Kinetics of codon recognition on the ribosome by tRNAs and release factors

Ribosomes translate messenger ribonucleic acid (mRNA) into proteins in all domains of life. During the elongation phase of protein synthesis, tRNAs bind to the ribosome in a codon dependent fashion as ternary complexes consisting of a protein elongation factor (EF-Tu), guanosine triphosphate (GTP), and an aminoacylated transfer ribonucleic acid (tRNA). The first step in the termination of protein synthesis is the recognition of stop codons by release factor 1 or 2 (RF1 or 2) in order to hydrolyze the completely synthesized protein from the tRNA bound in the peptidyl (P) site of the ribosome.;We have developed a fluorescence based method designed to monitor codon recognition by tRNAs and RFs in the aminoacyl (A) site of the 30S subunit of the ribosome. Using the change in fluorescence of a pyrene molecule attached to the 3' end of a short mRNA as a probe, we have investigated the kinetic mechanism of ternary complex and release factor binding to the A-site of the ribosome. Codon recognition by ternary complexes occurs as part of the second order association step between the ribosome and ternary complex. By interacting with the codon during the first encounter, competition between cognate and near or non-cognate ternary complexes is reduced and rapid screening of ternary complexes may occur. We have found that physiological concentrations of the polyamines spermine and spermidine stimulate ternary complex binding to the A-site of the ribosome at least as well as unphysiologically high concentrations of magnesium ions commonly used during in vitro translation experiments. We have also investigated the thermodynamics and kinetics of RF1 binding to the ribosome when a stop codon or a variety of sense codons were positioned in the A-site. The relative affinity of RF1 to different sense codons and the catalysis of peptide release by the RF were not directly related. The observed disparity between binding and catalysis indicates that RF1 employs an induced fit mechanism in the discrimination of stop codons from sense codons.