Studies of translational misreading in vivo show that the ribosome very efficiently discriminates against most potential errors

Manickam, Nandini; Nag, Nabanita; Abbasi, Aleeza; Patel, Kishan; Farabaugh, Philip J.

doi:10.1261/rna.039792.113

Cited by 86 publications

(149 citation statements)

References 31 publications

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“…The existence of two distinct proofreading steps may appear surprising, because the accuracy of initial codon selection by ternary complex normally is remarkably high (22,31). Therefore, we suggest that two-step proofreading has evolved to neutralize the deleterious effects of a small number of distinct error hot spots for initial codon selection (31) as observed in vitro (19) and in vivo (18). For instance, initial codon selection values near 100 were seen for Glu-tRNA Glu UUC reading GGA and His-tRNA His GUG reading CGC (31).…”

Section: Why Did Mother Nature Evolve Two Proofreading Steps In Geneticmentioning

confidence: 85%

“…We use the present results to discuss the molecular basis for proofreading of aminoacyl-tRNAs, which, until now, has remained obscure (17). We suggest that multistep proofreading in genetic code translation has evolved to neutralize potential error hot spots originating in error-prone initial selection of aa-tRNA in ternary complex with EF-Tu and GTP (18,19). Recent cryo-EM data of ribosomes from live human cells show two states of ribosome-bound preaccommodated aa-tRNA, one with aa-tRNA in complex with the EF-Tu homolog eEF1α in the GDP form, the other with only aa-tRNA after release of eEF1α·GDP (20).…”

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confidence: 87%

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Two proofreading steps amplify the accuracy of genetic code translation

Ieong

Uzun

Selmer

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Aminoacyl-tRNAs (aa-tRNAs) are selected by the messenger RNA programmed ribosome in ternary complex with elongation factor Tu (EF-Tu) and GTP and then, again, in a proofreading step after GTP hydrolysis on EF-Tu. We use tRNA mutants with different affinities for EF-Tu to demonstrate that proofreading of aatRNAs occurs in two consecutive steps. First, aa-tRNAs in ternary complex with EF-Tu·GDP are selected in a step where the accuracy increases linearly with increasing aa-tRNA affinity to EF-Tu. Then, following dissociation of EF-Tu·GDP from the ribosome, the accuracy is further increased in a second and apparently EFTu−independent step. Our findings identify the molecular basis of proofreading in bacteria, highlight the pivotal role of EF-Tu for fast and accurate protein synthesis, and illustrate the importance of multistep substrate selection in intracellular processing of genetic information.ribosome | error correction | fidelity | EF-Tu | ternary complex

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Section: Why Did Mother Nature Evolve Two Proofreading Steps In Geneticmentioning

confidence: 85%

mentioning

confidence: 87%

Two proofreading steps amplify the accuracy of genetic code translation

Ieong

Uzun

Selmer

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The relative levels of tRNAs and their activities set the balance of translation efficiency, fidelity, pausing, and polypeptide folding (Drummond et al 2005;Reynolds et al 2010;Spencer et al 2012;Manickam et al 2014;Arimbasseri1 et al 2015). Many nucleotides in tRNAs are post-transcriptionally modified and many affect tRNA stability or activity (Phizicky and Hopper 2010).…”

Section: Introductionmentioning

confidence: 99%

Lack of tRNA-i6A modification causes mitochondrial-like metabolic deficiency in S. pombe by limiting activity of cytosolic tRNA^Tyr, not mito-tRNA

Lamichhane

Arimbasseri

Rijal

et al. 2016

RNA

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tRNA-isopentenyl transferases (IPTases) are highly conserved enzymes that form isopentenyl-N 6 -A37 (i6A37) on subsets of tRNAs, enhancing their translation activity. Nuclear-encoded IPTases modify select cytosolic (cy-) and mitochondrial (mt-) tRNAs. Mutation in human IPTase, TRIT1, causes disease phenotypes characteristic of mitochondrial translation deficiency due to mt-tRNA dysfunction. Deletion of the Schizosaccharomyces pombe IPTase (tit1-Δ) causes slow growth in glycerol, as well as in rapamycin, an inhibitor of TOR kinase that maintains metabolic homeostasis. . We show that lower ATP levels in tit1-Δ relative to tit1 + cells are also more decreased by an inhibitor of oxidative phosphorylation, indicative of mitochondrial dysfunction. Here we asked if the tit1-Δ phenotypes are due to hypomodification of cy-tRNA or mt-tRNA. A cytosol-specific IPTase that modifies cy-tRNA, but not mt-tRNA, fully rescues the tit1-Δ phenotypes. Moreover, overexpression of cy-tRNAs also rescues the phenotypes, and cy-tRNA Tyr alone substantially does so. Bioinformatics indicate that cy-tRNA Tyr is most limiting for codon demand in tit1-Δ cells and that the cytosolic mRNAs most loaded with Tyr codons encode carbon metabolilizing enzymes, many of which are known to localize to mitochondria. Thus, S. pombe i6A37 hypomodificationassociated metabolic deficiency results from hypoactivity of cy-tRNA, mostly tRNA Tyr , and unlike human TRIT1-deficiency does not impair mitochondrial translation due to mt-tRNA hypomodification. We discuss species-specific aspects of i6A37. Specifically relevant to mitochondria, we show that its hypermodified version, ms2i6A37 (2-methylthiolated), which occurs on certain mammalian mt-tRNAs (but not cy-tRNAs), is not found in yeast.

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“…The accuracy of decoding is quite high, with measured error rates in bacterial cells on the order of 10 −3 -10 −5 , depending on the nature of the mismatch (Kramer and Farabaugh 2007;Manickam et al 2014). This level of accuracy relies in part on a kinetic proofreading mechanism (Hopfield 1974;Ninio 1975;Ruusala et al 1982).…”

Section: Introductionmentioning

confidence: 99%

Epistasis analysis of 16S rRNA ram mutations helps define the conformational dynamics of the ribosome that influence decoding

Ying

Fredrick

2016

RNA

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The ribosome actively participates in decoding, with a tRNA-dependent rearrangement of the 30S A site playing a key role. Ribosomal ambiguity (ram) mutations have mapped not only to the A site but also to the h12/S4/S5 region and intersubunit bridge B8, implicating other conformational changes such as 30S shoulder rotation and B8 disruption in the mechanism of decoding. Recent crystallographic data have revealed that mutation G299A in helix h12 allosterically promotes B8 disruption, raising the question of whether G299A and/or other ram mutations act mainly via B8. Here, we compared the effects of each of several ram mutations in the absence and presence of mutation h8Δ2, which effectively takes out bridge B8. The data obtained suggest that a subset of mutations including G299A act in part via B8 but predominantly through another mechanism. We also found that G299A in h12 and G347U in h14 each stabilize tRNA in the A site. Collectively, these data support a model in which rearrangement of the 30S A site, inward shoulder rotation, and bridge B8 disruption are loosely coupled events, all of which promote progression along the productive pathway toward peptide bond formation.

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Studies of translational misreading in vivo show that the ribosome very efficiently discriminates against most potential errors

Abstract: Protein synthesis must rapidly and repeatedly discriminate between a single correct and many incorrect aminoacyl-tRNAs. We have attempted to measure the frequencies of all possible missense errors by tRNA

Cited by 86 publications

References 31 publications

Two proofreading steps amplify the accuracy of genetic code translation

Two proofreading steps amplify the accuracy of genetic code translation

Lack of tRNA-i6A modification causes mitochondrial-like metabolic deficiency in S. pombe by limiting activity of cytosolic tRNA^Tyr, not mito-tRNA

Epistasis analysis of 16S rRNA ram mutations helps define the conformational dynamics of the ribosome that influence decoding

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Studies of translational misreading in vivo show that the ribosome very efficiently discriminates against most potential errors

Abstract: Protein synthesis must rapidly and repeatedly discriminate between a single correct and many incorrect aminoacyl-tRNAs. We have attempted to measure the frequencies of all possible missense errors by tRNA

Cited by 86 publications

References 31 publications

Two proofreading steps amplify the accuracy of genetic code translation

Two proofreading steps amplify the accuracy of genetic code translation

Lack of tRNA-i6A modification causes mitochondrial-like metabolic deficiency in S. pombe by limiting activity of cytosolic tRNATyr, not mito-tRNA

Epistasis analysis of 16S rRNA ram mutations helps define the conformational dynamics of the ribosome that influence decoding

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Lack of tRNA-i6A modification causes mitochondrial-like metabolic deficiency in S. pombe by limiting activity of cytosolic tRNA^Tyr, not mito-tRNA