STRUCTURAL INSIGHTS INTO PRE-TRANSLOCATION RIBOSOME MOTIONS

Subsequent to the peptidyl transfer step of the translation elongation cycle, the initially formed pre-translocation ribosome, which we refer to here as R1, undergoes a ratchet-like intersubunit rotation in order to sample a rotated conformation, referred to here as RF, that is an obligatory intermediate in the translocation of tRNAs and mRNA through the ribosome during the translocation step of the translation elongation cycle. RF and the R1 to RF transition are currently the subject of intense research, driven in part by the potential for developing novel antibiotics which trap RF or confound the R1 to RF transition. Currently lacking a 3D atomic structure of the RF endpoint of the transition, as well as a preliminary conformational trajectory connecting R1 and RF, the dynamics of the mechanistically crucial R1 to RF transition remain elusive. The current literature reports fitting of only a few ribosomal RNA (rRNA) and ribosomal protein (r-protein) components into cryogenic electron microscopy (cryo-EM) reconstructions of the Escherichia coli ribosome in RF. In this work we now fit the entire Thermus thermophilus 16S and 23S rRNAs and most of the remaining T. thermophilus r-proteins into a cryo-EM reconstruction of the E. coli ribosome in RF in order to build an almost complete model of the T. thermophilus ribosome in RF thus allowing a more detailed view of this crucial conformation. The resulting model validates key predictions from the published literature; in particular it recovers intersubunit bridges known to be maintained throughout the R1 to RF transition and results in new intersubunit bridges that ar