Biochemical characterization of ribosomal states involved in decoding and translocation

The process of translation elongation consists of sequential addition of amino acids to the growing peptide chain on the ribosome in a codon dependent manner. Each elongation cycle is composed of multiple steps. The ribosome:tRNA:mRNA complexes assume distinct conformations correlating with different functional states that characterize different steps of the elongation cycle. In the present thesis we characterize certain aspects of ribosomal states during decoding and preceding translocation.; Kinetic and structural studies argue for the existence of a productive state of the small ribosomal subunit that is required for the incorporation of incoming aminoacyl-tRNA. In the present study we show that the S12 residues that make intra-subunit contacts (K42 and R53---E. coli numbering) are critical for the incorporation of a near-cognate aminoacyl tRNA in the presence of the miscoding antibiotic paromomycin. We further demonstrate by foot-printing and biochemical analysis that paromomycin binds with high and low affinity to helices 44 and 27 of the 16S rRNA, respectively. Furthermore, the occupation of the low affinity helix 27 site by the antibiotic is associated with a productive state of the small subunit independent of contacts made by S 12 residues mentioned above. Thus distinct sets of intra-subunit interactions can result in a productive state of the small ribosomal subunit in terms of aminoacyl tRNA incorporation into the growing peptide chain.; Previous studies have suggested that following peptide bond formation the tRNAs occupy an intermediate (hybrid) state of binding where the acceptor ends of the tRNAs are shifted to their next sites of occupancy (the E and P sites) on the large ribosomal subunit but where their anticodon ends (and associated mRNA) remain fixed in their pre-peptidyl transferase binding states (the P and A sites) on the small subunit. Here we show that pre-translocation state ribosomes carrying a dipeptidyl-tRNA substrate efficiently react with the minimal A site substrate puromycin and that following this reaction, the pre-translocation state bound deacylated tRNA:mRNA complex remains untranslocated. These data establish that pre-translocation state ribosomes must sample or reside in an intermediate state of tRNA binding.