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Poster Session 10: Translation 21:00 - 22:00 Friday, 29Th May, 2020 Poster

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Poster Session 10: Translation 21:00 - 22:00 Friday, 29th May, 2020 Poster 66 Translational fidelity is maintained through precise aminoacyl-tRNA accommodation dynamics gated by Elongation Factor Tu Dylan Girodat1, Scott Blanchard2, Hans-Joachim Wieden3, Karissa Sanbonmatsu1 1Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, New Mexico, USA. 2Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. 3Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Alberta, Canada Abstract The fidelity of translation is enigmatic, as the efficiency of cognate aminoacyl(aa)-tRNA selection by the ribosome is greater than what can be predicted from Watson-Crick base-pairing between the codon in the mRNA and the anticodon in the tRNA. The complexity of this process arises from the fact that aa-tRNA selection is a multistep process aided by auxiliary proteins such as the GTPase elongation factor (EF)-Tu, responsible for delivery of aa-tRNA to the ribosome. As such, the precise structural mechanism of how the ribosome in complex with EF-Tu selects for cognate aa-tRNA remains to be fully resolved. Here, using all-atom molecular dynamics (MD) simulations, we identify subtle differences between cognate and near-cognate aa-tRNA movement into the ribosome and how conformational rearrangements of EF-Tu aid in tRNA selection. Near-cognate aa-tRNA accommodation follows an alternative trajectory, compared to cognate aa-tRNA, leading to a misaligned position within the A-site. The origins of the alternative trajectory originate from the perturbed base-pairing between the codon and anticodon of the mRNA and tRNA, respectively. The resulting position is ultimately not suitable for peptide bond formation. As aa-tRNA accommodation is initiated EF-Tu undergoes a conformational change involving the rapid conversion of the switch I element from an α- helix to a β-hairpin. The newly formed β-hairpin moves to interact with the acceptor stem of the aa-tRNA and in doing so gates the movement of the aa-tRNA during accommodation through steric and electrostatic interactions. Furthermore, switch I of EF-Tu traverses along either the acceptor stem or near the 3’-CCA end for cognate or near-cognate aa-tRNA, respectively. Ultimately, this is a result of near-cognate accommodating through a misaligned trajectory. Altogether, this project provides evidence for how the ribosome in complex with EF-Tu can sense the identity of the accommodating aa-tRNA and provides a structural description of tRNA selection. Presenting author email [email protected] Topic category Translation Mechanism 156 Co-translational translocon insertion and topogenesis of bacterial membrane proteins Evan Mercier, Marina Rodnina, Wolfgang Wintermeyer Max-Planck Institute for Biophysical Chemistry, Göttingen, Germany Abstract Integral membrane proteins insert into the bacterial inner membrane co-translationally via the translocon. Transmembrane segments (TM) of nascent proteins adopt their native topological arrangement with the N- terminus of TM1 oriented to the outside (type I) or the inside (type II) of the cell. Here we study TM1 topogenesis during ongoing mRNA translation in a bacterial in-vitro system, applying real-time FRET and protease protection assays. We find that TM1 of the type I protein LepB reaches the translocon immediately upon emerging from the ribosome. In contrast, the type II protein EmrD requires a longer nascent chain before TM1 reaches the translocon and adopts its topology by looping inside the ribosomal peptide exit tunnel early on in translation. Looping presumably is mediated by interactions between positive charges at the N-terminus of TM1 and negative charges in the wall of the peptide-exit tunnel. Early TM1 inversion is abrogated by charge reversal at the N-terminus. Kinetic analysis also shows that co-translational membrane insertion of TM1 is intrinsically rapid and rate-limited by mRNA translation. Thus, the ribosome and translation play vital roles in the insertion and topogenesis of newly synthesized membrane proteins in bacteria. Presenting author email [email protected] Topic category Translation Mechanism 185 How ArfB Rescues Stalled Ribosomes Christine Carbone, Gabriel Demo, Rohini Madireddy, Egor Svidritskiy, Andrei Korostelev University of Massachusetts Medical School, Worcester, MA, USA Abstract A translating ribosome stalls when it encounters the end of a non-stop mRNA, truncated during cellular stress or other conditions (Hayes and Keiler, 2010; Keiler, 2015). Alternative rescue factor B (ArfB) rescues stalled ribosomes by catalyzing peptide release from peptidyl-tRNA (Chadani et al., 2011). Crystallographic work (Gagnon et al., 2012) showed that the C-terminal α-helix of ArfB binds in the mRNA entry channel, allowing the catalytic N-terminal domain to reach the A site of the peptidyl transferase center. Thus, binding of ArfB should be incompatible with the mRNAs extending beyond the A-site. However, previous work showed that ArfB can act on ribosomes stalled on a rare codon cluster, and it remains unclear how the ribosome can accommodate both ArfB and mRNA in this case (Handa et al., 2011). In this work, we sought to clarify the mechanism for ArfB action on a broad range of mRNA substrates, using in vitro kinetics and cryogenic electron microscopy (cryo- EM). Surprisingly, ArfB remains highly efficient on mRNAs extending beyond the A site. Using single particle cryo-EM, we identified multiple states that suggest different mRNA-length-dependent structural mechanisms for ArfB-mediated ribosome rescue. Presenting author email [email protected] Topic category Translation Mechanism 389 Resurrection of a factorless internal ribosome entry site from Ancient Northwest Territories cripavirus Xinying Wang, Reid Warsaba, Eric Jan Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada Abstract Translation is a key step of gene expression in all organisms. The majority of eukaryotic mRNAs use a scanning cap-dependent mechanism that requires upwards of 12 factors to initiate translation. In contrast, the dicistrovirus intergenic region internal ribosome entry site (IGR IRES) uses an unprecedented streamlined mechanism whereby the IRES adopts a triple-pseudoknot (PK) structure to directly bind to the conserved core of the ribosome and drive translation from a non-AUG codon. The origin of this IRES mechanism is not known. Previously, a partial fragment of a divergent dicistrovirus RNA genome containing the IGR region was extracted from 700-year-old caribou feces trapped in a subarctic ice patch. This "ancient" virus was named ancient Northwest territories cripavirus (aNCV). Structural prediction of the aNCV IGR sequence generated a secondary structure similar to contemporary IGR IRES structures. There are, however, subtle differences that may impact IRES function. There are also 100 nucleotides upstream of the IRES with an unknown function. Using filter binding assays, we showed that the aNCV IGR IRES could bind to purified salt-washed human ribosomes. They could also compete with excess CrPV IGR IRESs for ribosomes. Toeprinting analysis using primer extension pinpointed the putative start site of the aNCV IGR: a GCU alanine codon adjacent to PKI. Using a bicistronic reporter RNA, the aNCV IGR IRES can direct internal ribosome entry in vitro in rabbit reticulocyte lysates that was dependent on the integrity of the PKI domain. Lastly, we generated a chimeric virus clone by swapping the aNCV IRES into the cricket paralysis virus infectious clone. The chimeric infectious clone with an aNCV IGR IRES supported translation and virus infection. The characterization and resurrection of a functional IGR IRES from a divergent 700-year-old virus points to the importance of this translational mechanism and will contribute to our understanding of viral RNA evolution. Presenting author email [email protected] Topic category Translation Mechanism 563 Investigating the role of DDX3X in translation initiation Kevin Wilkins1,2, Srivats Venkataramanan1, Lorenzo Calviello1, Bao Thai1, Malvika Tejura1, Stephen Floor1,3 1University of California, San Francisco, San Francisco, CA, USA. 2Graduate Division, University of California, San Francisco, San Francisco, CA, USA. 3Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Abstract Translation of mRNA into protein is an essential process in all cells, and its dysregulation is linked to human diseases. During translation initiation, ribosome scanning is influenced by the secondary structure of the 5′ untranslated region (UTR). A family of enzymes known as RNA helicases modulate ribosome scanning and hence protein synthesis by unwinding and resolving secondary RNA structures. The RNA helicase DDX3X is an ATP-dependent DEAD-box RNA helicase that is implicated in the translation of mRNAs that contain long and highly structured 5′ UTRs. Despite DDX3X being altered in several human cancers and developmental diseases, how DDX3X acts on mRNA during scanning and what makes a given transcript responsive to it is still incompletely understood. Using an in vitro translation assay, I determined that missense mutations in DDX3X lead to decreased translation initiation for mRNAs containing select 5′ UTRs. Mechanistic details regarding how RNA sequence and structure impact DDX3X sensitivity will be presented. We previously found that DDX3
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  • A C. Elegans Model of C9orf72-Associated ALS/FTD Uncovers a Conserved Role

    A C. Elegans Model of C9orf72-Associated ALS/FTD Uncovers a Conserved Role

    bioRxiv preprint doi: https://doi.org/10.1101/2020.06.13.150029; this version posted June 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 A C. elegans model of C9orf72-associated ALS/FTD uncovers a conserved role 2 for eIF2D in RAN translation 3 4 5 Yoshifumi Sonobe 1, 2, 3, Jihad Aburas 1, 3, 4, Priota Islam 5, 6, Tania F. Gendron 7, André E.X. 6 Brown 5, 6, Raymond P. Roos* 1, 2, 3, Paschalis Kratsios* 1, 3, 4 7 8 * These authors contributed equally to this study. 9 10 Correspondence: 11 R.P.R ([email protected]), P.K ([email protected]) 12 13 Affiliations: 14 1 University of Chicago Medical Center, 5841 S. Maryland Ave., Chicago, IL 60637 15 2 Department of Neurology, University of Chicago Medical Center, 5841 S. Maryland Ave., 16 Chicago, IL 60637 17 3 The Grossman Institute for Neuroscience, Quantitative Biology, and Human Behavior, 18 University of Chicago, Chicago, IL, USA 19 4 Department of Neurobiology, University of Chicago, Chicago, IL, USA 20 5 MRC London Institute of Medical Sciences, London, UK 21 6 Institute of Clinical Sciences, Imperial College London, London, UK 22 7 Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA 23 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.13.150029; this version posted June 15, 2020.