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.
Highlights
RelA/SpoT homolog (RSH) proteins play a central role in bacterial stringent response—a major stress-response pathway and key driver of bacterial virulence and antibiotic resistance (Neidhardt, 1987; Gentry et al, 2000; Pizarro-Cerda and Tedin, 2004; Dalebroux et al, 2010a; Gao et al, 2010; Nguyen et al, 2011; Dordel et al, 2014)
Accumulation of (p)ppGpp activates transcription of genes required for stress response, inhibits transcription of genes required for replication and growth, and reformats the transcription of metabolic genes according to the stress condition (Polakis et al, 1973; Mittenhuber, 2001; Magnusson et al, 2005; Jain et al, 2006a; Kuroda, 2006; Wang et al, 2007; Ferullo and Lovett, 2008; Potrykus and Cashel, 2008a; Traxler et al, 2008; Dalebroux et al, 2010a; Dalebroux et al, 2010b)
To understand how RelA activates stringent response on ribosomes bound with cognate deacyltRNA, we sought a high-resolution structure of the entire 70S.RelA.deacyl-tRNA complex
Summary
RelA/SpoT homolog (RSH) proteins play a central role in bacterial stringent response—a major stress-response pathway and key driver of bacterial virulence and antibiotic resistance (Neidhardt, 1987; Gentry et al, 2000; Pizarro-Cerda and Tedin, 2004; Dalebroux et al, 2010a; Gao et al, 2010; Nguyen et al, 2011; Dordel et al, 2014). When the supply of amino acids becomes limiting, binding of cognate but uncharged (deacylated) transfer RNA (tRNA) to the A (aminoacyl-tRNA) site of the 70S ribosome activates RelA (Haseltine and Block, 1973; Richter, 1976; Wendrich et al, 2002). To understand how RelA activates stringent response on ribosomes bound with cognate deacyltRNA, we sought a high-resolution structure of the entire 70S.RelA.deacyl-tRNA complex. The structures reveal large-scale conformational rearrangements in RelA when it binds deacyl-tRNA entering the 30S A site, suggesting a mechanism of activation of the (p)ppGpp synthetase. Distinct conformations of the deacyl-tRNA, 30S subunit, and the ribosomal decoding center reveal structural dynamics of tRNA binding in the presence of RelA and suggest why stringent response activation requires cognate tRNA
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