Folding of individual domains in large proteins occurs during translation, helping to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the G-domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway that initiates from the C-terminus and thus runs counter to synthesis. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.
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