Figure 7. 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 to the ribosomal A site, the decoding center controls the selection of cognate tRNA, coupled with domain closure of the 30S subunit. The codon recognition checkpoints are mediated by distinct positions of the universally conserved nucleotides of the decoding center A1492, A1493 and G530. Upon binding of cognate deacyl-tRNA to the ribosome, the RelA synthetase domain is exposed in the vicinity of the 30S spur and is activated for (p)ppGpp synthesis by alleviation of RelA autoinhibition and interactions with the ribosome.
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 70S•EF-Tu•GDP•kirromycin•Phe-tRNAPhe complex (PDB: 5AFI; [Fischer et al., 2015]) was obtained by structural alignment of the 16S rRNA. (B) The ACT domain of RelA sterically clashes with fully accommodated A-site tRNA. The 16S rRNA of Structures I, IV and a 70S•tRNA complex containing three tRNAs (PDB: 3I8H and 3I8I; [Jenner et al., 2010]) were superimposed. Structure I is shown in red. A/A tRNA from PDB: 3I8I is shown in blue. A/R tRNA from Structure IV is shown in green. (C) In the rotated (hybrid-state) conformation of the 70S ribosome sampled during translocation, the RIS domain interactions with S19 would be disrupted. Superposition of the 70S•RelA complex (Structure I) with the pre-translocation 70S•tRNA•EF-G•viomycin complex (PDB: 4V7C; [Brilot et al., 2013]) was obtained by structural alignment of 23S rRNA. The 50S subunit (cyan), RelA (red), S19 (yellow) and P tRNA (orange) are from Structure I. 30S subunit (gray) and S19 (black) are from PDB: 4V7C. (D) In the post-translocation state, elongation factor G (EF-G) sterically clashes with Linker Helix 2 of RelA. Superposition of the 70S•RelA•deacyl-tRNA complex (Structure IV) with a 70S•EF-G•GDP•fusidic acid complex (PDB: 2WRI; [Gao et al., 2009]) was obtained by structural alignment of the 16S rRNA. EF-G domain IV (blue, residues 483–603) and post-translocation tRNA (orange) are shown from PDB: 2WRI. RelA RIS and ACT domains and Linker Helix 2 (red) are shown from Structure I.