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bioRxiv · 10.1101/2023.09.04.555162

Single-Molecule Tracking Reveals Dynamic Regulation of Ribosomal Scanning

Hong, H. J.·Zhang, A. L.·Conn, A. B.·Blaha, G.·O'Leary, S. E.
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Abstract

To initiate protein synthesis, the eukaryotic ribosomal pre-initiation complex must survey a messenger RNA leader sequence to identify the correct start codon.1 This pre-initiation complex motion through the leader, termed scanning, is coordinated by an intricate and highly-dynamic assemblage of translation factors, mRNA, initiator tRNA, and the small ribosomal subunit.2,3,4 Fundamental aspects of scanning dynamics remain poorly understood: estimates of its rate vary widely, and mechanisms that establish and regulate the motion remain largely unknown. Here we show, at the single-molecule level, that the Saccharomyces cerevisiae pre-initiation complex scans a diverse set of mRNA leaders at a rate of 10 - 20 nt s-1. Our data quantitatively support a scanning mechanism in which the mRNA leader is inspected base by base, essentially unidirectionally, and with modest sensitivity to mRNA structure. Unexpectedly, scanning bypasses canonical start sites where the initiator tRNA is present but GTP hydrolysis in the pre-initiation complex is impaired. Conversely, binding of the S. cerevisiae poly(A)-binding protein Pab1p to its own mRNA leader hinders scanning in a concentration-dependent manner. At saturating, physiological concentrations, Pab1p prolongs scanning by more than four-fold, evoking an autoregulation mechanism for translation initiation. Our data provide a real-time mechanistic framework for scanning regulation and energetics.

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Hong, H. J., Zhang, A. L., Conn, A. B., Blaha, G., O'Leary, S. E.. 2023-09-05. Single-Molecule Tracking Reveals Dynamic Regulation of Ribosomal Scanning. https://doi.org/10.1101/2023.09.04.555162

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