of a bifunctional enzyme that regulates (p)ppGpp synthetase as well as a (p)ppGpp hydrolase when starved for amino acids or for sources of carbon, nitrogen and phosphate. When these nutrients are limited, linked conformational changes mechanisms lead to inhibition of the hydrolase and activation of the synthetase in the balance leading to (p)ppGpp accumulation. The hydrolase and synthetase activity domains are both located in the N-terminal (NTD) half of the protein, whose structures have been solved. These activities are simultaneously regulated by the C-terminal half protein (CTD), whose structure is not yet defined. This year we are constructing and testing a series of mutants of both of the E. coli and M. tuberculosis enzymes designed to abolish one or the other of the two opposing activities yet retain the capacity for conformational changes allowing regulation of the unaltered activity. These activities will be measured during different conditions to ask how starvation signals provoke transitions between the two activity states. In collaboration with Drs. Vinella and D'Ari of the Jacques Monod Institute, we have verified their prediction that iron starvation is another stress capable of provoking ppGpp accumulation. We are also exploring aspects of regulation of transcription by (p)ppGpp. These efforts include attempting to characterize RNA polymerase conformational differences between (p)ppGpp regulated and unregulated promoters during promoter clearance.