The molecular mechanics of start site selection during eukaryotic translation initiation

During eukaryotic translation initiation, ribosomal pre-initiation complexes scan the 5' untranslated region of the mRNA in search of the correct site at which to begin protein synthesis. Recognition of an AUG codon triggers irreversible changes in the complex, committing the ribosome to beginning translation at that point in the mRNA. Because this sets the reading frame, fidelity at this step is absolutely critical for proper gene expression. While yeast genetics and qualitative biochemistry have identified eukaryotic initiation factors (eIFs) 1, 1A, 2 and 5 as key players, the molecular mechanisms by which they work together to ensure fidelity in start site selection have remained mysterious.; We have reconstituted translation initiation in vitro from purified S. cerevisiae components. This system allows us to couple the power of yeast genetics and molecular biology with high-resolution, quantitative biochemical and biophysical approaches. By utilizing a number of novel fluorescence-based techniques, we have discovered that eIFs 1 and 1A interact energetically when bound to the ribosome. We have also discovered that recognition of an AUG codon by the pre-initiation complex triggers a conformational change that ejects eIF1 from the complex. In collaboration with my colleague, Mikkel Algire, we were able to demonstrate that this event facilitates the first irreversible step in the pathway, release of inorganic phosphate (Pi) after GTP hydrolysis by eIF2.; Additionally, by quantitatively analyzing the interactions between eIF1, eIF5 and mRNA within the 43S complex, we have demonstrated that recognition of an AUG codon triggers an interaction between the C-terminal domain of eIF1A and eIF5 that causes a conformational change in the complex. Our data suggest that this conformational change may serve to prevent further scanning once an AUG codon has been located, providing time for downstream events such as eIF1/Pi release to occur. Together these studies have provided significant insight into the mechanisms by which eIFs 1, 1A, 5 and 2 work together to ensure fidelity in start site selection during eukaryotic translation initiation.