Translation quality control is critical for bacterial responses to amino acid stress Tammy J. Bullwinklea and Michael Ibbaa,1 aDepartment of Microbiology, The Ohio State University, Columbus, OH 43210 Edited by Dieter Söll, Yale University, New Haven, CT, and approved January 14, 2016 (received for review December 22, 2015) Gene expression relies on quality control for accurate transmission Despite their role in accurately translating the genetic code, of genetic information. One mechanism that prevents amino acid aaRS editing pathways are not conserved, and their activities misincorporation errors during translation is editing of misacy- have varying effects on cell viability. Mycoplasma mobile, for lated tRNAs by aminoacyl-tRNA synthetases. In the absence of example, tolerates relatively high error rates during translation editing, growth is limited upon exposure to excess noncognate and apparently has lost both PheRS and leucyl-tRNA synthetase proofreading activities (4). Similarly, the editing activity of amino acid substrates and other stresses, but whether these physi- Streptococcus pneumoniae ological effects result solely from mistranslation remains unclear. To IleRS is not robust enough to com- pensate for its weak substrate specificity, leading to the formation explore if translation quality control influences cellular processes Ile Ile other than protein synthesis, an Escherichia coli strain defective in of misacylated Leu-tRNA and Val-tRNA species (5). Also, in contrast to its cytoplasmic and bacterial counterparts, Saccha- Tyr-tRNAPhe editing was used. In the absence of editing, cellular Phe romyces cerevisiae mitochondrial PheRS completely lacks an levels of aminoacylated tRNA were elevated during amino acid editing domain and instead appears to rely solely on stringent stress, whereas in the wild-type strain these levels declined under Phe/Tyr discrimination to maintain specificity (6). The divergent the same growth conditions. In the editing-defective strain, in- Phe range of mechanisms used to discriminate against noncognate creased levels of aminoacylated tRNA led to continued synthesis amino acids illustrates how the requirements for translation of the PheL leader peptide and attenuation of pheA transcription quality control vary with cellular physiology and among envi- under amino acid stress. Consequently, in the absence of editing, ronmental niches (7). Although translation quality control by activation of the phenylalanine biosynthetic operon becomes less aaRS editing has not been shown to be essential in any organ- responsive to phenylalanine limitation. In addition to raising amino- isms, both its presence and absence can be critical for optimal acylated tRNA levels, the absence of editing lowered the amount growth and cellular fitness under various stress conditions in of deacylated tRNAPhe in the cell. This reduction in deacylated different organisms (2). These highly varied effects on cellular tRNA was accompanied by decreased synthesis of the second mes- physiology suggest that quality control of misacylated tRNA syn- senger guanosine tetraphosphate and limited induction of strin- thesis may play other important roles in the cell in addition to gent response-dependent gene expression in editing-defective maintaining proteome integrity. cells during amino acid stress. These data show that a single quality- In addition to acting as substrates for protein synthesis, ami- control mechanism, the editing of misacylated aminoacyl-tRNAs, noacyl-tRNAs have numerous other functions in the cell, e.g., as provides a critical checkpoint both for maintaining the accuracy of amino acid donors in processes including lipid and protein mod- translation and for determining the sensitivity of transcriptional ification, antibiotic biosynthesis, and heme metabolism (8). An- responses to amino acid stress. other key role of aminoacyl-tRNA is to serve as signaling molecules for a number of starvation sensors (9). The levels of aminoacyl-tRNA and deacylated tRNA are, respectively, signals translation | quality control | stress | stringent response for stimulating and limiting translation in response to amino acid stress. Most bacteria contain transcriptional attenuation mecha- ccurate translation of mRNA into the corresponding amino nisms or translational controls that monitor the aminoacylation Aacid sequence is an essential step during gene expression. state of tRNA as the signal for regulating the expression of genes Translational fidelity depends on correct tRNA–codon pairing required for amino acid biosynthesis and import. These mechanisms by the ribosome as well as attachment of the proper amino acids to their respective tRNAs by aminoacyl-tRNA synthetases (aaRSs). Significance The ability of aaRS enzymes to distinguish between cognate and noncognate amino acids is a major determinant of the fidelity Recent studies showing that both high-accuracy protein syn- with which the genetic code is translated (1). Discrimination thesis and low-accuracy protein synthesis are critical for cellular against noncognate amino acid substrates is particularly chal- growth and survival under different growth conditions suggest lenging for aaRSs, because a near-cognate amino acid may differ that the translation quality-control machinery may function in from the cognate substrate by as little as a single methyl or hy- cellular processes other than protein synthesis. We explored droxyl group. Generally, highly specific aaRS enzymes and the this possibility and found that inhibition of misacylated-tRNA widespread existence of editing mechanisms that proofread quality control suppressed the synthesis of the starvation- noncognate amino acids prevent errors in amino acid recognition inducible second messenger guanosine tetraphosphate and from compromising the overall accuracy of translation (2). For limited the induction of stringent response-dependent gene example, in the class I isoleucyl-tRNA synthetase a posttransfer expression. These findings show that translation quality control Ile editing site for deacylation of misacylated Val-tRNA exists 35 Å is a single checkpoint for both accurate protein synthesis and from the active site. Similarly, the class II phenylalanine-tRNA transcriptional responses to stress. synthetase (PheRS) edits misacylated Tyr-tRNAPhe at a hydro- lytic editing site ∼30 Å from the synthetic active site. Bacterial Author contributions: T.J.B. and M.I. designed research; T.J.B. performed research; T.J.B. PheRS editing prevents meta-tyrosine (m-Tyr), a readily acti- and M.I. analyzed data; and T.J.B. and M.I. wrote the paper. vated nonprotein amino acid and product of Phe oxidation, from The authors declare no conflict of interest. being misincorporated into the proteome (3). These and other This article is a PNAS Direct Submission. aaRS proofreading activities provide quality-control checkpoints 1To whom correspondence should be addressed. Email: [email protected]. against noncognate amino acid incorporation during translation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and thereby help prevent the formation of an aberrant proteome. 1073/pnas.1525206113/-/DCSupplemental. 2252–2257 | PNAS | February 23, 2016 | vol. 113 | no. 8 www.pnas.org/cgi/doi/10.1073/pnas.1525206113 Downloaded by guest on September 24, 2021 include ribosomal attenuation, T-box riboswitches, and occlusion A of the ribosome binding site, such as the feedback regulation of the E. coli threonyl-tRNA synthetase gene (thrS) leader se- quence that resembles tRNAThr (10–12). An example of ri- bosome attenuation occurs during translation of the E. coli leader peptide encoded by pheL when ribosome stalling stimu- lates the transcription of pheA, which encodes the chorismate mutase required for phenylalanine biosynthesis. Deacylated tRNA is an important signaling molecule in bacte- B ria, most notably during activation of the highly conserved strin- gent response pathway (13). The stringent response in E. coli is activated under a number of stresses, including nutrient depri- vation, and helps promote cell survival under unfavorable growth conditions. The paralogous enzymes RelA and SpoT both mediate the stringent response through the synthesis of the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphos- phate (pppGpp) collectively known as “(p)ppGpp.” SpoT has been proposed to be responsible for synthesis of (p)ppGpp as part of the general cellular stress response to conditions in- cluding fatty acid starvation, carbon starvation, and osmotic shock. Alternatively amino acid starvation also may lead to ri- bosome stalling and binding of deacylated tRNA in the ribo- somal A site, resulting in the release of RelA and production of (p)ppGpp. Therefore, levels of deacylated tRNA are critical for regulating the activation of the stringent response under condi- tions of amino acid limitation in E. coli. The rise in alarmone levels in the cell is rapid and inhibits transcription initiation at the promoters of genes needed for the transcription and trans- Fig. 1. Aminoacylated and deacylated tRNA levels are altered in the ab- lation machineries, including rRNAs, aaRSs, and tRNAs, through sence of quality control by PheRS editing. (A) Representative Northern blot probed with a 32P-5′-end–labeled tRNAPhe probe in which aminoacylated direct binding to the β′–Ω subunit interface of E. coli RNA poly- – and nonaminoacylated species are separated by an acid urea gel before merase (14 16). The initiation of