Ribosome interactions of aminoacyl-tRNA and elongation factor Tu in the codon-recognition complex

Abstract

The mRNA codon in the ribosomal A-site is recognized by aminoacyl-tRNA (aa-tRNA) in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here we report the 13 A resolution three-dimensional reconstruction determined by cryo-electron microscopy of the kirromycin-stalled codon-recognition complex. The structure of the ternary complex is distorted by binding of the tRNA anticodon arm in the decoding center. The aa-tRNA interacts with 16S rRNA, helix 69 of 23S rRNA and proteins S12 and L11, while the sarcin-ricin loop of 23S rRNA contacts domain 1 of EF-Tu near the nucleotide-binding pocket. These results provide a detailed snapshot view of an important functional state of the ribosome and suggest mechanisms of decoding and GTPase activation.

The pathway to GTPase activation of elongation factor SelB on the ribosome

“…First, images were sorted according to global ribosome conformation, as described 58 (step 1), which allowed us to discard low quality particles and 50S subunits. Subsequently (step 2), particles were sorted according to ligand occupancy using supervised classification by projection matching 59 on the basis of a library of ribosome-ligand cryo-EM maps 34,60 . Importantly, this library entailed a cryo-EM map of the canonical ribosome-EF-Tu complex 60 as potential reference for the ribosome-SelB complex to avoid any reference bias for SelB-Sec-tRNA Sec , for which no structure was available.…”

“…Subsequently (step 2), particles were sorted according to ligand occupancy using supervised classification by projection matching 59 on the basis of a library of ribosome-ligand cryo-EM maps 34,60 . Importantly, this library entailed a cryo-EM map of the canonical ribosome-EF-Tu complex 60 as potential reference for the ribosome-SelB complex to avoid any reference bias for SelB-Sec-tRNA Sec , for which no structure was available. To further avoid any high-resolution reference bias, sorting in both steps was performed using low-pass filtered reference maps and particle images binned to about 6.9 Å per pixel.…”

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

“…In all subsequent steps, the resulting 1,339,775 contrast-transferfunction-corrected particle images were used. First, the particle images were sorted into groups of particles according to: (1) 30S body rotation, as described 7 ; and (2) ligand occupancy, using supervised classification by projection matching 35 on the basis of a structural library of different ribosome complexes 7,36 (Extended Data Fig. 2).…”

Recognition and selection of tRNA in translation

“…It is very likely, therefore, that the structural rearrangements of aa-tRNA found in the kirromycin-stalled complex by cryo-EM [9,10] appear as early as during GTPase activation. The anticodon arm of tRNA on Fig.…”

“…Crystal structures showed how the decoding center specifically interacts with the codon and anticodon at each position of the duplex [6][7][8]. Finally, cryoelectron microscopy (cryo-EM) provided a wealth of information about the arrangements of aa-tRNA and EF-Tu on the ribosome and the contacts and conformational rearrangements of these molecules during decoding [9,10]. The goal of this review is to summarize these recent findings and to present a unifying mechanism of tRNA selection on the ribosome.…”