Structural basis of l-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide

Abstract

In Escherichia coli, elevated levels of free l-tryptophan (l-Trp) promote translational arrest of the TnaC peptide by inhibiting its termination. However, the mechanism by which translation-termination by the UGA-specific decoding release factor 2 (RF2) is inhibited at the UGA stop codon of stalled TnaC-ribosome-nascent chain complexes has so far been ambiguous. This study presents cryo-EM structures for ribosomes stalled by TnaC in the absence and presence of RF2 at average resolutions of 2.9 and 3.5 Å, respectively. Stalled TnaC assumes a distinct conformation composed of two small α-helices that act together with residues in the peptide exit tunnel (PET) to coordinate a single L-Trp molecule. In addition, while the peptidyl-transferase center (PTC) is locked in a conformation that allows RF2 to adopt its canonical position in the ribosome, it prevents the conserved and catalytically essential GGQ motif of RF2 from adopting its active conformation in the PTC. This explains how translation of the TnaC peptide effectively allows the ribosome to function as a L-Trp-specific small-molecule sensor that regulates the tnaCAB operon.

Nascent MSKIK peptide cancels ribosomal stalling by arrest peptides in Escherichia coli

“…Using such a simulation approach, we postulate that explaining the translation-enhancing mechanism may be through mechanical forces generated between MSKIK and RAPs during translation. In addition, because many RAPs other than SecM or CmlA leader have been found ( 39 ) and a recent study showed a force-sensitive arrest by MifM in Bacillus subtilis was canceled by N terminally–adjacent dynamic nascent chain ( 40 ), we expect that there are still many translation-promoting peptides remain to be undiscovered.…”

Nascent MSKIK peptide prevents or releases translation arrest in Escherichia coli

“…Using such a simulation approach, we postulate that explaining the translation-enhancing mechanism may be through mechanical forces generated between MSKIK and RAPs during translation. In addition, because many RAPs other than SecM or CmlA leader have been found (Su et al, 2021),and a recent study showed a force-sensitive arrest by MifM in Bacillus subtilis was cancelled by N-terminally adjacent dynamic nascent chain (Fujiwara et al, 2020), we expect that there are still many translation-promoting peptides remain to be undiscovered.…”

Mapping Protein–Protein Interactions at Birth: Single-Particle Cryo-EM Analysis of a Ribosome–Nascent Globin Complex

“…Interestingly, identical RNC-interacting regions along the E. coli ribosome tunnel core were previously reported in the context of entirely different nascent chain sequences, i.e., all βsheet proteins and ribosome-stalling polypeptides. 33,55,79,80 The recurring presence of these interaction sites across a variety of RNCs highlights their likely importance, regardless of the nascent chain sequence. Finally, as the nascent chain traverses the ribosomal exit tunnel into the spatially wider vestibule region, an increase in RNC dynamics is observed, as detailed in the next section.…”

“…In addition, we detected interactions between RNCs and the 23S rRNA (rRNA) near the PTC site, including well-known interacting regions near the U2585 ribonucleotide 33,55,78−80 and near the C2063 and G2505 ribonucleotides. 33,79 It is worth noting that the nascent chain density within the tunnel core region close to the PTC is better resolved than that in regions further up across the ribosomal exit tunnel core, vestibule, and outer ribosomal surface. This increase in local motion is likely a direct result of the extensive interactions between the nascent chain and rRNA at the PTC.…”