Biography
Biography: Yuhong Wang
Abstract
The ribosomal frameshifting is a rare but ubiquitous process. The putative "-1" frameshifting motif includes a slippery sequence, a spacer, and a secondary mRNA structure. We report a new force-based method to direct observation of “-1” and “-2” frameshiftings with single nucleotide resolution. We reveal that EF-G•GTP is indispensable to frameshifting. The biological relevance of the in vitro results is verified by protein translations in the cell. The mechanistic insights provided by our assay demonstrated the application of this method to study the ribosome system. The ribosome pre- and post-translocation complexes are tethered to the surface by biotinylated mRNA. The 3'-mRNA uncovered by the ribosome forms duplexes with DNA probes with known sequences. The probes are labeled with magnetic beads. Under external forces, the duplexes dissociate step-wisely according to the base pairs in the duplexes. The dissociation is detected by an atomic magnetometer and reflects the ribosome position with single nucleotide resolution. Three consecutive translocation steps were tracked to unambiguously identify the total of nine possible ribosome positions on the mRNA under in vitro conditions. Mechanistic studies were carried out by modifying the motif, introducing a secondary structure, and varying other experimental conditions. Meanwhile, in vivo and in vitro protein synthesis experiments were performed to demonstrate the biological significance of the frameshifting results.
