Abstract
Translation initiation is a critical control point for eukaryotic gene expression. The molecular mechanism of initiation requires a highly dynamic and exquisitely coordinated interplay of mRNA with the translation machinery. Recognition of the mRNA 5ʹ cap structure by initiation factor eIF4F is a key early initiation step, and plays important roles in translational control. We have developed single-molecule fluorescence approaches to directly observe eIF4F–mRNA interaction in real time, both on individual mRNAs and at transcriptome scale with single-molecule resolution. Our data reveal order-of-magnitude variability across mRNAs for interaction of the cap-binding eIF4F subunit, eIF4E, and provide insights into how the eIF4F subunits eIF4G and eIF4A dynamically modulate cap recognition. The data implicate features throughout the mRNA as controlling cap recognition at the 5ʹ end, including a relationship between coding-sequence length and the accessibility of the cap for recognition. We have also developed a single-molecule assay to directly observe the initiation steps that follow eIF4F–mRNA recognition, i.e. 43S ribosomal pre-initiation complex recruitment and pre-initiation complex scanning to locate the start codon. Taken together, our results evoke a multi-layered, mRNA-centric mechanism to kinetically control translation initiation and confer differential sensitivity to distinct regulatory mechanisms.
Published Version
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