title: Ribosome•RelA structures reveal the mechanism of stringent response activation.
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 Å 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 'closure', providing insights into the dynamics of high-fidelity tRNA decoding.
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States.|Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States.|Department of Biochemistry, Brandeis University, Waltham, United States.|Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, United States.
authors.forename: Anna B
authors.initials: AB
authors.lastname: Loveland
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States.|Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States.
authors.forename: Eugene
authors.initials: E
authors.lastname: Bah
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States.|Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States.
authors.forename: Rohini
authors.initials: R
authors.lastname: Madireddy
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States.|Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, United States.|Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, United States.
authors.forename: Axel F
authors.initials: AF
authors.lastname: Brilot
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, United States.|Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, United States.|Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States.
authors.forename: Nikolaus
authors.identifier: 0000-0002-1506-909X
authors.initials: N
authors.lastname: Grigorieff
authors.affiliation: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States.|Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States.
authors.forename: Andrei A
authors.identifier: 0000-0003-1588-717X
authors.initials: AA
authors.lastname: Korostelev
mesh_terms: D020285:Cryoelectron Microscopy; D004926:Escherichia coli / Q000502:physiology*; D008025:Ligases / Q000378:metabolism* / Q000648:ultrastructure*; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism* / Q000648:ultrastructure*; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure*; D013312:Stress, Physiological
chemical_list: D012343:RNA, Transfer; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases
keywords: E. coli; RelA; biophysics; cognate tRNA; cryo-EM; decoding; ribosome; stringent response; structural biology