Observing Prokaryotic Translation Elongation in Real-Time using Single-Molecule Fluorescence

Abstract

Translation elongation is a highly dynamic and energy intensive process where the ribosome must processively decode each codon encoded by the mRNA and add the corresponding amino acid via the correct tRNA. The process achieves a delicate balance between speed, proceeding at up to 20 amino acids per second in vivo, and accuracy, with an error rate of 1 error in 10,000 amino acids, while translating mRNAs with potentially complex sequences and structures. The ribosome must undergo significant conformational changes during elongation and move the mRNA in precise codon-sized steps to maintain reading frame. Accordingly, this process is the target of many clinically important antibiotics which increase the energy barrier to various steps of elongation in order to inhibit peptide synthesis. Nevertheless, the detailed effects of those antibiotics are challenging to probe using conventional biochemical techniques over the stochastic and processive process of elongation. To overcome the limited parameters simultaneously observable by one single-molecule experiment, we employed multiple single-molecule fluorescence techniques to probe elongation in real-time over multiple codons from multiple perspectives. We observed the mechanistically distinct effects of various aminoglycosides and other antibiotics on the steps of elongation. We additionally are extending our single-molecule methods from simple model mRNAs to ones with realistic coding sequences using a commercially available in vitro translation system. This opens the door for observing elongation dynamics over complex mRNA sequences to observe how complex mRNA sequences, such as secondary structures, internal Shine-Dalgarno-like sequences, or frame-shifting sequences, affect elongation dynamics. These studies demonstrate the power and potential of real-time single-molecule fluorescence techniques in dissecting the mechanisms of complex processes during translation and probing the global energy landscape of elongation under various conditions.