Partial spontaneous intersubunit rotations in actively translating ribosomes

The ribosome is a molecular machine that adopts at least two global states during translation. Two main steps of translation, peptidyl transfer and translocation, are accompanied by counterclockwise and clockwise rotations of the two ribosomal subunits. However, when and why the ribosome alternates between these states remains unclear, with two well supported but conflicting hypotheses. Ribosomes may undergo a single cycle of forward and backward rotations per codon read. Alternatively, in addition to rotations caused by peptidyl transfer and translocation, ribosomes may undergo multiple full spontaneous rotations, with these rotations playing a critical role in elongation and specifically in translocation mechanism. We applied high-speed single-molecule TIRF microscopy to follow translation in real-time. Actively translating ribosomes undergo partial spontaneous rotations between three different rotational states. Spontaneous rotations are restricted prior to A-site tRNA decoding. Peptidyl transfer unlocks spontaneous rotations. Consequently, translocation proceeds via a novel rotational state induced by EF-G. Our results bridge both models and provide a coherent view of ribosome dynamics during translation.