Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection

McClory, Sean P.; Leisring, Joshua M.; Qin, Daoming; Fredrick, Kurt

doi:10.1261/rna.2228510

Cited by 57 publications

(138 citation statements)

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“…However, other contacts in the decoding com-plex may specifically affect the stringency of decoding, e.g. helix 14 and helix 8 of 16S rRNA that negatively regulate GTP hydrolysis (36), or the interactions between helix 5 and domain 2 of EF-Tu (37). The structural basis for the very strong effect of ribosomal protein L7/12 on the GTPase activity of EF-Tu (38,39) remains to be clarified.…”

Section: Discussionmentioning

confidence: 99%

Distortion of tRNA upon Near-cognate Codon Recognition on the Ribosome

Mittelstaet

Konevega

Rodnina

2011

Journal of Biological Chemistry

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The accurate decoding of the genetic information by the ribosome relies on the communication between the decoding center of the ribosome, where the tRNA anticodon interacts with the codon, and the GTPase center of EF-Tu, where GTP hydrolysis takes place. In the A/T state of decoding, the tRNA undergoes a large conformational change that results in a more open, distorted tRNA structure. Here we use a real-time transient fluorescence quenching approach to monitor the timing and the extent of the tRNA distortion upon reading cognate or nearcognate codons. The tRNA is distorted upon codon recognition and remains in that conformation until the tRNA is released from EF-Tu, although the extent of distortion gradually changes upon transition from the pre-to the post-hydrolysis steps of decoding. The timing and extent of the rearrangement is similar on cognate and near-cognate codons, suggesting that the tRNA distortion alone does not provide a specific switch for the preferential activation of GTP hydrolysis on the cognate codon. Thus, although the tRNA plays an active role in signal transmission between the decoding and GTPase centers, other regulators of signaling must be involved.Proteins are synthesized from aminoacyl-tRNAs (aa-tRNAs) 2 that are delivered to the ribosome in ternary complexes with elongation factor Tu (EF-Tu) and GTP. The ribosome selects aa-tRNAs according to the sequence of codons in the mRNA template and rejects the bulk of aa-tRNAs with anticodons that do not match the given codon in each round of elongation. Correct base pairing between the mRNA codon and the anticodon of the tRNA on the 30S subunit of the ribosome provides a signal that is then transmitted to the GTPase center of EF-Tu on the 50S subunit and results in the activation of GTP hydrolysis by EF-Tu. Mismatches in the codon-anticodon complex impair GTPase activation, thereby allowing the ribosome to reject incorrect ternary complexes prior to GTP hydrolysis. Deciphering the mechanism and the specificity of signal transmission between the decoding center and the GTPase center of EF-Tu is one of the central questions in understanding the fidelity of translation.Decoding entails a number of elemental steps. Initial binding of the ternary complex EF-Tu⅐GTP⅐aa-tRNA to the ribosome takes place codon-independently, mainly through contacts of EF-Tu with ribosomal protein L7/12, and is followed by rapid and reversible codon reading ( Fig. 1A; reviewed in Refs. 1-4). The formation of the fully complementary codon-anticodon duplex induces local and global conformational changes at the decoding center of the ribosome, which lock the aa-tRNA in the codon-bound state and activate EF-Tu for rapid GTP hydrolysis (5-8). Binding of near-cognate ternary complexes that entail single mismatches between codon and anticodon does not induce these structural rearrangements or rapid GTP hydrolysis, explaining why initial tRNA selection is more accurate than can be accounted for by the energetic differences between fully matched and mismatched codon-anticodon ...

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Section: Discussionmentioning

confidence: 99%

Distortion of tRNA upon Near-cognate Codon Recognition on the Ribosome

Mittelstaet

Konevega

Rodnina

2011

Journal of Biological Chemistry

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“…1B,C), and destabilizes bridge B8 allosterically (Fagan et al 2013). All four of these mutations increase both missense and nonsense suppression in vivo (McClory et al 2010). Mutation G886A is located in h27 (Fig.…”

Section: Resultsmentioning

confidence: 95%

“…1A), near the binding sites of several error-promoting antibiotics (Carter et al 2000). While conferring more modest effects on missense and nonsense suppression (McClory et al 2010), G886A was the only mutation to be additionally identified in a screen for translation initiation errors (Qin and Fredrick 2009). Four mutations (C1054U, C1054A, C1200U, and G1491A) are located in or near the A site (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We previously isolated many ribosomal ambiguity (ram) mutations in the 16S rRNA (McClory et al 2010). These mutations increase errors during translation elongation and cluster in several distinct regions of the ribosome.…”

Section: Introductionmentioning

confidence: 99%

“…Analysis of a subset of these 16S ram mutations has shown that bridge B8 increases the stringency of decoding by negatively regulating GTPase activation (McClory et al 2010;Fagan et al 2013). Mutations predicted to disrupt bridge B8 (i.e., h8Δ3, a 3-bp truncation of h8; h14Δ2, a 2-bp truncation of h14; and G347U in h14)-as well as mutation G299A in h12 -increase the rate of EF-Tu-dependent GTP hydrolysis, particularly in the near-cognate case.…”

Section: Introductionmentioning

confidence: 99%

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Distinct functional classes oframmutations in 16S rRNA

McClory

Devaraj

Fredrick

2014

RNA

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During decoding, the ribosome selects the correct (cognate) aminoacyl-tRNA (aa-tRNA) from a large pool of incorrect aa-tRNAs through a two-stage mechanism. In the initial selection stage, aa-tRNA is delivered to the ribosome as part of a ternary complex with elongation factor EF-Tu and GTP. Interactions between codon and anticodon lead to activation of the GTPase domain of EFTu and GTP hydrolysis. Then, in the proofreading stage, aa-tRNA is released from EF-Tu and either moves fully into the A/A site (a step termed "accommodation") or dissociates from the ribosome. Cognate codon-anticodon pairing not only stabilizes aa-tRNA at both stages of decoding but also stimulates GTP hydrolysis and accommodation, allowing the process to be both accurate and fast. In previous work, we isolated a number of ribosomal ambiguity (ram) mutations in 16S rRNA, implicating particular regions of the ribosome in the mechanism of decoding. Here, we analyze a representative subset of these mutations with respect to initial selection, proofreading, RF2-dependent termination, and overall miscoding in various contexts. We find that mutations that disrupt inter-subunit bridge B8 increase miscoding in a general way, causing defects in both initial selection and proofreading. Mutations in or near the A site behave differently, increasing miscoding in a codon-anticodon-dependent manner. These latter mutations may create spurious favorable interactions in the A site for certain near-cognate aa-tRNAs, providing an explanation for their context-dependent phenotypes in the cell.

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Nonsense Mutations and Suppression

Herrington

2018

Encyclopedia of Life Sciences

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A nonsense mutation occurs when a sense codon, one that codes for an amino acid, is changed to a chain‐termination codon, UAG, UAA or UGA. A nonsense suppressor can result from a second mutation affecting the translational apparatus. This mutation enables the cell to insert an amino acid in response to the nonsense codon, resulting in a wild‐type or near wild‐type phenotype. Some suppressor mutations change the anticodon of a transfer ribonucleic acid (tRNA) so that it can pair with the nonsense codon. Other suppressors increase the readthrough of the nonsense mutation. Readthrough occurs at low levels but changes in the ribosome, tRNA or in translation factors can increase readthrough by altering the initial selection steps, proofreading or quality control in decoding the messenger RNA. Manipulation of the accuracy of translation holds promise as a method for the treatment of genetic diseases, many of which result from nonsense mutations. Key Concepts Suppression results when one mutation counteracts the effect of another mutation to give a wild‐type phenotype. The nonsense codons UAG, UAA and UGA do not code for amino acids, but signal the end of the protein‐coding sequence in the mRNA. Nonsense suppression competes with chain termination. Errors occur during translation and include reading a nonsense codon as sense as well as misreading and frameshifting. Decoding during elongation involves conformational changes in the ribosome, tRNA and EF‐Tu. Changes in the ribosome or other components of the translational apparatus can modify decoding and thus enhance or reduce readthrough of nonsense codons.

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Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection

Cited by 57 publications

References 51 publications

Distortion of tRNA upon Near-cognate Codon Recognition on the Ribosome

Distortion of tRNA upon Near-cognate Codon Recognition on the Ribosome

Distinct functional classes oframmutations in 16S rRNA

Nonsense Mutations and Suppression

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