Ribosome•RelA structures reveal the mechanism of stringent response activation

  1. Anna B Loveland
  2. Eugene Bah
  3. Rohini Madireddy
  4. Ying Zhang
  5. Axel F Brilot
  6. Nikolaus Grigorieff  Is a corresponding author
  7. Andrei A Korostelev  Is a corresponding author
  1. University of Massachusetts Medical School, United States
  2. Mayo Medical School, United States
  3. Brandeis University, United States

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 stresses. 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.

Data availability

The following data sets were generated

Article and author information

Author details

  1. Anna B Loveland

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  2. Eugene Bah

    Mayo Medical School, Rochester, United States
    Competing interests
    No competing interests declared.
  3. Rohini Madireddy

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  4. Ying Zhang

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  5. Axel F Brilot

    Department of Biochemistry, Brandeis University, Waltham, United States
    Competing interests
    No competing interests declared.
  6. Nikolaus Grigorieff

    Department of Biochemistry, Brandeis University, Waltham, United States
    For correspondence
    niko@grigorieff.org
    Competing interests
    Nikolaus Grigorieff, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1506-909X
  7. Andrei A Korostelev

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    For correspondence
    andrei.korostelev@umassmed.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1588-717X

Funding

Howard Hughes Medical Institute

  • Nikolaus Grigorieff

National Institutes of Health (RO1 GM106105)

  • Andrei A Korostelev

National Institutes of Health (PO1 GM62580)

  • Nikolaus Grigorieff

Helen Hay Whitney Foundation

  • Anna B Loveland

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2016, Loveland et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 5,616
    views
  • 931
    downloads
  • 143
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Anna B Loveland
  2. Eugene Bah
  3. Rohini Madireddy
  4. Ying Zhang
  5. Axel F Brilot
  6. Nikolaus Grigorieff
  7. Andrei A Korostelev
(2016)
Ribosome•RelA structures reveal the mechanism of stringent response activation
eLife 5:e17029.
https://doi.org/10.7554/eLife.17029

Share this article

https://doi.org/10.7554/eLife.17029

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Yamato Niitani, Kohei Matsuzaki ... Michio Tomishige
    Research Article

    The two identical motor domains (heads) of dimeric kinesin-1 move in a hand-over-hand process along a microtubule, coordinating their ATPase cycles such that each ATP hydrolysis is tightly coupled to a step and enabling the motor to take many steps without dissociating. The neck linker, a structural element that connects the two heads, has been shown to be essential for head–head coordination; however, which kinetic step(s) in the chemomechanical cycle is ‘gated’ by the neck linker remains unresolved. Here, we employed pre-steady-state kinetics and single-molecule assays to investigate how the neck-linker conformation affects kinesin’s motility cycle. We show that the backward-pointing configuration of the neck linker in the front kinesin head confers higher affinity for microtubule, but does not change ATP binding and dissociation rates. In contrast, the forward-pointing configuration of the neck linker in the rear kinesin head decreases the ATP dissociation rate but has little effect on microtubule dissociation. In combination, these conformation-specific effects of the neck linker favor ATP hydrolysis and dissociation of the rear head prior to microtubule detachment of the front head, thereby providing a kinetic explanation for the coordinated walking mechanism of dimeric kinesin.

    1. Structural Biology and Molecular Biophysics
    Christopher T Schafer, Raymond F Pauszek III ... David P Millar
    Research Article

    The canonical chemokine receptor CXCR4 and atypical receptor ACKR3 both respond to CXCL12 but induce different effector responses to regulate cell migration. While CXCR4 couples to G proteins and directly promotes cell migration, ACKR3 is G-protein-independent and scavenges CXCL12 to regulate extracellular chemokine levels and maintain CXCR4 responsiveness, thereby indirectly influencing migration. The receptors also have distinct activation requirements. CXCR4 only responds to wild-type CXCL12 and is sensitive to mutation of the chemokine. By contrast, ACKR3 recruits GPCR kinases (GRKs) and β-arrestins and promiscuously responds to CXCL12, CXCL12 variants, other peptides and proteins, and is relatively insensitive to mutation. To investigate the role of conformational dynamics in the distinct pharmacological behaviors of CXCR4 and ACKR3, we employed single-molecule FRET to track discrete conformational states of the receptors in real-time. The data revealed that apo-CXCR4 preferentially populates a high-FRET inactive state, while apo-ACKR3 shows little conformational preference and high transition probabilities among multiple inactive, intermediate and active conformations, consistent with its propensity for activation. Multiple active-like ACKR3 conformations are populated in response to agonists, compared to the single CXCR4 active-state. This and the markedly different conformational landscapes of the receptors suggest that activation of ACKR3 may be achieved by a broader distribution of conformational states than CXCR4. Much of the conformational heterogeneity of ACKR3 is linked to a single residue that differs between ACKR3 and CXCR4. The dynamic properties of ACKR3 may underly its inability to form productive interactions with G proteins that would drive canonical GPCR signaling.