Protein Translation Mechanism Revealed by Next-Generation Single Molecule Technology
Abstract: Translation by the ribosome occurs by a complex mechanism involving the coordinated interaction of multiple nucleic acid and protein ligands. Here we have used zero-mode waveguides (ZMWs) and sophisticated detection instrumentation to allow real-time observation of translation at physiologically-releveant (µM) ligand concentrations. Translation at each codon is monitored by stable binding of tRNAs – labeled with distinct fluorophores – to translating ribosomes, allowing direct detection of the identity of tRNA molecules bound to the ribosome, and therefore, the underlying mRNA sequence. We observe the transit of tRNAs on single translating ribosomes and have determined the number of tRNA molecules bound, or occupancy of the ribosome, in real time. The methods outlined here have broad application to the study of mRNA sequences, and the mechnanism and regulation of translation.
Key words: Protein translation, Single molecule measurement, Zero-Mode Waveguides, Translation initiation, Ribosome