The S box system is a novel global regulatory mechanism for control of genes involved in methionine metabolism in Gram-positive bacteria. Genes in the S box family contain a complex set of conserved primary sequence and structural elements upstream of the start of the coding sequence. These elements act in the nascent RNA to bind S-adenosylmethionine (SAM), and interaction with SAM results in a structural switch in the RNA that promotes premature termination of transcription. Lysine biosynthesis genes use a similar mechanism, with binding of lysine to nascent RNAs in the L box family to terminate transcription or prevent translation initiation. A new SAM-binding RNA element has now been identified in the upstream region of SAM synthetase genes of certain Gram-positive organisms, and this element, like the S box element, is predicted to regulate expression of the downstream coding sequence in response to SAM. These and related systems are widely used to regulate gene expression in Gram-positive bacteria, including important pathogens. The overall goals of this project are to investigate the molecular basis for specific recognition of effector molecules by the nascent RNA, and for calibration of the affinity of each class of RNA to physiologically relevant concentrations of the effector. A variety of genetic, biochemical, and structural biology approaches will be employed to elucidate both the RNA-effector interaction and the structural rearrangement necessary for the appropriate regulatory response to effector binding. The interaction between methionine gene regulation and the stringent response will also be explored. Work will focus on Bacillus subtilis as a model for analysis of these systems, and will also include analysis of the new SAM binding element from Enterococcus faecalis. This project will provide basic information about novel RNA-based mechanisms of gene regulation, and will also provide insight into metabolic regulation in pathogenic organisms that use these mechanisms. Gram-positive pathogens generally use regulatory mechanisms closely related to those found in Bacillus subtilis. Expression of determinants for pathogenicity are often regulated in response to physiological signals, and understanding how the cell monitors these signals is important for understanding bacterial virulence. [unreadable] [unreadable] [unreadable]