Ribosome•RelA structures reveal the mechanism of stringent response activation
- University of Massachusetts Medical School, United States
- Brandeis University, United States
- Janelia Research Campus, Howard Hughes Medical Institute, United States
Figures
Figure 1 with 4 supplements
Cryo-EM structures of the 70S•RelA complexes.
(A) Structure of the 70S•RelA complex lacking deacyl-tRNA in the A site (Structure I) reveals the C-terminal superdomain comprising the RIS and ACT domains (red). This superdomain binds near the A …
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Figure 1—source data 1
Structure I-IV map resolution and refinement statistics.
- https://doi.org/10.7554/eLife.17029.003
Figure 1—figure supplement 1
Schematic of cryo-EM refinement and classification procedures.
All particles were initially aligned to a single model, resulting in a 3.5 Å resolution averaged map. 3D classification into 15 classes yielded 7 high-resolution classes of which 4 were found to …
Figure 1—figure supplement 2
Cryo-EM density in Structures I-IV.
For panels (A–D), the maps were B-factor sharpened by applying a B-factor of 42 Å2 (Structure II) or -65 Å2 (Structure I, III and IV). (A) Cryo-EM map of Structure I, 70S•RelA complex in the absence …
Figure 1—figure supplement 3
Interactions of the L11 stalk with A/R tRNA.
The N-terminal portion of L11 contacts the elbow of A/R tRNA. Structure IV is shown colored as in Figure 1.
Figure 1—figure supplement 4
Domain organization of RelA.
Domains are shown in different colors, the numbers denote approximate amino acid positions demarcating the domains. The view of the structures of RelA and A/R tRNA (green) in the lower panel shows …
Figure 2 with 2 supplements
The C-terminal superdomain of RelA binds at the intersubunit bridge B1a.
(A) In the absence of A-site tRNA (Structure I), the C-terminal superdomain of RelA interacts with the intersubunit bridge B1a. The ACT domain interacts with the A-site finger (helix 38 of 23S rRNA) …
Figure 2—figure supplement 1
Cryo-EM density for the RIS, ACT and AH domains.
(A) Cryo-EM map (gray) and the structural model of the C-terminal ACT domain and its interaction with the bulged A896 of the A-site finger (ASF) of 23S ribosomal RNA (Structure III). The cryo-EM map …
Figure 2—figure supplement 2
Re-evaluation of the previously reported 10.8 Å cryo-EM map.
(A) EMD-2373 (Agirrezabala et al., 2013) was segmented according to the atomic model for Structure IV. The segmentation reveals density for the RIS and ACT domains at the subunit interface, as …
Figure 3 with 1 supplement
Interactions of the TGS domain of RelA with the A/R tRNA and 16S rRNA.
(A) The 3´ CCA end of A/R tRNA pins the TGS domain against helix 5 of 16S rRNA. (B). Interactions of the terminal nucleotides of the A/R tRNA with the TGS domain. (C) Comparison of the TGS domain …
Figure 3—figure supplement 1
Interactions of the TGS domain with the A/R tRNA.
(A) The conformations of the TGS domain and interactions with the CCA end of A/R tRNA are similar in Structures II, III and IV. RelA TGS domains from Structures II, III, and IV (aa 404–487) were …
Figure 4 with 2 supplements
Positions and interactions of the N-terminal domains of RelA.
(A) Pseudo-hydrolase (PH; pink) and synthetase (Synth; red) domains are in the intersubunit space between the sarcin-ricin loop (SRL) of the 23S rRNA and the spur of the 16S rRNA. The N-terminal …
Figure 4—figure supplement 1
Cryo-EM densities for the N-terminal domains, obtained by sub-classification of Structures II, III and IV.
(A−F).Two predominant conformations of the N-terminal domains are shown, obtained by sub-classification of each Structure into three classes. In one class, the synthetase domain is in the vicinity …
Figure 4—figure supplement 2
Comparison of the synthetase domain of RelA with metazoan innate immune sensors OAS1 and cGAS.
(A) Position of the synthetase domain of RelA (red) in Structure IV near the tip of the spur of the 30S subunit (yellow). (B) Interaction of OAS1 (blue) with an RNA helix (magenta) results in …
Figure 5 with 1 supplement
A/R tRNA and RelA rearrange toward the 30S subunit in Structures II to IV.
(A) A/R tRNA settles into the decoding center of the 30S subunit between Structures II (grey) and IV (colored as in Figure 1). Structures II and IV were aligned on the 16S rRNA. RelA is not shown. …
Figure 5—figure supplement 1
Comparison of A/R tRNA to A/T and A/A tRNA.
(A) The position of A/R tRNA in Structure II (green) is compared with the A/T tRNA (purple) in E. coli 70S•EF-Tu•GDP•kirromycin•Phe-tRNAPhe complex (PDB: 5AFI; [Fischer et al., 2015]) and A/A tRNA …
Figure 6 with 3 supplements
Closure of the 30S subunit and decoding-center rearrangements in Structures II, III and IV.
(A) A view down on the 30S subunit from the inter-subunit interface shows the position of the decoding center (boxed). The 50S subunit (except for helix 69), small ribosomal proteins and RelA are …
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Figure 6—source data 1
Distances between Structures II, III and IV, reflecting the movement of the 30S shoulder domain from Structures II to III to IV, relative to the head and the body of the 30S subunit.
- https://doi.org/10.7554/eLife.17029.020
Figure 6—figure supplement 1
Comparison of the 30S subunits of Structures II, III and IV reveals domain closure of the 30S subunit from Structure II to IV.
The 30S subunit adopts the most open conformation in Structure II, intermediate conformation in Structure III, and most closed conformation in Structure IV. In Structure IV, the shoulder of the 30S …
Figure 6—figure supplement 2
The nucleotides at the decoding center and vicinity are resolved in the cryo-EM density.
(A) Example of cryo-EM density near the decoding center in Structure III. The map was sharpened by applying a B-factor of -100 Å2 and is shown at 3.2 σ. (B) Example of cryo-EM density showing the …
Figure 6—figure supplement 3
Conformational differences between the decoding centers of Structures I through IV.
(A) Cryo-EM density (shown as mesh) of the decoding center in Structure I. The map was sharpened by applying a B-factor of -120 Å2 and density is shown at 2.0 σ for mRNA, 4.0 σ for G530, 2.0 σ for …
Figure 7 with 1 supplement
Schematic of the mechanism of RelA activation by the ribosome and cognate deacyl-tRNA.
The C-terminal domains of RelA, the RIS and the ACT, bind the ribosome at the intersubunit bridge B1a near the vacant A site, but the synthetase remains unbound and inactive. When deacyl-tRNA binds …
Figure 7—figure supplement 1
Superpositions with structures of 70S-ribosome complexes suggest that RelA is displaced from ribosomes during tRNA accommodation and translocation.
(A) In the absence of deacyl-tRNA, a RelA-bound 70S ribosome can accommodate EF-Tu ternary complex. Superposition of the 70S•RelA complex, Structure I, with the E. coli …
Videos
Video 1
An animation showing transitions between Structures I, II, III and IV.
Three views (scenes) are shown: (1) A view of the complete 70S complex, as in Figure 1; two conformations of the N-terminal domain of RelA are shown for Structures II, III and IV. In Structure I, …
