Translation factor LepA contributes to tellurite resistance in Escherichia coli but plays no apparent role in the fidelity of protein synthesis

Abstract

LepA is a translational GTPase highly conserved in bacterial lineages. While it has been shown that LepA can catalyze reverse ribosomal translocation in vitro, the role of LepA in the cell remains unclear. Here, we show that deletion of the lepA gene (DeltalepA) in Escherichia coli causes hypersensitivity to potassium tellurite and penicillin G, but has no appreciable effect on growth under many other conditions. DeltalepA does not increase miscoding or frameshifting errors under normal or stress conditions, indicating that LepA does not contribute to the fidelity of translation. Overexpression of LepA interferes with tmRNA-mediated peptide tagging and A-site mRNA cleavage, suggesting that LepA is a bona fide translation factor that can act on stalled ribosomes with a vacant A site in vivo. Together these results lead us to hypothesize that LepA is involved in co-translational folding of proteins that are otherwise vulnerable to tellurite oxidation.

Figure 1. Phenotypes of a ΔlepA strain

(A) RT-PCR of the lep transcript in ΔlepA cells. Genomic organization of the lep operon is shown in the left panel. Total RNA was extracted from control or ΔlepA cells in mid-log phase and amplified by gene-specific primers (indicated by thin arrows on each gene). (B) Growth curves of a wild-type and ΔlepA strain in LB medium at 37°C in the absence (control) or presence of 0.05 μg/ml potassium tellurite, 20 μg/ml penicillin G, 40 μg/ml paraquat, 15 μg/ml phenazine methosulfate, or 100 mM MgCl₂ (as indicated). In the latter case, the pH of media was adjusted to 5 with HCl prior to inoculation. All data points in the figure include error bars; many of them are smaller than the symbols. (C) Complementation of phenotypes of ΔlepA cells by plasmid pLEPA harboring the lepA gene. Overnight culture of wild-type and ΔlepA strains carrying either empty vector or pLEPA were diluted in LB medium by a factor of 10², 10⁴, 10⁵, 10⁶, and 10⁷. A 10 μl aliquot of each dilution was spotted on LB plates containing 0.05 or 0.4 μg/ml potassium tellurite, and the plates were incubated at 37°C until colonies became visible.

Figure 2. Effects of LepA and BipA on the tmRNA quality control system

(A) Analysis of tmRNA(DD)-mediated tagging of protein expressed from a non-stop mRNA. The N-terminal domain of cI repressor was purified by Ni²⁺-affinity chromatography and analyzed by SDS-PAGE and Coomassie blue staining. Untagged cI repressor accumulates in ΔtmRNA cells, whereas most of the repressor protein is tagged in tmRNA(DD) cells. The percentage (mean ± SEM) of tmRNA(DD)-tagged protein for each strain is indicated. (B) Effect of LepA and BipA overexpression on tmRNA(DD) tagging. The N-terminal domain of cI repressor was purified from cells containing plasmid-borne P_BAD-lepA (left panel) or P_BAD-bipA (right panel) grown in the absence (−) or presence (+) of arabinose. The percentage (mean ± SEM) of tmRNA(DD)-tagged protein for each strain is indicated. (C) Northern blot analysis of flag-(m)ybeL(PP) mRNA for A-site mRNA cleavage. RNA was isolated from strains carrying plasmid-borne P_BAD-lepA (left panel) or P_BAD-bipA (right panel) grown in the absence (−) or presence (+) of arabinose. Two mRNA species corresponding to full-length and A-site cleaved messages were detected (as indicated). An in vitro transcript corresponding to A-site cleaved mRNA was used as a size marker (lanes 1 and 4). (D) Analysis of tmRNA(DD)-mediated tagging of protein expressed from ybeL(PP) mRNA. His₆-YbeL(PP) was purified by Ni²⁺-affinity chromatography, followed by SDS-PAGE analysis and Coomassie blue staining.