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. 2016 Jul 27;44(13):6471-81.
doi: 10.1093/nar/gkw470. Epub 2016 May 25.

The Stringent Factor RelA Adopts an Open Conformation on the Ribosome to Stimulate ppGpp Synthesis

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Free PMC article

The Stringent Factor RelA Adopts an Open Conformation on the Ribosome to Stimulate ppGpp Synthesis

Stefan Arenz et al. Nucleic Acids Res. .
Free PMC article

Abstract

Under stress conditions, such as nutrient starvation, deacylated tRNAs bound within the ribosomal A-site are recognized by the stringent factor RelA, which converts ATP and GTP/GDP to (p)ppGpp. The signaling molecules (p)ppGpp globally rewire the cellular transcriptional program and general metabolism, leading to stress adaptation. Despite the additional importance of the stringent response for regulation of bacterial virulence, antibiotic resistance and persistence, structural insight into how the ribosome and deacylated-tRNA stimulate RelA-mediated (p)ppGpp has been lacking. Here, we present a cryo-EM structure of RelA in complex with the Escherichia coli 70S ribosome with an average resolution of 3.7 Å and local resolution of 4 to >10 Å for RelA. The structure reveals that RelA adopts a unique 'open' conformation, where the C-terminal domain (CTD) is intertwined around an A/T-like tRNA within the intersubunit cavity of the ribosome and the N-terminal domain (NTD) extends into the solvent. We propose that the open conformation of RelA on the ribosome relieves the autoinhibitory effect of the CTD on the NTD, thus leading to stimulation of (p)ppGpp synthesis by RelA.

Figures

Figure 1.
Figure 1.
Generation of a RelA-stalled ribosome complex. (A) The bicistronic 2XermCL_S10K mRNA was translated in the presence of 10 μM erythromycin (ERY) in order to generate (B) ErmCL-S10K_SRC disomes. (C) ErmCL_S10K-SRC disomes were converted to monosomes by antisense DNA-mediated RNase H cleavage, as shown by sucrose density gradient centrifugation and negative stain electron microscopy (EM). (D) A-tRNA deficient ErmCL_S10K-SRCs were used as substrate for (E) RelA binding in the presence of deacylated tRNALys, GDP and α, β-methylene-ATP.
Figure 2.
Figure 2.
Novel binding site for RelA on the ribosome. (A and B) Overview of the cryo-EM reconstruction of the RelA-SRC filtered to (A) 4 Å and (B) 12 Å resolution showing 30S subunit (yellow), 50S subunit (grey), P-tRNA (cyan), A/R-tRNA (blue) and RelA (red). (C) Overview of EF-Tu-bound E. coli 70S ribosomes with P-tRNA (cyan), A/T-tRNA (green) and EF-Tu (orange) (29). (D) Isolated cryo-electron density with fitted model for the A/R-tRNA (blue). (E and F) Comparison of (E) H43 and H44 of the 23S rRNA and (F) A/R-tRNA of RelA-SRC (blue) and A/T-tRNA of EF-Tu-70S (green) (29).
Figure 3.
Figure 3.
RelA adopts an extended conformation on the ribosome. (A) Schematic showing the domain organization of E. coli RelA with HD (magenta), SYNTH (pink), TGS (green), HELICAL (blue), CC (yellow) and ACT (orange) subdomains. (B) Overview of RelA-SRC with subdomains colored according to (A) and A/R-tRNA (light blue). (C and D) Complementary views showing isolated electron density of RelA with fitted homology models for TGS (green, PDB2EKI) and ACT (orange, PDB2KO1) subdomains, as well as model poly-Alanine helices fitted to the helical linker region (blue) and density for CC colored in yellow. (EG) As (BD) with 12 Å filtered maps and additional fitted homology models for HD (magenta) and SYNTH (pink) based on PDB1VJ7 (24).
Figure 4.
Figure 4.
Interactions of RelA with the ribosome and A/R-tRNA. (A) Cryo-electron density of the CC subdomain of RelA (yellow) suggests interaction with the β-sheet of r-protein S19 (blue) and the minor groove of H38 in the 23S rRNA (grey). (B) Interaction of the C-terminal RelA ACT domain with Arg59 of r-protein L16 (blue) as well as 23S rRNA residues A896 of H38 and the tip of H43. (C) The β4-strand of the TGS domain of RelA (green) approaches the minor groove of h5 of the 16S rRNA, where residues in the vicinity of Ile448 (light green) appear to interact with the base-pair formed by nucleotides A55 and U368 (yellow), which are flipped-out of helices h5 and h15, respectively. (D) Tentative placement of the A/R-tRNA CCA-end (blue) shows vicinity of C74 of the A/R-tRNA with His432 located at the kink in α-helix α1, whereas C75 and A76 enter into a pocket formed by the β1–β2 hairpin and β5-strand of TGS (green).
Figure 5.
Figure 5.
Model for RelA action during the stringent response. (A and B) Under optimal conditions, aminoacyl-tRNAs (aa-tRNAs) are delivered by EF-Tu (orange) to the A-site of the ribosome (green pathway). (CG) Under starvation conditions (yellow pathway), the interaction of RelA (red) with deacylated A/R-tRNA at the A-site of the ribosome leads to the conversion of RelA from a ‘closed’ to an ‘open’ conformation and thereby stimulating high levels of (p)ppGpp synthesis. For more details, please refer to the text.

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