Direct measurements of mRNA translation kinetics in living cells

ABSTRACT Ribosome mediated mRNA translation is central to life as we know it. The cycle of translation has, however, not been characterized in a living cell. Here we have developed a live-cell ribosome-labeling method, which allows us to characterize the whole processes of finding an mRNA and translating it, using single-molecule tracking techniques. We find that more than 90% of both bacterial ribosomal subunits are engaged in elongation at any particular time, and that neither of the subunits, in general, continues translation from one open reading frame to the next on a poly-cistronic mRNA. Furthermore, we find that a variety of previously published orthogonal ribosomes, with altered anti-Shine-Dalgarno sequences, show significant binding to endogenous mRNAs, with the rate of translation initiation only modestly affected. Hence, our results suggest that other mRNA elements than the SD sequence play major roles in directing the ribosome to the correct translation start sites.