Structures of the Bacterial Ribosome in Classical and Hybrid States of tRNA Binding

Abstract

During protein synthesis, the ribosome controls the movement of transfer RNA (tRNA) and messenger RNA (mRNA) by means of large-scale structural rearrangements. We describe structures of the intact bacterial ribosome from Escherichia coli that reveal how the ribosome binds tRNA in two functionally distinct states, determined to a resolution of ~3.2 Å by x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor (RRF) and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit (P/E) site. The structures help to explain how the ratchet-like motion of the two ribosomal subunits contributes to the mechanisms of translocation, termination, and ribosome recycling.

Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM

“…Map normalization (mean and standard deviation of density values are 0 and 1.0, respectively) was performed using MAPMAN (Rave package). The initial fit of an atomic model of the E.coli ribosome assembled from various crystal structures (PDB codes: 4GD2 49 (30S) 49 , 3R8T (50S) 49 , 2J00 (mRNA, tRNA fMet ) 50 , 3L0U (tRNA Phe ) 51 , 1OB2 (EF-Tu) (R. C. Nielsen et al unpublished data)) was performed using Chimera, followed by rigid body refinement in the PHENIX program. The atomic model was refined with deformable elastic network (DEN) restraints 52 in CNS with alternating cycles of manual rebuilding in Coot 53 and monitoring the local fit to the density with RESOLVE 54 .…”

Elongation Factor Ts Directly Facilitates the Formation and Disassembly of the Escherichia coli Elongation Factor Tu·GTP·Aminoacyl-tRNA Ternary Complex

“…Purification and Labeling of E. coli tRNA Phe -Wild-type E. coli tRNA Phe was purified from an RNase-deficient bacterial strain (MRE600) (44). Native tRNA Phe molecules were site-specifically labeled with the small molecule organic fluorophore Cy3 via the naturally occurring modified nucleotide acp 3 U present at position 47 (43,45).…”

Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-EM

“…Bridge B4, composed primarily of helix 34 in the 50S subunit, has not been seen to be disordered in known 50S structures, and atomic models of the isolated 50S do not require any rearrangement of helix 34 to be fitted into the cryo-EM maps of the 70S ribosome. However, density for helix 34 is not visible in our reconstructions of 70S at a reaction time of 9.4 ms and is only visible in a subpopulation of images at 43 ms. On the basis of an atomic model of the 70S ribosome (25) (Fig. 3), it appears that helix 34 of the 50S subunit fits into a notch on the 30S subunit formed by helix 24 of the 16S RNA and protein S15.…”

“…The inset at the top shows the 70S ribosome with two subunits (30S, yellow; 50S, blue) identified. X-ray crystallographic structures of the two ribosomal subunits [Protein Data Bank ID codes 3R8O and 3R8T (25)] were used to display the known locations of canonical intersubunit bridges. Locations of the bridges (1) were superimposed.…”