@article {65018, title = {Ensemble cryo-EM elucidates the mechanism of translation fidelity.}, journal = {Nature}, volume = {546}, year = {2017}, month = {2017 Jun 01}, pages = {113-117}, abstract = {

Gene translation depends on accurate decoding of mRNA, the structural mechanism of which remains poorly understood. Ribosomes decode mRNA codons by selecting cognate aminoacyl-tRNAs delivered by elongation factor Tu (EF-Tu). Here we present high-resolution structural ensembles of ribosomes with cognate or near-cognate aminoacyl-tRNAs delivered by EF-Tu. Both cognate and near-cognate tRNA anticodons explore the aminoacyl-tRNA-binding site (A site) of an open 30S subunit, while inactive EF-Tu is separated from the 50S subunit. A transient conformation of decoding-centre nucleotide G530 stabilizes the cognate codon-anticodon helix, initiating step-wise {\textquoteright}latching{\textquoteright} of the decoding centre. The resulting closure of the 30S subunit docks EF-Tu at the sarcin-ricin loop of the 50S subunit, activating EF-Tu for GTP hydrolysis and enabling accommodation of the aminoacyl-tRNA. By contrast, near-cognate complexes fail to induce the G530 latch, thus favouring open 30S pre-accommodation intermediates with inactive EF-Tu. This work reveals long-sought structural differences between the pre-accommodation of cognate and near-cognate tRNAs that elucidate the mechanism of accurate decoding.

}, keywords = {Anticodon, Codon, Cryoelectron Microscopy, Escherichia coli, GTP Phosphohydrolases, Guanosine Triphosphate, Hydrolysis, Models, Molecular, Peptide Elongation Factor Tu, Protein Biosynthesis, Protein Domains, Ribosome Subunits, Ribosomes, RNA, Ribosomal, 16S, RNA, Transfer, Amino Acyl}, issn = {1476-4687}, doi = {10.1038/nature22397}, author = {Loveland, Anna B and Demo, Gabriel and Grigorieff, Nikolaus and Korostelev, Andrei A} } @article {48344, title = {Mechanism of ribosome rescue by ArfA and RF2.}, journal = {eLife}, volume = {6}, year = {2017}, month = {2017 Mar 16}, pages = {e23687}, abstract = {

ArfA rescues ribosomes stalled on truncated mRNAs by recruiting release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2-{\r A} resolution cryo-EM structures - determined from a single sample - of the 70S ribosome with ArfA{\textbullet}RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA "senses" the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.

}, issn = {2050-084X}, doi = {10.7554/eLife.23687}, author = {Demo, Gabriel and Svidritskiy, Egor and Madireddy, Rohini and Diaz-Avalos, Ruben and Grant, Timothy and Grigorieff, Nikolaus and Sousa, Duncan and Korostelev, Andrei A} } @article {48865, title = {Structure of RNA polymerase bound to ribosomal 30S subunit.}, journal = {eLife}, volume = {6}, year = {2017}, month = {2017 Oct 13}, abstract = {

In bacteria, mRNA transcription and translation are coupled to coordinate optimal gene expression and maintain genome stability. Coupling is thought to involve direct interactions between RNA polymerase (RNAP) and the translational machinery. We present cryo-EM structures of E. coli RNAP core bound to the small ribosomal 30S subunit. The complex is stable under cell-like ionic conditions, consistent with functional interaction between RNAP and the 30S subunit. The RNA exit tunnel of RNAP aligns with the Shine-Dalgarno-binding site of the 30S subunit. Ribosomal protein S1 forms a wall of the tunnel between RNAP and the 30S subunit, consistent with its role in directing mRNAs onto the ribosome. The nucleic-acid-binding cleft of RNAP samples distinct conformations, suggesting different functional states during transcription-translation coupling. The architecture of the 30S{\textbullet}RNAP complex provides a structural basis for co-localization of the transcriptional and translational machineries, and inform future mechanistic studies of coupled transcription and translation.

}, issn = {2050-084X}, doi = {10.7554/eLife.28560}, author = {Demo, Gabriel and Rasouly, Aviram and Vasilyev, Nikita and Svetlov, Vladimir and Loveland, Anna B and Diaz-Avalos, Ruben and Grigorieff, Nikolaus and Nudler, Evgeny and Korostelev, Andrei A} }