The Mechanism of Ribosomal Translocation Revealed by Single-molecule FRET and Super-resolution Force Spectroscopy (SURFS)




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During protein synthesis, peptidyl tRNA at the A site moves to the P site, and deacylated tRNA at the P site moves to the E site. This step in protein translation is called translocation and is catalyzed by a conserved GTPase--elongation factor G (EF-G). This dissertation addresses the role of EF-G and EF-G-induced GTP hydrolysis in the typical translocation event. In addition, we evaluate the role of EF-G in abnormal translocation events like ribosomal stalling and frameshifting during the translation process. The rate of tRNA translocation during protein translation is accelerated 50-fold by the EF-G-induced GTP hydrolysis. It is not clear that the GTP hydrolysis causes some conformational changes in EF-G or powers tRNAs translocation directly. To achieve our experimental objectives, we developed a dual FRET system to track the internal interaction of two EF-G domains while simultaneously tracking the interaction of ribosomes and tRNAs. The FRET data shows that GTP hydrolysis and ribosome are required for EF-G conformational change but are not coupled directly with tRNA translocation. Meanwhile, translocation can be paused when the ribosome stalls on an mRNA encoding a polyproline sequence. One possible reason for ribosomal stalling is the slow peptide bond formation between prolines. The FRET system was used to track sensitive changes in the interaction of tRNAs and ribosomes to detect the kinetics of the prolyl peptide bond formation. Our observations suggest that ribosomal stalling is caused by the destabilization of A-site tRNAs in the cases where several consecutive proline codons enter the mRNA decoding center. Furthermore, translocation can also be misled by ribosomal frameshifting. This process is influenced by both the mRNA sequence and secondary mRNA structure. Super-resolution force spectroscopy (SURFS) was used to determine the force in the process of ribosomal translocation precisely. Neomycin was applied to trap different phases of ribosomal frameshifting. Surprisingly, four stages were observed when studying the movements at the 5'-end of mRNA by SURFS. It was found that during mRNA translation, its 5' terminus is always more motile than the 3' terminus. It was concluded that EF-G rescues the frameshifting by stabilizing the ribosome with compromised conformational changes.



Elongation factor G, tRNA translocation, Ribosomal Frameshifting, Ribosomal Stalling, FRET, SURFS