The long-term, major objective of this research is to understand how guanosine tetraphosphate (ppGpp), a general signal molecule for amino acid deprivation in bacteria interacts at the molecular level to repress transcription of some genes (e.g., tRNA and rRNA operons) and to activate transcription of other genes (e.g., amino acid biosynthetic operons). ppGpp appears to be part of a global regulatory system that adjusts many cellular processes in response to the cell's need for amino acids. The specific goals are to identify the target DNA sequences at which the regulatory effects of ppGpp are exerted in the promoter regions of regulated genes and to characterize the protein components with which ppGpp interacts to regulate gene expression. The approach being used to analyze the mechanism of ppGpp regulation at susceptible promoters combines molecular-genetic and biochemical methods of analysis. The activable histidine biosynthetic operon and the repressible tRNALeul operon are being analyzed as model systems of positive and negative control by ppGpp, respectively. The promoters of these genes are being dissected through analysis of mutations isolated by classical genetic techniques, site-directed in vitro mutagenesis techniques and by direct synthesis of mutant promoters with a DNA synthesizing machine. Effects of the promoter mutations on ppGpp-mediated regulation is being assessed with an S30 transcription-translation system in vitro and with physiological experiments in vivo employing amino acid shiftdown conditions. Gene replacement and retrieval techniques have been developed to shuttle the promoter mutations between plasmids and the bacterial chromosome. Potential protein factors with which ppGpp interacts are being isolated from bacterial extracts and mutants having lesions in potential factor genes are being selected. The research problem addressed in this application is part of the broader, basic issue of biological signal transduction. Defects in biological signal transduction are apparently part of the basis of the causation of certain genetic and regulatory disorders such as cancer. How ppGpp acts and what components are involved in its actions remain important questions. It is anticipated that the answers will have general significance in the understanding of biological regulatory mechanisms.
