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Elongation Factor P Is Required to Maintain Proteome Homeostasis at High Growth Rate

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Elongation Factor P Is Required to Maintain Proteome Homeostasis at High Growth Rate Elongation factor P is required to maintain proteome homeostasis at high growth rate Rodney Tollerson IIa, Anne Witzkyb,c, and Michael Ibbaa,c,1 aDepartment of Microbiology, The Ohio State University, Columbus, OH 43210; bDepartment of Molecular Genetics, The Ohio State University, Columbus, OH 43210; and cCenter for RNA Biology, The Ohio State University, Columbus, OH 43210 Edited by Dieter Söll, Yale University, New Haven, CT, and approved September 11, 2018 (received for review July 12, 2018) Elongation factor P (EF-P) is a universally conserved translation factor Previous studies have shown that distal sequences, specifically that alleviates ribosome pausing at polyproline (PPX) motifs by the Shine-Dalgarno (SD) sequence and the start codon, influence facilitating peptide bond formation. In the absence of EF-P, PPX ribosome queuing at PPX motifs. In general, in the presence of peptide bond formation can limit translation rate, leading to EF-P, translation initiation is the limiting factor in protein syn- pleotropic phenotypes including slowed growth, increased antibi- thesis rate. When translation initiation rates are high in the ab- otic sensitivity, and loss of virulence. In this study, we observe that sence of EF-P, pausing at PPX motifs can cause ribosome queuing many of these phenotypes are dependent on growth rate. and induce a translational bottleneck. When translation initiation Limiting growth rate suppresses a variety of detrimental pheno- rates are decreased by weakening the SD sequence or start codon, types associated with ribosome pausing at PPX motifs in the fewer ribosomes load on the transcript, reducing ribosome absence of EF-P. Polysome levels are also similar to wild-type queuing at PPX motifs (18, 19). This effect can be best observed in under slow growth conditions, consistent with global changes in the context of the ATP synthase proteins AtpA and AtpD. Both ribosome queuing in cells without EF-P when growth rate is AtpA and AtpD have the same PPX motif (PPE) and similar decreased. Inversely, under high protein synthesis demands, we immediately neighboring sequences, whereas the SD of AtpD is observe that Escherichia coli lacking EF-P have reduced fitness. Our closer to the consensus sequence than that of AtpA. Ribosome data demonstrate that EF-P-mediated relief of ribosome queuing is profiling data reveal that atpD transcripts contain ribosome required to maintain proteome homeostasis under conditions of queuing in the absence of EF-P, whereas atpA transcripts do not, high translational demands. indicating that the major determinant influencing the result of EF-P dependence on PPX containing protein production is the protein synthesis | cellular physiology | elongation factor P | rate of translation initiation (18). translation elongation | adaptation When studying EF-P in E. coli, assays have been regularly performed in conditions in which E. coli is growing rapidly and he average translation elongation rate in Escherichia coli is translation is occurring at a high rate (14, 20). Recent studies Tbetween 10 and 20 amino acids per second in rapid growth have shown that translation rates in E. coli can vary depending conditions (1). This rate can fluctuate, depending on environ- on the growth conditions (2, 21). In this study, we determine that mental conditions. For example, different temperatures and reducing translation rates by limiting growth rate in E. coli masks nutrient conditions can increase or decrease translational speed slow PPX peptide bond formation in the absence of EF-P, sug- (2). Specific transcripts are known to have programmed trans- gesting that high protein synthesis rates under rapid growth lation rate tuning and use ribosome pausing for regulatory and conditions exacerbate the effects of ribosome queuing because of functional roles (3). Stretches of proline are also known to cause slow PPX synthesis (6). These results highlight a role for EF-P in ribosome pausing (4, 5). Proline contains a unique constrained ring structure, which is important for making turns and in- Significance creasing stability of proteins, but makes it a poor peptide bond donor and acceptor (6, 7). Translation elongation factor P (EF-P) Adaptation to rapidly changing environments is essential for alleviates ribosome pausing at polyproline (PPX; Pro-Proany cellular fitness and is a critical component in pathogenesis. amino acid) motifs by facilitating peptide bond formation be- Translational control has important roles for fitness in a range tween prolines (4, 5, 8). EF-P and its homologs are conserved of environments and requires multiple factors for efficient re- throughout all domains of life (eIF5A and aIF5A in eukaryotes sponses. One such factor is the translation elongation factor and archaea, respectively). Although all EF-P family members (EF)-P, which alleviates ribosome pausing at polyproline motifs. stimulate translation of PPX motifs, eIF5A has recently been Our findings show that EF-P-mediated relief of ribosome queu- shown to also have roles in translation termination, which is likely ing is integral in environmentally driven changes to translation the cause of eIF5A essentiality in eukaryotes (9, 10). rates. We observe that ribosome pausing leads to changes in To perform its function, EF-P enters paused ribosomes through protein yield only under rapid growth conditions, demonstrating the E-site and facilitates peptide bond formation through inter- that effects resulting from ribosome queuing correlate directly actions with the P-site tRNA (11, 12). This interaction is aided by Pro fMet with translational demand. These results provide physiological the D-arm of tRNA , which is shared with tRNA (13). Fur- context to previous studies establishing EF-P as a critical factor in ther, EF-P requires posttranslational modification (PTM) at a cell growth and virulence. conserved residue to efficiently facilitate peptide bond formation within the peptidyl transfer center of the ribosome (14–17). β Author contributions: R.T., A.W., and M.I. designed research; R.T. performed research; PTMs range from linear structures such as (R)- -lysine and 5- R.T., A.W., and M.I. analyzed data; and R.T. and M.I. wrote the paper. aminopentanol in E. coli and Bacillus subtilis, respectively, to The authors declare no conflict of interest. glycosylation with rhamnose in Pseudomonas aeruginosa.Al- though modification structures are diverse, the general function of This article is a PNAS Direct Submission. the modification is to interact and stabilize proline ProtRNAPro Published under the PNAS license. within the peptidyl transfer center to aid peptide bond formation 1To whom correspondence should be addressed. Email: [email protected]. (12). Loss of PTM leads to comparable phenotypes to efp deletion This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in all organisms in which EF-P has been studied, demonstrating 1073/pnas.1812025115/-/DCSupplemental. the importance of PTM in EF-P function. Published online October 8, 2018. 11072–11077 | PNAS | October 23, 2018 | vol. 115 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1812025115 Downloaded by guest on September 29, 2021 blotting (SI Appendix, Fig. S1). This data show that in the ab- sence of EF-P, addition of the 4P motif to GFP results in a greater than 95% decrease in protein yield. When this experi- ment is performed with cells cultured at 25 °C, we observe that insertion of a 4P motif has no change on the relative protein yield in the absence of EF-P. In our system, we observe that at 25 °C, the effect of slow PPX peptide bond formation on protein yield is relieved consistent with that seen with weakening the SD and translation start sequences (18). These data suggest that slowing translation through slow growth conditions suppresses the effect of slow peptide bond formation at PPX motifs. In the absence of EF-P, peptide bond formation at PPX motifs limits translation rate, causing ribosome queuing and decreasing pro- tein output potential (18). When E. coli grows at 25 °C, the re- duced rate of translation prevents ribosome queuing in the absence of EF-P, leaving total protein yield unaffected. Slowed Translation Rate at 25 °C Masks Global Translation Defect in Δefp Cells. Ribosome profiling studies have shown that loss of EF-P can cause significant pausing in up to 50% of PPX motifs in E. coli, which affects cellular concentrations of PPX containing proteins (19, 22). To assess the effect of slow PPX synthesis on global protein production, we use biorthogonal noncanonical amino acid tagging (BONCAT). BONCAT uses a noncanonical Fig. 1. Total yield of GFP with four proline residues at the N terminus (4P- amino acid, which has an available substrate to interact with a GFP) is unaffected by the absence of EF-P at 25 °C. (Top) Graphical represen- fluorescent dye in a copper-mediated alkyne-azide cycloaddition tation of 4P-GFP construct. 4X-Pro represents four consecutive prolines at the (23). This technique has been used to detect translational activity N terminus of GFP. (Bottom) Bars indicate the average ratio of 4P-GFP to GFP over time in both pure and mixed cultures (24). In this study, we fluorescence after 1 h of induction with 1 mM IPTG between three biological use BONCAT to measure changes in global protein synthesis in the Δ replicates in WT or efp::kan ( efp) E. coli BW25113. Values normalized to absence of EF-P. We labeled WT and Δefp E. coli with L-azido- OD600 at time of reading. Error bars represent the SD between ratios for each homoalanine (AHA), a substrate for methionine tRNA synthetase replicate. Experiment was performed at either 37 °C or 25 °C, as indicated. ’ ≤ > that can be readily used in translation, at both 37 °C and 25 °C. By Significance was determined using Student s t test (*P 0.05; N.S., P 0.05). determining the incorporation of AHA into the proteome over time through conjugation with the dye Cy3, we can correlate an maintaining proteome homeostasis in conditions that demand increase in fluorescence over time to protein synthesis rates.
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