We study the coordination of global patterns of bacterial gene expression by nutrient availability, continuing to 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 (amino acids, phosphate, nitrogen or energy sources) leads to changes (p)ppGpp levels that provoke regulatory adjustments at levels of transcription, translation, and metabolism. This year we have learned more of the molecular details of how (p)ppGpp abundance is regulated. Nearly all eubacteria (and probably plants) regulate (p)ppGpp synthesis by a protein (approximately 75 kDa) that can sense when charge tRNA is limiting on the ribosome and responds by undergoing a conformational change that activates (p)ppGpp synthetase. The same protein contains a (p)ppGpp hydrolase domain and can by sense starvation for nutrients other than amino acids, such as sources of carbon, nitrogen and phosphate. When these nutrients are limited, the hydrolase is inhibited, also leading to (p)ppGpp accumulation. The hydrolase and synthetase activity domains are both located in the N-terminal (NTD) half of the protein. These activities are simultaneously regulated by the C-terminal half protein (CTD). We find that the reciprocal switch of hydrolase and synthetase activities govern (p)ppGpp abundance is an intrinsic property of the protein insuring that both opposing reactions are not simultaneously activated. This year, Drs. Hogg & Hilgenfeld, our collaborators in Germany, have solved the crystal structure at 2.1 Angstroms resolution of the bifunctional catalytic portion of the protein. These structures allow detailed proposals for understanding the switch. The lab of our collaborator in France, Dr. Schneider, has discovered that new mutants in the CTD that surprisingly suppress topoisomerase mutants. Using a bacterial two hybrid analyses with the CTD as bait, we have found two proteins that interact with the CTD and are therefore potential regulators (p)ppGpp. Using immobilized ribosomes, we have also shown that the small terminal portion of the CTD, containing the ACT domain, is virtually solely responsible for binding of the protein to ribosomes in the absence of mRNA and tRNA. A molecular understanding of how (p)ppGpp is beginning to emerge after many years of effort by several laboratories. Unfortunately, the computer system will not allow the addition of this year's bibliography!
