Ribosomes catalyze protein synthesis via the translation cycle. Studies of bacterial ribosome structure provide strong evidence that functional complexes of the bacterial ribosome undergo large conformational changes during the course of this cycle. At the same time, studies of partial reactions of the translation cycle have shown these processes to be complex, multistep reactions, about which our knowledge remains quite incomplete. Our general approach, involves combining the results of several measurements made in parallel to allow formulation of quantitative kinetic schemes describing such partial reactions. Such quantitative schemes allow elucidation of the timing of conformational changes within a process, and the precise mechanisms by which important variables affect ribosome catalytic function. Resolving the translation cycle into its component parts permits determination of the specific effect(s) of a given antibiotic on ribosomal function. Such knowledge, linked to known sites of antibiotic binding, is useful for the design and evaluation of new antibiotics.
Our specific aims are: 1. Preparation and utilization of fluorescent reagents. We will prepare fluorescent ribosomal ligands and fluorescent ribosomes labeled in specific proteins. These fluorescent reagents will be used in rapid kinetics experiments in Aims 2-5. 2. The elongation cycle. We will determine how specific steps within the cycle are affected by a) E-site occupation, b) the identities of the tRNAs participating in peptide bond formation, c) the size and sequence of the nascent peptide chain bound to the ribosome, d) mutations in tRNA and ribosomal RNA, and e) added antibiotics. We also will use cryoelectronmicroscopy (cryoEM) to elucidate the structures of intermediates in translocation and formulate a quantitative mechanism for LepA catalysis of back translocation. 3. Initiation. A key step in initiation is the formation of a 70S initiation complex (70SIC). We will i) determine how the kinetic scheme for 70SIC formation is affected by changes in the initiation codon; ii) elucidate the timing and magnitude of a key conformational change within the process of 70SIC formation; and iii) compare the effects of thiazole antibiotics on 70SIC formation with those already found for thiostrepton, as part of the process of developing new therapeutics. 4. Simultaneous binding of G-factor proteins to the ribosome. We will employ cryoEM and modeling to obtain a structure of the complex that is formed when two G-proteins, IF2 and EF-Tu bind to the ribosome simultaneously during the transition from initiation to elongation, and couple such studies with dynamic measurements designed to both capture the movement of IF2 as EF-Tu binds. We also will determine the dependence of complex formation on the stoichiometry of ribosomal protein dimer L7/L12. Similar studies will be conducted on EF-G and EF-Tu to test whether they might bind simultaneously to the ribosome to form a short-lived intermediate during elongation. 5. Single molecule FRET experiments will be used to identify transient intermediates in reaction pathways studied as parts of Aims 2 and 4.
The ribosome catalyzes protein synthesis via the translation cycle, and its fundamental importance in cellular function is evidenced by its universality and fundamental conservation throughout all forms of life. The studies we propose will contribute importantly to the further elucidation of the dynamic mechanism of action of the ribosome, and provide a paradigm for determining the specific effect(s) of a given antibiotic on ribosomal function. Such knowledge is of clear utility in the design and evaluation of new antibiotics that are urgently needed due to the widespread development of resistance to antibiotics currently in clinical use. ? ? ?
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