Translation quality control is critical for bacterial responses to amino acid stress

Abstract

Gene expression relies on quality control for accurate transmission of genetic information. One mechanism that prevents amino acid misincorporation errors during translation is editing of misacylated tRNAs by aminoacyl-tRNA synthetases. In the absence of editing, growth is limited upon exposure to excess noncognate amino acid substrates and other stresses, but whether these physiological effects result solely from mistranslation remains unclear. To explore if translation quality control influences cellular processes other than protein synthesis, an Escherichia coli strain defective in Tyr-tRNA(Phe) editing was used. In the absence of editing, cellular levels of aminoacylated tRNA(Phe) were elevated during amino acid stress, whereas in the wild-type strain these levels declined under the same growth conditions. In the editing-defective strain, increased levels of aminoacylated tRNA(Phe) led to continued synthesis of the PheL leader peptide and attenuation of pheA transcription under amino acid stress. Consequently, in the absence of editing, activation of the phenylalanine biosynthetic operon becomes less responsive to phenylalanine limitation. In addition to raising aminoacylated tRNA levels, the absence of editing lowered the amount of deacylated tRNA(Phe) in the cell. This reduction in deacylated tRNA was accompanied by decreased synthesis of the second messenger guanosine tetraphosphate and limited induction of stringent response-dependent gene expression in editing-defective cells during amino acid stress. These data show that a single quality-control mechanism, the editing of misacylated aminoacyl-tRNAs, provides a critical checkpoint both for maintaining the accuracy of translation and for determining the sensitivity of transcriptional responses to amino acid stress.

Keywords: quality control; stress; stringent response; translation.

Fig. 1.

Aminoacylated and deacylated tRNA levels are altered in the absence of quality control by PheRS editing. (A) Representative Northern blot probed with a ³²P-5′-end–labeled tRNA^Phe probe in which aminoacylated and nonaminoacylated species are separated by an acid urea gel before transfer. (B) Percent tRNA charging levels in vivo in wild-type cells (solid bars) and in editing-deficient pheT(G318W) PheRS cells (checkered bars) grown to late log in M9 minimal medium containing no Phe, 100 µM Phe, or 10 µM m-Tyr. Error bars represent the SD from three independent experiments.

Fig. 2.

Transcription of the pheA leader under amino acid stress is reduced in the absence of quality control by PheRS editing. β-Galactosidase activity from an E. coli pheA leader-lacZ expression transcriptional reporter was monitored in wild-type PheT and editing-defective PheT(G318W) strains. β-Gal activity is reported as rate (Δ absorbance at 420 nm time) as a function of OD₆₀₀ and was determined for both wild-type PheT (solid bars) and PheT(G318W) (checkered bars) strains at late log phase. Activity from cultures grown in M9 minimal medium containing no Phe, 50 µM and 100 µM Phe, and 4 µM and 10 µM m-Tyr are shown. Error bars represent the SD from three independent experiments.

Fig. S1.

Transcription of the pheA leader under amino acid stress is reduced in the absence of quality control by PheRS editing. β-Galactosidase activity was monitored as described in the Materials and Methods and was determined for both wild-type pheT (solid bars) and pheT (G318W) (checkered bars) strains at late log phase. Activity from cultures grown in M9 minimal medium containing 20 µM, 200 µM Phe, and 2 mM p-Tyr are shown. Error bars represent the SD from three independent experiments.

Fig. S2.

PheRS editing-dependent reduction of pheA expression is caused by the translation of the PheL gene past residue 6. β-Galactosidase activity from a F6/stop mutant pheA′-lacZ reporter was monitored as described in the Materials and Methods and was determined for both the wild-type pheT (solid bars) and pheT(G318W) (checkered bars) strains at late log phase. Activity from cultures grown in M9 minimal medium containing 100 µM Phe or 10 µM m-Tyr is shown. Error bars represent the SD from three independent experiments.

Fig. 3.

ppGpp synthesis is reduced in the absence of quality control by PheRS editing. ppGpp levels were normalized to OD₆₀₀ in pheA auxotrophic strains with either wild-type PheT (solid bars) or editing-deficient PheT(G318W) (checkered bars) strains grown to early log phase in dropout medium containing high Phe (0.5 mM), low Phe (10 µM), or low Phe and m-Tyr (0.2 or 0.4 mM). Error bars represent the SD from three independent experiments.

Fig. S3.

Reduction in ppGpp synthesis in the absence of quality control by PheRS editing is dependent on RelA. (Left) Representative TLC showing undetectable in vivo ³²P-ppGpp levels in ΔrelA, pheA auxotrophic strains with either wild-type pheT or editing-deficient pheT G318W) strains grown to early log phase in dropout medium containing high Phe (0.5 mM), low Phe (10 µM), or low Phe and m-Tyr (0.4 mM). (Right) Cold standards were run in parallel and visualized with UV light.

Fig. 4.

Downstream transcriptional effects of the stringent response are repressed by tRNA mischarging. qRT-PCR analyses of iraP and rrnB P1 transcripts are normalized to ompA levels and are shown relative to levels in the wild-type pheT strain in high-Phe conditions. Cultures of pheA auxotrophic strains with either wild-type PheT (solid bars) or editing-deficient PheT(G318W) (checkered bars) strains grown to early log phase in Mops minimal medium containing high Phe (0.5 mM) (green bars), low Phe (10 µM) (blue bars), or low Phe and m-Tyr (0.4 mM) (red bars). Errors bars represent the SD of three independent biological replicates.