Intermediate states during mRNA–tRNA translocation

Recent studies support the notion that the pre-translocation (PRE) ribosomal complex functions, at least in part, as a Brownian machine, stochastically fluctuating among multiple conformations and transfer RNA (tRNA) binding configurations. Apart from the relatively more energetically stable conformational states of the PRE complex, termed macrostate I (MS I) and macrostate II (MS II), several additional intermediate states have been recently discovered. Structural and kinetic analyses of these states, made possible by cryogenic-electron microscopy (cryo-EM), X-ray crystallography, and single-molecule fluorescence resonance energy transfer (smFRET), have provided important insights into the translocation process, which is now understood to proceed, at least in the first step of the process, as a Brownian machine that is transiently stabilized in the ‘productive’ MS II conformation by the binding of the translocase elongation factor G (EF-G).

► During elongation, mRNA–tRNA translocation entails large-scale movements of the ribosomal subunits, mRNA, and two tRNAs.
► Data from cryo-EM, X-ray crystallography, and – to some extent – smFRET point to the existence of intermediate states.
► Visualization of such intermediates equilibrating in a factor-free sample is possible by cryo-EM with the aid of classification.
► Analysis of atomic structures shows that a peripheral intersubunit bridge may control the intersubunit motion.
► The model of the ribosome as a Brownian machine is supported by T-dependence of intersubunit rotation reported by smFRET.