Dissection of the Mechanism for the Stringent Factor RelA

During conditions of nutrient deprivation, ribosomes are blocked by uncharged tRNA at the A site. The stringent factor RelA binds to blocked ribosomes and catalyzes synthesis of (p)ppGpp, a secondary messenger that induces the stringent response. We demonstrate that binding of RelA and (p)ppGpp synthesis are inversely coupled, i.e., (p)ppGpp synthesis decreases the affinity of RelA for the ribosome. RelA binding to ribosomes is governed primarily by mRNA, but independently of ribosomal protein L11, while (p)ppGpp synthesis strictly requires uncharged tRNA at the A site and the presence of L11. A model is proposed whereby RelA hops between blocked ribosomes, providing an explanation for how low intracellular concentrations of RelA (1/200 ribosomes) can synthesize (p)ppGpp at levels that accurately reflect the starved ribosome population.