BPS2026 – Mechanistic insights into human scanning during translation initiation

During translation initiation in human cells, the 40S ribosomal subunit binds to the 5′ end of messenger RNA (mRNA) and “scans” through the 5′ untranslated region (UTR) to locate the correct start codon. Scanning to the correct start codon is critical for setting the reading frame for translation, and defects in the same have been associated with dysregulation of translation causing neurological disorders and cancers. Despite its importance, the factors governing scanning in humans remain poorly understood. Here, we developed a real-time single-molecule FRET approach to monitor scanning kinetics directly in an in vitro reconstituted human translation system. Human 40S ribosomal subunits were purified, site-specifically labeled, and validated for FRET with probes on the mRNA using TIRF microscopy. Real-time delivery of labeled ribosomes to mRNA on zero-mode waveguides enabled us to observe and quantify scanning kinetics. We also examined the effect of ATP concentration on scanning times, elucidating the energy dependence of scanning. Ongoing work focuses on how 5′ UTR structure and length modulate scanning. This approach establishes a powerful tool to study the human scanning mechanism and provides a framework for understanding the dysregulation of translation in disease.