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 R(1), undergoes a ratchet-like intersubunit rotation in order to sample a rotated conformation, referred to here as R(F), that is an obligatory intermediate in the translocation of tRNAs and mRNA through the ribosome during the translocation step of the translation elongation cycle. R(F) and the R(1) to R(F) transition are currently the subject of intense research, driven in part by the potential for developing novel antibiotics which trap R(F) or confound the R(1) to R(F) transition. Currently lacking a 3D atomic structure of the R(F) endpoint of the transition, as well as a preliminary conformational trajectory connecting R(1) and R(F), the dynamics of the mechanistically crucial R(1) to R(F) 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 R(F) in order to build an almost complete model of the T. thermophilus ribosome in R(F) 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 R(1) to R(F) transition and results in new intersubunit bridges that are predicted to exist only in R(F). In addition, we use a recently reported E. coli ribosome structure, apparently trapped in an intermediate state along the R(1) to R(F) transition pathway, referred to here as R(2), as a guide to generate a T. thermophilus ribosome in the R(2) state. This demonstrates a multiresolution method for morphing large complexes and provides us with a structural model of R(2) in the species of interest. The generated structural models form the basis for probing the motion of the deacylated tRNA bound at the peptidyl-tRNA binding site (P site) of the pre-translocation ribosome as it moves from its so-called classical P/P configuration to its so-called hybrid P/E configuration as part of the R(1) to R(F) transition. We create a dynamic model of this process which provides structural insights into the functional significance of R(2) as well as detailed atomic information to guide the design of further experiments. The results suggest extensibility to other steps of protein synthesis as well as to spatially larger systems.
Bioengineering Department, Stanford University, James H Clark Center S172 MC:5448, Stanford, California 94305, USA. samuelflorec@gmail.com
Samuel Coulbourn
SC
Flores
Russ
R
Altman
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D019295:Computational Biology; D020285:Cryoelectron Microscopy; D004926:Escherichia coli / Q000378:metabolism / Q000648:ultrastructure; D007091:Image Processing, Computer-Assisted; D008958:Models, Molecular; D008968:Molecular Conformation; D009068:Movement; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D016963:Thermus thermophilus / Q000378:metabolism / Q000648:ultrastructure
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins