Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan

Martínez, Allyson K.; Gordon, Emily R.; Sengupta, Arnab; Shirole, Nitin H.; Klepacki, Dorota; Martínez-Garriga, Blanca; Brown, Lewis M.; Benedik, Michael J.; Yanofsky, Charles; Mankin, Alexander S.; Vázquez‐Laslop, Nora; Sachs, Matthew S.; Cruz-Vera, Luis R.

doi:10.1093/nar/gkt923

Cited by 34 publications

(39 citation statements)

References 43 publications

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“…Earlier biochemical studies suggested that the binding site of the free L-Trp may be located directly in the A-site of the PTC and that it may overlap with the binding site of the antibiotic sparsomycin (Cruz-Vera et al, 2006Cruz-Vera and Yanofsky, 2008). However, this view was recently challenged in a careful mutational analysis (Martínez et al, 2014). Indeed, our cryo-EM reconstruction cannot provide any evidence for tryptophan binding within the PTC.…”

Section: Cryo-em Structure Of a Tnac-stalled Ribosomementioning

confidence: 80%

“…Consistent with the composition of these binding pockets, mutation of U2609 and A752 and mutations of A2058 have been shown to severely reduce the efficiency of TnaC stalling (Cruz-Vera et al, 2005;Cruz-Vera and Yanofsky, 2008;Gong and Yanofsky, 2002). Moreover, mutation of I19 of the TnaC peptide to amino acids other than the chemically very similar leucine also affects stalling (Martínez et al, 2014). Although distinct density enabled the majority of side chains in the TnaC nascent peptide to be assigned, I19 and I15 lack a clearly defined conformation.…”

Section: Interactions Of Tnac With Components Of the Ribosomal Tunnelmentioning

confidence: 83%

“…When comparing our structure with the crystal structure of an RF2-bound ribosome (Jin et al, 2010), it is apparent that U2585 as well as A2602 would clash with the GGQ motif of RF2 ( Figures 3D and 3E). In contrast to RF2, accommodated aminoacyl-tRNA in the A-site would not clash with the observed conformation of U2585 and A2602, thus explaining why replacing the stop codon in the TnaC peptide with a canonical (nonrare) sense codon alleviates stalling (Cruz-Vera et al, 2006Martínez et al, 2014). However, placing the rare isoleucine codon AUA in place of the stop codon induces stalling on the TnaC leader peptide (Cruz-Vera et al, 2006).…”

Section: Inactivation Of the Peptidyl Transferase Centermentioning

confidence: 96%

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Molecular Basis for the Ribosome Functioning as an L-Tryptophan Sensor

Bischoff¹,

Berninghausen²,

Beckmann³

2014

Cell Reports

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Elevated levels of the free amino acid L-tryptophan (L-Trp) trigger expression of the tryptophanase tnaCAB operon in E. coli. Activation depends on tryptophan-dependent ribosomal stalling during translation of the upstream TnaC peptide. Here, we present a cryoelectron microscopy (cryo-EM) reconstruction at 3.8 Å resolution of a ribosome stalled by the TnaC peptide. Unexpectedly, we observe two L-Trp molecules in the ribosomal exit tunnel coordinated within composite hydrophobic pockets formed by the nascent TnaC peptide and the tunnel wall. As a result, the peptidyl transferase center (PTC) adopts a distinct conformation that precludes productive accommodation of release factor 2 (RF2), thereby inducing translational stalling. Collectively, our results demonstrate how the translating ribosome can act as a small molecule sensor for gene regulation.

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Section: Cryo-em Structure Of a Tnac-stalled Ribosomementioning

confidence: 80%

Section: Interactions Of Tnac With Components Of the Ribosomal Tunnelmentioning

confidence: 83%

Section: Inactivation Of the Peptidyl Transferase Centermentioning

confidence: 96%

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Molecular Basis for the Ribosome Functioning as an L-Tryptophan Sensor

Bischoff¹,

Berninghausen²,

Beckmann³

2014

Cell Reports

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“…In this model, slow translation of CGA codons closer to the initiation site (in which stalled ribosomes would directly affect the next initiating ribosome) would be more inhibitory than slow translation further from the initiation site; a feedback mechanism of this type would be independent of Asc1 protein and might only operate near the initiation site. The second model is based on the idea that the efficiency of the peptidyl transferase reaction is modulated by the nascent polypeptide within the exit tunnel, an idea for which there is precedent from nascent peptide sequences that stall the ribosome (Ramu et al 2011;Martinez et al 2014), and evidence for contact between the nascent polypeptide and 28S rRNA at the peptidyl transferase center (Bhushan et al 2010). In this model, which is similar to the promoter clearance step in transcription, the ribosome becomes committed to continued elongation as the exit tunnel fills, and thus CGA codons become less inhibitory as the exit tunnel fills.…”

Section: Discussionmentioning

confidence: 99%

Asc1, homolog of human RACK1, prevents frameshifting in yeast by ribosomes stalled at CGA codon repeats

Wolf

Grayhack

2015

RNA

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Quality control systems monitor and stop translation at some ribosomal stalls, but it is unknown if halting translation at such stalls actually prevents synthesis of abnormal polypeptides. In yeast, ribosome stalling occurs at Arg CGA codon repeats, with even two consecutive CGA codons able to reduce translation by up to 50%. The conserved eukaryotic Asc1 protein limits translation through internal Arg CGA codon repeats. We show that, in the absence of Asc1 protein, ribosomes continue translating at CGA codons, but undergo substantial frameshifting with dramatically higher levels of frameshifting occurring with additional repeats of CGA codons. Frameshifting depends upon the slow or inefficient decoding of these codons, since frameshifting is suppressed by increased expression of the native tRNA Arg(ICG) that decodes CGA codons by wobble decoding. Moreover, the extent of frameshifting is modulated by the position of the CGA codon repeat relative to the translation start site. Thus, translation fidelity depends upon Asc1-mediated quality control.

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“…Because the active localization reaction cancels the elongation arrest, their arrest propensity is inversely correlated with the cellular activity of the respective translocation machinery; this allows them to compensate for the loss of activity of the cellular machinery by upregulating the synthesis of the core component of the machinery. Another regulatory nascent polypeptide, TnaC, undergoes termination arrest in response to increased concentration of tryptophan (Martinez et al 2013). The arrested ribosome then interferes with recruitment of the transcription termination factor ρ onto the mRNA and, consequently, enhances transcription of the target gene encoding tryptophanase (Chap.…”

Section: Regulatory Nascent Polypeptides Monitor and Control Cellularmentioning

confidence: 99%

Biological Significance of Nascent Polypeptides That Stall the Ribosome

Ito

Chiba

2014

Regulatory Nascent Polypeptides

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The local speed of polypeptide elongation by the ribosome is not constant. One of the elements that modulate the rates of elongation or termination in translation is the amino acid sequence of the nascent polypeptide, the product of translation. Regulatory nascent polypeptides contain a segment that interacts with the ribosomal components from the peptidyl-transferase center (PTC) through the exit tunnel and arrests translation continuation beyond it. In many cases, translation arrest is either inducible with a specific effector molecule (such as a metabolite and an antibiotic) or subject to release by the engagement of the nascent peptide in a specific cellular event such as targeting and translocation. The stalling of the ribosome affects the cellular states of mRNA, leading to specific biological outputs, including induction or repression of target genes, recoding, and regulated mRNA localization in the cell. Arrest sequences are divergent in the amino acid sequences, and each of them interacts with the ribosome in a distinct way. Near the PTC, amino acids close to the arrest point interact with PTC-proximal residues of the largesubunit ribosomal RNA, whereas near the constricted region of the tunnel, more N-terminal amino acids of the nascent chain interact with ribosomal proteins L22 (also called L17 in eukaryotes) and L4, as well as with some ribosomal RNA residues. These interactions seem to create a specific configuration of the nascent chain and the ribosomal components to cis-specifically interfere with the translation reactions. Regulatory nascent polypeptides, which monitor cellular physiology and control gene expression in unique ways, have thus provided new concepts about the transformation of genetic information into cellular functions.

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Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan

Cited by 34 publications

References 43 publications

Molecular Basis for the Ribosome Functioning as an L-Tryptophan Sensor

Molecular Basis for the Ribosome Functioning as an L-Tryptophan Sensor

Asc1, homolog of human RACK1, prevents frameshifting in yeast by ribosomes stalled at CGA codon repeats

Biological Significance of Nascent Polypeptides That Stall the Ribosome

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