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Elongation factor-G-catalyzed translocation of mRNA and tRNAs during protein synthesis involves large-scale conformational changes in the ribosome. Formation of hybrid-state intermediates is coupled to counterclockwise (forward) rotation of the body of the 30S subunit. Recent structural studies implicate intrasubunit rotation of the 30S head in translocation. Here, we observe rotation of the head during translocation in real time using ensemble stopped-flow FRET with ribosomes containing fluorescent probes attached to specific positions in the head and body of the 30S subunit. Our results allow ordering of the rates of movement of the 30S subunit body and head during translocation: body forward &gt; head forward &gt; head reverse ≥ body reverse. The rate of quenching of pyrene-labeled mRNA is consistent with coupling of mRNA translocation to head rotation.

Rotation of the head of the 30S ribosomal subunit during mRNA translocation