Ribosome-dependent activation of stringent control

In order to survive, bacteria continually sense, and respond to, environmental fluctuations. Stringent control represents a key bacterial stress response to nutrient starvation1,2 that leads to a rapid and comprehensive reprogramming of metabolic and transcriptional patterns3. In general, transcription of genes for growth and proliferation are down-regulated, while those important for survival and virulence are favored4. Amino acid starvation is sensed by depletion of the aminoacyl-tRNA pools5, which results in accumulation of ribosomes stalled with non-aminoacylated (uncharged) tRNA in the ribosomal A-site6,7. RelA is recruited to stalled ribosomes, and activated to synthesize a hyperphosphorylated guanosine analog, (p)ppGpp8, which acts as a pleiotropic second messenger. However, structural information for how RelA recognizes stalled ribosomes and discriminates against aminoacylated tRNAs is missing. Here, we present the electron cryo-microscopy (cryo-EM) structure of RelA bound to the bacterial ribosome stalled with uncharged tRNA. The structure reveals that RelA utilizes a distinct binding site compared to the translational factors, with a multi-domain architecture that wraps around a highly distorted A-site tRNA. The TGS domain of RelA binds the CCA tail to orient the free 3’ hydroxyl group of the terminal adenosine towards a β-strand, such that an aminoacylated tRNA at this position would be sterically precluded. The structure supports a model where association of RelA with the ribosome suppresses auto-inhibition to activate synthesis of (p)ppGpp and initiate the stringent response. Since stringent control is responsible for the survival of pathogenic bacteria under stress conditions, and contributes to chronic infections and antibiotic tolerance, RelA represents a good target for the development of novel antibacterial therapeutics.