The fine control of global gene expression allows bacteria to maintain a balanced metabolism and to adjust rapidly to environmental challenges and changing nutrient conditions. The control of the genes involved in tryptophan biosynthesis or utilization has served as one of the most important paradigms for the discovery and atomic level characterization of novel genetic regulatory mechanisms in bacteria. The diverse mechanisms sense both the availability of free tryptophan and the level of charging (aminoacylation) of tRNATrp. This project will continue these investigations by investigating the control of the tna operon (tryptophan utilization) in Escherichia coli and the at operon (tryptophan regulation) in Bacillus licheniformis. Structural and regulatory studies will be performed using the tna operon to identify the molecular features in the nascent TnaC peptidyl-tRNA and in the ribosome exit tunnel that are responsible for tryptophan binding to the peptidyl transferase center of the ribosome and the concomitant inhibition of peptidyl-tRNA cleavage at the tnaC stop codon. Specific nucleotide substitutions or amino acid replacements will be introduced in rRNA and r-proteins to identify the ribosomal determinants essential for recognition of the features of the nascent peptide and free tryptophan. An efficient in vitro system will be used in which purified ribosomes are examined. This project is revealing that the translating ribosome is not a non-specific protein-synthesizing machine. Rather, the amino acid sequence of a polypeptide can influence a ribosome's ability to catalyze polypeptide elongation, or cleavage at a stop codon. Studies will also be performed with the gram-positive bacterium Bacillus licheniformis to determine the regulatory features of the leader peptide coding region of the at operon. This coding region contains three dispersed tryptophan codons at which a translating ribosome could stall if tryptophan-charged tRNATrp is unavailable. Ribosome stalling at either of the first two tryptophan codons is predicted to increase synthesis of the AT regulatory protein whereas ribosome stalling at the third tryptophan codon is predicted to reduce AT synthesis. This project provides training for post doctoral students and is establishing new paradigms for the post transcriptional regulation of gene expression in microorganisms.
