Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling

Nat Commun. 2021 Sep 9;12(1):5340. doi: 10.1038/s41467-021-25663-8.


Free L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive characterization, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a TnaC variant (R23F) with greatly enhanced sensitivity for L-Trp. We show that the TnaC-ribosome complex captures a single L-Trp molecule to undergo termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. Importantly, the L-Trp binding site is not altered by the R23F mutation, suggesting that the relative rates of L-Trp binding and peptidyl-tRNA cleavage determine the tryptophan sensitivity of each variant. Thus, our study reveals a strategy whereby a nascent peptide assists the ribosome in detecting a small metabolite.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Substitution
  • Binding Sites
  • Cryoelectron Microscopy
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Gene Expression Regulation, Bacterial*
  • Models, Molecular
  • Mutation
  • Operon
  • Peptide Chain Initiation, Translational*
  • Peptide Chain Termination, Translational
  • Peptide Termination Factors / genetics
  • Peptide Termination Factors / metabolism
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • RNA, Transfer, Amino Acyl / genetics
  • RNA, Transfer, Amino Acyl / metabolism
  • Ribosomes / genetics*
  • Ribosomes / metabolism
  • Ribosomes / ultrastructure
  • Tryptophan / chemistry*
  • Tryptophan / metabolism


  • Escherichia coli Proteins
  • Peptide Termination Factors
  • RNA, Transfer, Amino Acyl
  • tRNA, peptidyl-
  • tnaC protein, E coli
  • Tryptophan