Spontaneous Vs. Allosteric Dissociation of E-Site tRNA During Polypeptide Elongation

During ribosome-catalyzed polypeptide chain elongation, dissociation of the deacylated tRNA from the E-site has been proposed to be either spontaneous or triggered allosterically by binding of the next cognate ternary complex to the A-site. Using fluorescent labeled tRNAs, we have measured single molecule fluorescence intensities and single molecule FRET between adjacent tRNAs in the ribosome. From these measurements we have been able to determine tRNA occupancy in the ribosome, and thus whether E-site tRNA dissociates before or after A-site occupancy. In the former case there are a maximum of 2 simultaneously bound tRNAs, while the latter case results in transient binding of 3 tRNAs simultaneously. In a total internal reflection florescence microscope, ribosomes were attached to glass microscopes slides via a biotinylated mRNA coding for MRFFRFY.... (single letter amino acid sequence). When synthesis was initiated with tRNAfMet fully charged with formylated-Met, 60-70% of the ribosomes lost their E-site tRNA prior to ternary complex binding (2-tRNA pathway) at the 2nd and 3rd elongation cycles (R and F respectively). In contrast, for synthesis initiated with uncharged initiator tRNAfMet, >90% of the ribosomes followed the 2-tRNA pathway in the 2nd cycle, but only ∼15% in the 3rd cycle (∼75% following the 3-tRNA route, 10% not categorized). In cycles 4 and 5, almost all ribosomes followed the 2-tRNA pathway. Thus, the length of the peptide chain and/or the specific amino acids bound to the P-site tRNA strongly influence the allostery of E-site tRNA dissociation. Such allostery is also sensitive to the presence or absence of polyamines, Mg2+ concentration, and the specific codons in positions 2 and 3. Supported by NIH R01grant GM080376 and NIST ATP grant 70NANB7H7011 through Anima Cell Metrology, Inc.