Dynamics of ribosome scanning during translation initiation.

Abstract

Faithful recognition of the mRNA start codon at the initial stage of translation is an essential process during regulation of gene expression. For this purpose, eukaryotes utilize a 43S pre-initiation complex (PIC), which moves along the mRNA 5′ untranslated region (UTR) to locate the start codon; this movement is termed “scanning” and is thought to proceed linearly with significantly processive 5′-to-3′ directionality. Although the scanning model was proposed many decades ago, the exact molecular mechanism of the motion, and how it may vary between different mRNAs and under various cellular conditions, remain unclear due to the highly dynamic nature of the process. We developed a multi-color single molecule fluorescence method to observe PIC dynamics in real time on the translation initiation timescale. Using this technology, we aim to directly measure the rate of scanning on full-length yeast mRNA, in order to understand the dynamics of PIC movement along different UTR lengths and structural contexts. Furthermore, we seek to parse the roles of key initiation factors and their biochemical activities in the scanning mechanism. Our data provide direct insights into this vital process in translational control.