Abstract
Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNA(Phe). Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNA(Phe), an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol.
Highlights
Editing mechanism in IleRS that hydrolyzes both misactivated Val-AMP and misaminoacylated Val-tRNAIle [13, 14]
The H158A replacement had no effect, introduction of D243A led to a loss in Tyr-tRNAPhe deacylation activity (Fig. 3A) similar to that observed for E. coli variants defective in editing [25]
High-resolution crystal structures suggested that the substantial differences between the canonical (␣)2 phenylalanyl-tRNA synthetase (PheRS) and the mitochondrial ␣-type might not significantly impact tRNA aminoacylation, as the additional domains in the former were presumed to have alternative non-canonical functions
Summary
Plasmids, and General Methods—Plasmids carrying cytosolic or mitochondrial yeast tRNAPhe genes for T7 runoff transcription. Post-transfer Editing Assay—The cytosolic or mitochondrial TyrtRNAPhe and Phe-tRNAPhe (S. cerevisiae transcripts) were prepared as described previously [25] in an aminoacylation reaction containing 30 M [3H]Tyr (180 cpm/pmol), 0.5 M mitochondrial PheRS A333G, and 5 M corresponding in vitro transcribed tRNAPhe. Comparison to the total tRNA concentration allowed us to estimate that purified PhetRNAPhe yields were about 20% (i.e. these preparations contained 80% uncharged tRNA), whereas Tyr-tRNAPhe yields were 15% (85% uncharged), within the typical range expected for aminoacyl-tRNA preparations. Purified organelles were obtained by centrifugation at 100,000 ϫ g (Beckman SW41) in a PercollTM step gradient (Amersham Biosciences) (40% PercollTM in 0.6 M sorbitol (Fluka), 10 mM Tris-HCl (pH 7.4), overlaid with 20% PercollTM in the same buffer) These procedures yielded cytosolic and mitochondrial fractions with less than 0.5% cross-contamination as judged by Western blot analysis with antibodies specific for proteins in the NOVEMBER 18, 2005 VOLUME 280 NUMBER 46. The trees were rooted using the nine known sequences of o-phosphoseryl-tRNA synthetase found in methanogenic archaea that were previously identified as paralogues of the ␣-subunit of PheRS [41]
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