Activation of the Stringent Response by Loading of RelA-tRNA Complexes at the Ribosomal A-Site

Mol Cell. 2018 Apr 5;70(1):95-105.e4. doi: 10.1016/j.molcel.2018.02.033.

Abstract

RelA/SpoT homologs (RSHs) are ubiquitous bacterial enzymes that synthesize and hydrolyze (p)ppGpp in response to environmental challenges. Bacteria cannot survive in hosts and produce infection without activating the (p)ppGpp-mediated stringent response, but it is not yet understood how the enzymatic activities of RSHs are controlled. Using UV crosslinking and deep sequencing, we show that Escherichia coli RelA ((p)ppGpp synthetase I) interacts with uncharged tRNA without being activated. Amino acid starvation leads to loading of cognate tRNA⋅RelA complexes at vacant ribosomal A-sites. In turn, RelA is activated and synthesizes (p)ppGpp. Mutation of a single, conserved residue in RelA simultaneously prevents tRNA binding, ribosome binding, and activation of RelA, showing that all three processes are interdependent. Our results support a model in which (p)ppGpp synthesis occurs by ribosome-bound RelA interacting with the Sarcin-Ricin loop of 23S rRNA.

Keywords: (p)ppGpp; A-site finger; RSH; RelA; Sarcin-Ricin loop; rRNA; ribosome; stringent response; tRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / deficiency
  • Binding Sites
  • Escherichia coli K12 / enzymology*
  • Escherichia coli K12 / genetics
  • Guanosine Tetraphosphate / biosynthesis*
  • Ligases / genetics
  • Ligases / metabolism*
  • Mutation
  • Nucleic Acid Conformation
  • Protein Binding
  • Protein Biosynthesis
  • Protein Conformation
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism*
  • RNA, Ribosomal, 23S / genetics
  • RNA, Ribosomal, 23S / metabolism*
  • RNA, Transfer / genetics
  • RNA, Transfer / metabolism*
  • Ribosomes / enzymology*
  • Ribosomes / genetics

Substances

  • Amino Acids
  • RNA, Bacterial
  • RNA, Ribosomal, 23S
  • Guanosine Tetraphosphate
  • RNA, Transfer
  • Ligases
  • guanosine 3',5'-polyphosphate synthetases