The broad, long-term objective is to understand the molecular basis of the function of the ribosome in protein synthesis. In the near-term the aim is to learn how information is transmitted from one ribosomal domain to another in order to coordinate the partial reactions of protein synthesis. We postulate that signals are conveyed by local or regional changes in rRNA conformation; the result of breaking and remaking tertiary contacts between domains.
In aim 1, we shall test the hypothesis by constructing site-specific, single nucleotide (nt) substitutions in putative tertiary interactions between the sarcin/ricin domain, the peptidyl transferase center, and the GTPase center in E. coli 23S rRNA; we will be guided in this by the atomic structure of the 50S subunit. The intent is to uncover mutations that have a phenotype and, hence, are likely to be important for function. Next, we shall attempt to isolate suppressors of deleterious mutations by constructing a second mutation in the nt partner in the proposed tertiary interaction. We shall also assess the efficiency with which ribosomes with these mutations catalyze the partial reactions of elongation. Finally, we shall analyze the effect of mutations on the conformation of the RNA in the three domains using chemical reagents and Fe (II)-EDTA. The question is whether a mutation in one functional center has an effect on the structure of one or both of the other two centers.
In aim 2 we shall attempt to determine the molecular basis of the cyclic, sequential binding of the elongation factors Tu and G to the ribosome. One possibility, the one we shall investigate first, is that the order is determined by the location on the ribosome of peptidyl-tRNA: that the EF-Tu ternary complex only binds to the ribosome when peptidyl-tRNA is in the P-site, that is to sayin the post translocation state; whereas EF-G.GTP binds when peptidyl-tRNA is in the A-site, i.e., the pretranslocation state. The possibility we espouse is that the position of the peptidyl-tRNA in the A- or P-site, is monitored in the peptidyl transferase center and that a signal generated there is relayed, through a series of tertiary interactions, to the sarcin/ricin domain, initiating in the latter a conformational transition. This is the possibility we shall test using the experimental design and the methods of aim 1.
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