@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 {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} } @article {65009, title = {Ribosome{\textbullet}RelA structures reveal the mechanism of stringent response activation.}, journal = {Elife}, volume = {5}, year = {2016}, month = {2016 07 19}, abstract = {

Stringent response is a conserved bacterial stress response underlying virulence and antibiotic resistance. RelA/SpoT-homolog proteins synthesize transcriptional modulators (p)ppGpp, allowing bacteria to adapt to stress. RelA is activated during amino-acid starvation, when cognate deacyl-tRNA binds to the ribosomal A (aminoacyl-tRNA) site. We report four cryo-EM structures of E. coli RelA bound to the 70S ribosome, in the absence and presence of deacyl-tRNA accommodating in the 30S A site. The boomerang-shaped RelA with a wingspan of more than 100 {\r A} wraps around the A/R (30S A-site/RelA-bound) tRNA. The CCA end of the A/R tRNA pins the central TGS domain against the 30S subunit, presenting the (p)ppGpp-synthetase domain near the 30S spur. The ribosome and A/R tRNA are captured in three conformations, revealing hitherto elusive states of tRNA engagement with the ribosomal decoding center. Decoding-center rearrangements are coupled with the step-wise 30S-subunit {\textquoteright}closure{\textquoteright}, providing insights into the dynamics of high-fidelity tRNA decoding.

}, keywords = {Cryoelectron Microscopy, Escherichia coli, Ligases, Protein Binding, Ribosomes, RNA, Transfer, Stress, Physiological}, issn = {2050-084X}, doi = {10.7554/eLife.17029}, author = {Loveland, Anna B and Bah, Eugene and Madireddy, Rohini and Zhang, Ying and Brilot, Axel F and Grigorieff, Nikolaus and Korostelev, Andrei A} }