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The Section on Molecular Regulation investigates how nutrient availability coordinates global patterns of gene expression as well as pathway-specific regulation in bacteria. These complex networks serve to integrate synthesis of macromolecules as well as to regulate expression of the whole genomic repertoire. We focus on the role of two regulatory nucleotides that occur almost exclusively in bacteria. These are analogs of GTP and GDP with pyrophosphate residues on the ribose 3'-hydroxyl, collectively called (p)ppGpp. Nutrient limitation causes fluctuations of (p)ppGpp levels, whether starving for amino acids, phosphate, nitrogen or energy sources. A regulatory role is assigned to (p)ppGpp because artificial induction of (p)ppGpp without nutrient limitation gives many of the same regulatory effects as starvation itself. Fluctuation of (p)ppGpp can also be a key element in adaptive responses to starvation, such as insuring survival by induction of the stationary phase-specific sigma factor of RNA polymerase. Most regulatory responses to (p)ppGpp are thought to occur by affecting transcription but verification of this notion with pure transcription components has proven elusive. We have taken the alternative genetic approach of isolating mutants that suppress cellular phenotypes due to a complete deficiency of (p)ppGpp. This has led to identification of over 50 distinct lesions in the two largest RNA polymerase subunits. Interestingly, the sites of these amino acid changes (shown as large spheres in the home page figure) are found almost exclusively on enzyme surfaces deduced to involve DNA contacts.
We are also very interested in how (p)ppGpp is made and regulated in response to nutrient starvation. Most bacteria contain a single enzyme capable of both (p)ppGpp synthesis and (p)ppGpp degradation. This means that net activity requires a switch within the enzyme that activates one activity while simultaneously curtailing the opposing activity. To understand this switch, we have isolated single intragenic missense suppressor mutations conferring reciprocal activation and repression of opposing activities. A bifunctional domain from this sort of enzyme has been crystallized and its structure is being determined in collaboration with another laboratory. In contrast, E. coli contains two enzymes, each largely dedicated to opposing activity with one enzyme bound to ribosomes where it can sense a aminoacyl tRNA deficiency during protein synthesis. We are exploring these and other fundamental similarities between (p)ppGpp biosynthesis and degradation well as other steps in (p)ppGpp metabolism.