Cell-specific differences in the requirements for translation quality control - PubMed (original) (raw)

Cell-specific differences in the requirements for translation quality control

Noah M Reynolds et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Protein synthesis has an overall error rate of approximately 10(-4) for each mRNA codon translated. The fidelity of translation is mainly determined by two events: synthesis of cognate amino acid:tRNA pairs by aminoacyl-tRNA synthetases (aaRSs) and accurate selection of aminoacyl-tRNAs (aa-tRNAs) by the ribosome. To ensure faithful aa-tRNA synthesis, many aaRSs employ a proofreading ("editing") activity, such as phenylalanyl-tRNA synthetases (PheRS) that hydrolyze mischarged Tyr-tRNA(Phe). Eukaryotes maintain two distinct PheRS enzymes, a cytoplasmic (ctPheRS) and an organellar form. CtPheRS is similar to bacterial enzymes in that it consists of a heterotetramer in which the alpha-subunits contain the active site and the beta-subunits harbor the editing site. In contrast, mitochondrial PheRS (mtPheRS) is an alpha-subunit monomer that does not edit Tyr-tRNA(Phe), and a comparable transacting activity does not exist in organelles. Although mtPheRS does not edit, it is extremely specific as only one Tyr-tRNA(Phe) is synthesized for every approximately 7,300 Phe-tRNA(Phe), compatible with an error rate in translation of approximately 10(-4). When the error rate of mtPheRS was increased 17-fold, the corresponding strain could not grow on respiratory media and the mitochondrial genome was rapidly lost. In contrast, error-prone mtPheRS, editing-deficient ctPheRS, and their wild-type counterparts all supported cytoplasmic protein synthesis and cell growth. These striking differences reveal unexpectedly divergent requirements for quality control in different cell compartments and suggest that the limits of translational accuracy may be largely determined by cellular physiology.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Active site specificity of mitochondrial and cytosolic PheRS. (A) Structure of active site of the α-subunit of T. thermophilus PheRS in complex with phenylalanyl-adenylate (22). The optimal size of the Phe binding pocket is provided by residue Ala314 (residue A333 of S. cerevisiae mitochondrial PheRS). Motifs 1, 2, and 3 forming the active site of PheRS are colored in green, red, and blue, respectively. (B) Alignment summary of PheRS motif 3. The secondary structure elements of motif 3 are indicated on the top of the alignment, and position Ala314 is indicated by the arrowhead. Residues displaying more than 85% identity or similarity are depicted on black or gray background, respectively. (C) Secondary structural analysis of mtPheRS. Circular dichroism was measured at 25 °C with 5 μM samples of S. cerevisiae wild-type mtPheRS or the A333G variant.

Fig. 2.

Fig. 2.

Growth of S. cerevisiae msf1-1. (A) Streak plates showing growth of diploid and haploid strains on YPDA and ethanol plus glycerol medium. (B) Serial dilutions of strains obtained from dissection of MSF1/msf1-1 on YPDA; MSF1 msf1-1, and msf1Δ on YPDA and ethanol plus glycerol medium. Plates were incubated at 30 °C for 3 days. (C) Dissection of tetrads from MSF1/MSF1, MSF1/msf1Δ, and MSF1/msf1-1 strains on ethanol plus glycerol medium. Plates were incubated at 30 °C for 3 (MSF1/MSF1) or 4 days (MSF1/msf1Δ and MSF1/msf1-1).

Fig. 3.

Fig. 3.

Function of A333G mtPheRS in E. coli. Rescue of the growth phenotype of E. coli NP37 transformed with (A) yeast MSF1, msf1-1, or msf1 with a stop codon at A333 and (B) human FARS2, fars2-1, or fars2 with a stop codon at A308. (C) Transediting activity of E. coli wild-type and NP37 FARS2 complemented cell extracts grown at 30 or 42 °C. Reactions were carried out at 42 °C. Data points are an average of three independent experiments with errors bars representing 1 SD.

Fig. 4.

Fig. 4.

Function of βD243A ctPheRS in S. cerevisiae. (A) Dissection of tetrads from S. cerevisiae FRS1/frsΔ complemented with pFL36-FRS1, pFL36-frs1-1, or pFL36 onto YPD and replica plated onto G418 and CSM-Leu. In liquid YPD doubling times for frsΔ pFL36-FRS1 and pFL36-frs1-1 were 1.37 ± 0.05 and 1.34 ± 0.09 h, respectively. (B) Posttransfer editing activity of S. cerevisiae frs1Δ pFL36-FRS1 and frs1Δ pFL36-frs1-1 extracts at 37 °C. Data points are an average of at least three independent experiments, with errors bars representing 1 SD.

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