Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling
Anne‐Xander van der Stel, Emily R. Gordon, Arnab Sengupta, Allyson K. Martínez, Dorota Klepacki, Thomas N. Perry, Alba Herrero del Valle, Nora Vázquez‐Laslop, Matthew S. Sachs, Luis R. Cruz-Vera, C.A. Innis
Abstract
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.