Operative Binding of Class I Release Factors and YaeJ Stabilizes the Ribosome in the Nonrotated State.

During translation, the small subunit of the ribosome rotates with respect to the large subunit primarily between two states as mRNA is being translated into a protein. At the termination of bacterial translation, class I release factors (RFs) bind to a stop codon in the A-site and catalyze the release of the peptide chain from the ribosome. Periodically, mRNA is truncated prematurely, and the translating ribosome stalls at the end of the mRNA forming a nonstop complex requiring one of several ribosome rescue factors to intervene. One factor, YaeJ, is structurally homologous with the catalytic region of RFs but differs by binding to the ribosome directly through its C-terminal tail. Structures of the ribosome show that the ribosome adopts the nonrotated state conformation when these factors are bound. However, these studies do not elucidate the influence of binding to cognate or noncognate codons on the dynamics of intersubunit rotation. Here, we investigate the effects of wild-type and mutant forms of RF1, RF2, and YaeJ binding on ribosome intersubunit rotation using single-molecule Förster resonance energy transfer. We show that both RF1 binding and RF2 binding are sufficient to shift the population of posthydrolysis ribosome complexes from primarily the rotated to the nonrotated state only when a cognate stop codon is present in the A-site. Similarly, YaeJ binding stabilizes nonstop ribosomal complexes in the nonrotated state. Along with previous studies, these results are consistent with the idea that directed conformational changes and binding of subsequent factors to the ribosome are requisite for efficient termination and ribosome recycling.