@article {65613, title = {mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding.}, journal = {eLife}, volume = {9}, year = {2020}, month = {2020 May 19}, abstract = {

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule F{\"o}rster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from mRNA and the transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape the ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.

}, issn = {2050-084X}, doi = {10.7554/eLife.55799}, author = {Bao, Chen and Loerch, Sarah and Ling, Clarence and Korostelev, Andrei A and Grigorieff, Nikolaus and Ermolenko, Dmitri N} } @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} } @article {47465, title = {Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016 May 9}, abstract = {

Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2{\textbullet}GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. The structures suggest missing links in our understanding of tRNA translocation.

}, issn = {2050-084X}, doi = {10.7554/eLife.14874}, author = {Abeyrathne, Priyanka D and Koh, Cha San and Grant, Timothy and Grigorieff, Nikolaus 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} } @article {65021, title = {Taura syndrome virus IRES initiates translation by binding its tRNA-mRNA-like structural element in the ribosomal decoding center.}, journal = {Proc Natl Acad Sci U S A}, volume = {111}, year = {2014}, month = {2014 Jun 24}, pages = {9139-44}, abstract = {

In cap-dependent translation initiation, the open reading frame (ORF) of mRNA is established by the placement of the AUG start codon and initiator tRNA in the ribosomal peptidyl (P) site. Internal ribosome entry sites (IRESs) promote translation of mRNAs in a cap-independent manner. We report two structures of the ribosome-bound Taura syndrome virus (TSV) IRES belonging to the family of Dicistroviridae intergenic IRESs. Intersubunit rotational states differ in these structures, suggesting that ribosome dynamics play a role in IRES translocation. Pseudoknot I of the IRES occupies the ribosomal decoding center at the aminoacyl (A) site in a manner resembling that of the tRNA anticodon-mRNA codon. The structures reveal that the TSV IRES initiates translation by a previously unseen mechanism, which is conceptually distinct from initiator tRNA-dependent mechanisms. Specifically, the ORF of the IRES-driven mRNA is established by the placement of the preceding tRNA-mRNA-like structure in the A site, whereas the 40S P site remains unoccupied during this initial step.

}, keywords = {Nucleic Acid Conformation, Open Reading Frames, Peptide Chain Initiation, Translational, Picornaviridae, Ribosomes, RNA, Messenger, RNA, Transfer, RNA, Viral}, issn = {1091-6490}, doi = {10.1073/pnas.1406335111}, author = {Koh, Cha San and Brilot, Axel F and Grigorieff, Nikolaus and Korostelev, Andrei A} }