Amino acid–dependent stability of the acyl linkage in aminoacyl-tRNA

Peacock, Anna C.; Walvoord, Ryan R.; Ay, Chang; Mc, Kozlowski; Gamper, Howard; Hou, Ya‐Ming

doi:10.1261/rna.044123.113

Cited by 43 publications

(48 citation statements)

References 40 publications

(46 reference statements)

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“…Consequently, the in vitro synthesized, orthogonal tRNA might display a lower translation efficiency compared to native tRNAs. Third, amino acid hydrolysis from the tRNA is pH and temperature dependent and might occur during incubation of the injected cells prior to forming a complex with the endogenous elongation factors (Peacock et al, 2014; Stepanov and Nyborg, 2002). …”

Section: Resultsmentioning

confidence: 99%

Cellular encoding of Cy dyes for single-molecule imaging

Leisle

Chadda

Lueck

et al. 2016

eLife

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A general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and Cy5) are tolerated by the eukaryotic ribosome in cell-free and whole-cell environments and can be incorporated into soluble and membrane proteins. In the context of the Xenopus laevis oocyte expression system, this technique yields ion channels with encoded Cy-ncAAs that are trafficked to the plasma membrane where they display robust function and distinct fluorescent signals as detected by TIRF microscopy. This is the first demonstration of an encoded cyanine dye as a ncAA in a eukaryotic expression system and opens the door for the analysis of proteins with single-molecule resolution in a cellular environment.DOI: http://dx.doi.org/10.7554/eLife.19088.001

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Section: Resultsmentioning

confidence: 99%

Cellular encoding of Cy dyes for single-molecule imaging

Leisle

Chadda

Lueck

et al. 2016

eLife

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“…tRNA Phe was labeled at acp 3 U47 position with Cy5 using NHS chemistry as previously described 54 , with Cy5-NHS-ester purchased from GE Healthcare. Synthesis and purification of activated Ala-and Val-DBE (3,5-dinitrobenzyl esters) derivatives was done using detailed protocol 55 .…”

Section: Zmw-based Single-molecule Fluorescence Assay To Monitor Tranmentioning

confidence: 99%

Short translational ramp determines efficiency of protein synthesis

Verma¹,

Choi

Cottrell³

et al. 2019

Preprint

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It is generally assumed that translation efficiency is governed by translation initiation. However, the efficiency of protein synthesis is regulated by multiple factors including tRNA abundance, codon composition, mRNA motifs and amino-acid sequence 1-4 . These factors influence the rate of protein synthesis beyond the initiation phase of translation, typically by modulating the rate of peptide-bond formation and to a lesser extent that of translocation. The slowdown in translation during the early elongation phase, known as the 5' translational ramp, likely contributes to the efficiency of protein synthesis 5-9 . Multiple mechanisms, which could explain the molecular basis for this translational ramp, have been proposed that include tRNA abundance bias 6,9 , the rate of translation initiation 10-15 , mRNA and ribosome structure 11,12,14,[16][17][18] , or retention of initiation factors during early elongation events 19 .Here, we show that the amount of synthesized protein (translation efficiency) depends on a short translational ramp that comprises the first 5 codons in mRNA. Using a library of more than 250,000 reporter sequences combined with in vitro and in vivo protein expression assays, we show that differences in the short ramp can lead to 3 to 4 orders of magnitude changes in protein abundance. The observed difference is not dependent on tRNA abundance, efficiency of translation initiation, or overall mRNA structure. Instead, we show that translation is regulated by amino-acid-sequence composition and local mRNA sequence. Single-molecule measurements of translation kinetics indicate substantial pausing of ribosome and abortion of protein synthesis on the 4 th or 5 th codon for distinct amino acid or nucleotide compositions. Introduction of preferred sequence motifs, only at the exact positions within the mRNA, improves protein synthesis for recombinant proteins, indicating an evolutionarily conserved mechanism for controlling translational efficiency.

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“…First, discrimination between correct and non-cognate aa-tRNAs occurs through interaction with translation factors. The prokaryotic and eukaryotic EFs, EF-Tu and eEF1a, respectively form a ternary complex with aa-tRNAs and guanosine triphosphate (GTP), which protects against premature deacylation and facilitates delivery to the ribosome 45 . The interaction between the aa-tRNA and EF is thermodynamically tuned to bind cognate amino acid:tRNA pairs, while misaminoacylation perturbs this interaction such that an increase in binding affinity may prevent release of the aa-tRNA, or a decrease may lead to premature release of the aa-tRNA 46–50 before ribosome delivery.…”

Section: Mechanisms Of Translational Fidelity and Errormentioning

confidence: 99%

Translational fidelity and mistranslation in the cellular response to stress

2017

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Faithful translation of mRNA into the corresponding polypeptide is a complex multistep process, requiring accurate amino acid selection, transfer RNA (tRNA) charging and mRNA decoding on the ribosome. Key players in this process are aminoacyl-tRNA synthetases (aaRSs), which not only catalyse the attachment of cognate amino acids to their respective tRNAs, but also selectively hydrolyse incorrectly activated non-cognate amino acids and/or misaminoacylated tRNAs. This aaRS proofreading provides quality control checkpoints that exclude non-cognate amino acids during translation, and in so doing helps to prevent the formation of an aberrant proteome. However, despite the intrinsic need for high accuracy during translation, and the widespread evolutionary conservation of aaRS proofreading pathways, requirements for translation quality control vary depending on cellular physiology and changes in growth conditions, and translation errors are not always detrimental. Recent work has demonstrated that mistranslation can also be beneficial to cells, and some organisms have selected for a higher degree of mistranslation than others. The aims of this Review Article are to summarize the known mechanisms of protein translational fidelity and explore the diversity and impact of mistranslation events as a potentially beneficial response to environmental and cellular stress.

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Amino acid–dependent stability of the acyl linkage in aminoacyl-tRNA

Cited by 43 publications

References 40 publications

Cellular encoding of Cy dyes for single-molecule imaging

Cellular encoding of Cy dyes for single-molecule imaging

Short translational ramp determines efficiency of protein synthesis

Translational fidelity and mistranslation in the cellular response to stress

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