Friday, March 14, 2025
3/14/2025
Project Summary This project focuses on understanding the molecular mechanisms underlying the coupled translocation of mRNA and tRNAs during protein synthesis. It includes the important related problems of how the translational reading frame is preserved (or shifted) and how the ribosomal helicase unwinds structured mRNAs. Our laboratory uniquely uses a combination of biochemistry, structural biology, genetics, FRET and computational methods to address these challenging problems. We are also extending our approaches to include single-molecule optical tweezer methods, in collaboration with the Bustamante laboratory (UC Berkeley) and single- molecule FRET, in collaboration with the Ermolenko laboratory (Univ. of Rochester), as well as cryo-electron microscopy, in collaboration with the Chiu laboratory (Stanford/SLAC). In previous studies, we have determined the structures of trapped translocation intermediates, which have provided unexpected insights into how the movements of mRNA and tRNA through the ribosome are coupled to large- and small-scale conformational changes in the structure of the ribosome itself. We then created FRET pairs that allowed us to correlate intersubunit rotation, movement of the L1 stalk and rotation of the 30S subunit head domain with movements of mRNA and tRNA. We plan to extend this search to discover new intermediate states. Having exhausted previous strategies for trapping translocation intermediates, we will use a new approach which exploits a set of dominant-lethal mutations in all five structural domains of elongation factor EF-G that we expect will block translocation at different steps. Development of a novel fluorescent labeling approach that will allow site-specific labeling of FRET pairs directly to ribosomal RNA will overcome technical barriers to single-molecule studies of ribosome dynamics, including studies using simultaneous measurement of molecular forces and FRET changes in the ribosome, in collaboration with the Bustamante group. Finally, we have designed model structured mRNAs that will provide the basis for studying the mechanism of the mRNA helicase and for determination of the structures of translocation complexes stalled in the act of encountering and unwinding an mRNA helix.
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