Elsevier

Biochimie

Volume 92, Issue 2, February 2010, Pages 157-163
Biochimie

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

https://doi.org/10.1016/j.biochi.2009.11.002Get rights and content

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 (ΔlepA) in Escherichia coli causes hypersensitivity to potassium tellurite and penicillin G, but has no appreciable effect on growth under many other conditions. ΔlepA 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.

Introduction

Protein synthesis relies on several related GTPases (translational GTPases) that have in common two tandem structural domains, designated I and II [1], [2]. In bacteria, four translational GTPases—initiation factor IF2, elongation factors EF-Tu and EF-G, and release factor RF3—carry out well-established roles in protein synthesis. With the exception of RF3, these factors are essential for cell viability and conserved in all three domains of life.

A number of other translational GTPases have been identified in bacteria whose physiological roles are less clear. LepA (or EF4) is an EF-G paralog encoded upstream of the leader peptidase gene (lepB) in Escherichia coli [3], [4]. Although LepA is widely conserved among bacteria and bacterium-derived organelles, the lepA gene can be deleted from the chromosome of E. coli without an obvious effect in growth rate [5]. LepA bears considerable similarity to EF-G but localizes to the membrane [6]. Sequence and structural data show that the protein domains of LepA are homologous to domains I, II, III, and V of EF-G [7]. LepA lacks regions corresponding to domain IV and sub-domain G′, but has a unique C-terminal domain which is unlike any other known proteins. In 2006, Nierhaus and coworkers showed that LepA can catalyze reverse translocation in vitro (i.e., movement of tRNAs from the P and E sites to the A and P sites, respectively) [8]. Cryo-EM reconstructions revealed that LepA binds the ribosome in the same orientation as EF-G, but the lack of domain IV allows LepA to bind simultaneously with A-site tRNA [9]. The C-terminal domain of LepA has a positively-charged surface that interacts extensively with A-site tRNA in the complex, and this interaction may bias tRNA-mRNA movement in the reverse direction [7], [9]. It was also shown by the Nierhaus group that addition of purified LepA can increase the fraction of active Green Fluorescent Protein (GFP) synthesized in a coupled in vitro transcription/translation system [8]. The authors proposed that the increase in GFP activity resulted from an increase in the translation fidelity. According to their model, by promoting reverse translocation, LepA can rescue “defective” posttranslocation complexes that are particularly prone to miscoding. A separate study indicated that LepA of mitochondria (Guf1) plays an important role in the biogenesis of functional cytochrome oxidase in S. cerevisiae [10]. Under suboptimal growth conditions, the absence of LepA/Guf1 decreased the activity of cytochrome oxidase to 10% of the control strain without a substantial decrease in the protein level. These authors suggested that this loss of activity could be due to errors in protein synthesis, consistent with the Nierhaus model, or to a defect in co-translational protein folding and assembly. But the question of whether LepA contributes to translational fidelity in vivo was not experimentally addressed in either study.

BipA is another paralog of EF-G conserved in most bacterial lineages [1], [11]. BipA has the same domain architecture as LepA but a distinct C-terminal domain. BipA exhibits ribosome-stimulated GTPase activity, binds 70S ribosomes in the presence GDPNP, and binds 30S subunits in the presence of the ppGpp [12], [13]. The ΔbipA phenotype is pleiotropic, with defects in cell motility, expression of the K5 capsule system, growth at low temperatures, and resistance to certain antimicrobial peptides [11], [14], [15], [16], [17]. While it is clear that BipA influences several processes and the expression of various genes in the cell, the mechanism by which it acts remains unknown. At least two phenotypes of ΔbipA (cold sensitivity and altered capsule synthesis) are suppressed by mutations in rluC, which is responsible for pseudouridylation at positions 955, 2504, and 2580 of 23S rRNA [18]. Base substitutions at these three positions of 23S rRNA are sufficient for suppression, suggesting that the physiological effects of BipA depend on its interaction with the ribosome.

In this study, we find that ΔlepA causes hypersensitivity to potassium tellurite but not to other oxidizing agents, suggesting that ΔlepA increases the accessibility of thiol groups in the cell. Neither ΔlepA nor ΔbipA has an appreciable effect on missense, nonsense, or frameshift suppression, indicating that the accuracy of translation depends on neither LepA nor BipA. Both of these factors can inhibit tmRNA-tagging in vivo, providing evidence that they act as elongation factors, but only LepA inhibits A-site mRNA cleavage. Based on these results, potential functions of LepA in regulating protein biogenesis are discussed.

Section snippets

Strains, plasmids, and growth conditions

ΔlepA and ΔbipA strains (JW2553 and JW5771, respectively) and their parental strain BW25113 (rrnB3 ΔlacZ4787 hsdR514 Δ[araBAD]567 Δ[rhaBAD]568 rph-1) were obtained from the Keio collection (National Institute of Genetics, Japan) [19]. In each case, the gene was replaced by an in-frame kanamycin resistance gene. For complementation experiments, a ∼2.9-kb HindIII fragment of the genomic region encompassing lepA, but not lepB, was cloned into pSC101-derived low-copy-number vector pWSK29 [20] to

ΔlepA confers hypersensitivity to potassium tellurite

Only a few phenotypes have been attributed to null mutations of lepA, including lethality at low pH in Helicobacter pylori [28] and heat/cold sensitivity in yeast [10], despite wide conservation of the gene [8]. To further investigate the role of LepA, E. coli strains BW25113 (control) and JW2553 (ΔlepA) were analyzed by high-throughput Phenotype Microarrays (Biolog, Inc., Hayward, CA), testing ∼1200 independent growth conditions [29]. Strain JW2553 contains an in-frame kanamycin resistance

Discussion

It was shown previously that addition of purified LepA to an in vitro transcription/translation system increased the percentage of active protein product from ∼50% to >90% [8]. The authors attributed this effect to miscoding in the absence of added LepA. They proposed that, by catalyzing reverse translocation, LepA rescues "defective" posttranslocation complexes that are highly prone to decoding errors in the next round of elongation. However, no evidence for “defective” posttranslocation

Acknowledgement

We thank B. Ahmer for vector pWSK29, R. Dalbey for helpful suggestions, C. H. Yang for experimental support, and S. Popova-Butler and K. Green-Church for proteomic analysis of the ΔlepA mutant. This work was supported by the National Institutes of Health [grant numbers GM072528 (to K.F.) & GM078634 (to C.S.H.)].

References (52)

  • F. Baneyx

    Recombinant protein expression in Escherichia coli

    Curr. Opin. Biotechnol.

    (1999)
  • V.R. Agashe et al.

    Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed

    Cell

    (2004)
  • H.C. Chang et al.

    De novo folding of GFP fusion proteins: high efficiency in eukaryotes but not in bacteria

    J. Mol. Biol.

    (2005)
  • M.V. Berridge et al.

    Translation of Xenopus liver messenger RNA in Xenopus oocytes: vitellogenin synthesis and conversion to yolk platelet proteins

    Cell

    (1976)
  • M. Dyllick-Brenzinger et al.

    The role of cysteine residues in tellurite resistance mediated by the TehAB determinant

    Biochem. Biophys. Res. Commun.

    (2000)
  • D.M. Rojas et al.

    Sensitivity to potassium tellurite of Escherichia coli cells deficient in CSD, CsdB and IscS cysteine desulfurases

    Res. Microbiol.

    (2005)
  • T. Margus et al.

    Phylogenetic distribution of translational GTPases in bacteria

    BMC Genomics

    (2007)
  • T. Date et al.

    Isolation of the Escherichia coli leader peptidase gene and effects of leader peptidase overproduction in vivo

    Proc. Natl. Acad. Sci. USA

    (1981)
  • N.J. Dibb et al.

    lep operon proximal gene is not required for growth or secretion by Escherichia coli

    J. Bacteriol.

    (1986)
  • P.E. March et al.

    GTP-binding membrane protein of Escherichia coli with sequence homology to initiation factor 2 and elongation factors Tu and G

    Proc. Natl. Acad. Sci. USA

    (1985)
  • R.N. Evans et al.

    The structure of LepA, the ribosomal back translocase

    Proc. Natl. Acad. Sci. USA

    (2008)
  • S.R. Connell et al.

    A new tRNA intermediate revealed on the ribosome during EF4-mediated back-translocation

    Nat. Struct. Mol. Biol.

    (2008)
  • M. Farris et al.

    BipA: a tyrosine-phosphorylated GTPase that mediates interactions between enteropathogenic Escherichia coli (EPEC) and epithelial cells

    Mol. Microbiol.

    (1998)
  • M.A. deLivron et al.

    A novel domain in translational GTPase BipA mediates interaction with the 70S ribosome and influences GTP hydrolysis

    Biochemistry

    (2009)
  • M.A. deLivron et al.

    Salmonella enterica serovar Typhimurium BipA exhibits two distinct ribosome binding modes

    J. Bacteriol.

    (2008)
  • A.J. Grant et al.

    Co-ordination of pathogenicity island expression by the BipA GTPase in enteropathogenic Escherichia coli (EPEC)

    Mol. Microbiol.

    (2003)
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