Sears, R. M.; Aguilera, L. U.; Bunting, T.; Zhao, N.

Translation elongation and protein folding have long been proposed to coordinate during co-translational folding, yet the lack of technologies capable of simultaneously tracking both processes in live cells has hindered mechanistic understanding of this relationship. Here, we developed co-translational folding tracking (coTFT), a live-cell imaging platform that directly and simultaneously tracks translation and folding from individual mRNAs. Using reporters with distinct folding kinetics, we found that differences in folding kinetics were accompanied by corresponding changes in translation elongation rates. Conversely, altering translation elongation markedly affected protein folding outcomes. Combining coTFT with mathematical modeling enabled estimation of reporter folding times on translating ribosomes in live cells, confirming their distinct folding kinetics. Together, our results reveal that translation elongation and folding are bidirectionally coupled during co-translational folding.