The regulatory TnaC nascent peptide preferentially inhibits release factor 2-mediated hydrolysis of peptidyl-tRNA

Emmanuel, Jerusha Salome; Sengupta, Arnab; Gordon, Emily R.; Noble, Joseph Thomas; Cruz-Vera, Luis R.

doi:10.1074/jbc.ra119.011313

Cited by 11 publications

(14 citation statements)

References 43 publications

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“…A possible explanation is that the GGQ motif and its RF1-specific flanking regions are capable of a stronger interaction with the ribosome, thereby overcoming the steric hindrance posed by the TnaC-stabilized PTC bases. This would be consistent with recent mutational analyses ( 53 ) and the known higher affinity of RF1 for ribosomes ( 54 ).…”

Section: Discussionsupporting

confidence: 91%

“…Interestingly, in the case of the eukaryotic CMV-AP, which prevents termination by eRF1, the GGQ loop can adopt its active conformation, but the PTC is still inhibited due to a complete repositioning of the base equivalent to E. coli U2585 ( 52 ). It is more difficult to understand however, how the release of the TnaC AP is efficiently catalysed by RF1, which also carries a GGQ loop ( 53 ). A possible explanation is that the GGQ motif and its RF1-specific flanking regions are capable of a stronger interaction with the ribosome, thereby overcoming the steric hindrance posed by the TnaC-stabilized PTC bases.…”

Section: Discussionmentioning

confidence: 99%

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

Su¹,

Kudva

Becker³

et al. 2021

Nucleic Acids Research

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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.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

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

Su¹,

Kudva

Becker³

et al. 2021

Nucleic Acids Research

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“…Thus, it appears that the presence of TnaC inside the exit tunnel does not prevent domain III from crossing the "accommodation gate" leading to the ribosomal A-site 32 , but stops the GGQ loop from engaging with the PTC. This is consistent with biochemical data showing that the peptidyl-hydrolase activity of RF2 is blocked within the TnaC-ribosome complex 33 . In contrast, interactions between domain II of RF2 and the A-site codon in the mRNA are maintained, indicating that TnaC(R23F)-mediated ribosome stalling does not interfere with RF2 recognition of the UGA stop codon, in agreement with earlier observations 8,16,28 (Supplementary Fig.…”

Section: Rf2 Binds To the Tnac-ribosome Complex But Cannot Reach The Ptcsupporting

confidence: 92%

“…6b). This hypothesis is supported by the previous observation that RF2 protein variants that are known to be less effective at catalyzing hydrolysis are better inhibited by L-Trp 33 . For this to be the case, RF2 would have to compete with L-Trp for binding to the ribosome, implying that ligand binding would only occur once TnaC synthesis is complete.…”

Section: Discussionsupporting

confidence: 63%

“…Reactions performed in the presence of 20 nM of each RF2 variant were resolved by electrophoresis on 10% Tris-tricine gels and transferred to nylon membranes. RF2 protein variants were produced from bacteria expressing the plasmids indicated in Supplementary Table 3 and purified as described previously 33 . rRNA from the isolated complexes were obtained using standard phenol-chloroform extractions and resolved in 1% non-denaturing agarose gels.…”

Section: Model Building and Refinementmentioning

confidence: 99%

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

Stel

Gordon

Sengupta

et al. 2021

Preprint

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Free L-tryptophan (L-Trp) induces the expression of the Escherichia coli tryptophanase operon, leading to the production of indole from L-Trp. Tryptophanase operon expression is controlled via a mechanism involving the tryptophan-dependent stalling of ribosomes engaged in translation of tnaC, a leader sequence upstream of tnaA that encodes a 24-residue peptide functioning as a sensor for L-Trp. Although extensive biochemical characterization has revealed the elements of the TnaC peptide and the ribosome that are responsible for translational arrest, 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 variant of TnaC (R23F) in which stalling by L-Trp is enhanced because of reduced cleavage of TnaC(R23F)-peptidyl-tRNA. In contrast to previous data originated from lower resolution structural studies, we show that the TnaC-ribosome complex captures a single L-Trp molecule to undergo tryptophan-dependent 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. In addition, we show that the conformation of the L-Trp binding site is not altered by the R23F mutation. This leads us to propose a model in which rates of TnaC-peptidyl-tRNA cleavage by release factor and binding of the L-Trp ligand to the translating ribosome determine the tryptophan sensitivity of the wild-type and mutant TnaC variants. Thus, our study reveals a strategy whereby a nascent peptide assists the bacterial ribosome in sensing a small metabolite.

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

Ojima-Kato¹,

Nishikawa²,

Yuki³

et al. 2023

Journal of Biological Chemistry

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The regulatory TnaC nascent peptide preferentially inhibits release factor 2-mediated hydrolysis of peptidyl-tRNA

Cited by 11 publications

References 43 publications

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

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

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

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

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