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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.
Highlights
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
Our structural data reveal how the ribosome and nascent TnaC co-operate to capture a single molecule of L-Trp and explain the role of various functionally important residues, such as the strictly conserved Trp[12] and Asp[16] of TnaC, the 23S ribosomal RNA (rRNA) base pair A752:U2609 and residue Lys[90] of uL22
Visualization of a stalled TnaC(R23F)–70S–release factor 2 (RF2) complex yields fresh insights into the mechanism by which TnaC inhibits its own hydrolysis from tRNAPro, with 23S rRNA residue U2585 and residues 23 and 24 of TnaC proposed to interfere with the correct folding of the RF2 catalytic loop in a manner reminiscent of the inhibition of RF1 action by nascent SpeFL, an arrest peptide that functions as an L-ornithine sensor in certain γ-proteobacteria[23]
Summary
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. In the presence of inducing L-Trp levels, translation termination is inhibited when Pro[24] of TnaC is in the ribosomal P-site and the stop codon is in the A-site, causing the ribosome to stall This blocks Rho’s access to the rut site, allowing transcription of tnaA and tnaB to proceed[1,6,7]. Our structural and biochemical analyses reveal the molecular details leading to the capture of a single L-Trp molecule by the TnaC–ribosome complex This allows us to propose a model for tryptophan-dependent translational arrest in which a relatively low rate of TnaC peptidyl-tRNA hydrolysis by release factor 2 (RF2) could give sufficient time for free L-Trp to bind to the ribosome and stabilize a TnaC conformation that silences the PTC, resulting in ribosome stalling
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