Hervé Roy

PoxA, YjeK, and elongation factor P coordinately modulate virulence and drug resistance in Salmonella enterica

We report an interaction between poxA, encoding a paralog of lysyl tRNA-synthetase, and the closely linked yjeK gene, encoding a putative 2,3-beta-lysine aminomutase, that is critical for virulence and stress resistance in Salmonella enterica. Salmonella poxA and yjeK mutants share extensive phenotypic pleiotropy, including attenuated virulence in mice, an increased ability to respire under nutrient-limiting conditions, hypersusceptibility to a variety of diverse growth inhibitors, and altered expression of multiple proteins, including several encoded on the SPI-1 pathogenicity island. PoxA mediates posttranslational modification of bacterial elongation factor P (EF-P), analogous to the modification of the eukaryotic EF-P homolog, eIF5A, with hypusine. The modification of EF-P is a mechanism of regulation whereby PoxA acts as an aminoacyl-tRNA synthetase that attaches an amino acid to a protein resembling tRNA rather than to a tRNA.

Post‐transfer editing in vitro and in vivo by the β subunit of phenylalanyl‐tRNA synthetase

Translation of the genetic code requires attachment of tRNAs to their cognate amino acids. Errors during amino‐acid activation and tRNA esterification are corrected by aminoacyl‐tRNA synthetase‐catalyzed editing reactions, as extensively described for aliphatic amino acids. The contribution of editing to aromatic amino‐acid discrimination is less well understood. We show that phenylalanyl‐tRNA synthetase misactivates tyrosine and that it subsequently corrects such errors through hydrolysis of tyrosyl‐adenylate and Tyr‐tRNAPhe. Structural modeling combined with an in vivo genetic screen identified the editing site in the B3/B4 domain of the β subunit, 40 Å from the active site in the α subunit. Replacements of residues within the editing site had no effect on Phe‐tRNAPhe synthesis, but abolished hydrolysis of Tyr‐tRNAPhein vitro. Expression of the corresponding mutants in Escherichia coli significantly slowed growth, and changed the activity of a recoded β‐galactosidase variant by misincorporating tyrosine in place of phenylalanine. This loss in aromatic amino‐acid discrimination in vivo revealed that editing by phenylalanyl‐tRNA synthetase is essential for faithful translation of the genetic code.

RNA-dependent lipid remodeling by bacterial multiple peptide resistance factors

Multiple peptide resistance (MprF) virulence factors control cellular permeability to cationic antibiotics by aminoacylating inner membrane lipids. It has been shown previously that one class of MprF can use Lys-tRNALys to modify phosphatidylglycerol (PG), but the mechanism of recognition and possible role of other MprFs are unknown. Here, we used an in vitro reconstituted lipid aminoacylation system to investigate the two phylogenetically distinct MprF paralogs (MprF1 and MprF2) found in the bacterial pathogen Clostridium perfringens. Although both forms of MprF aminoacylate PG, they do so with different amino acids; MprF1 is specific for Ala-tRNAAla, and MprF2 utilizes Lys-tRNALys. This provides a mechanism by which the cell can fine tune the charge of the inner membrane by using the neutral amino acid alanine, potentially providing resistance to a broader range of antibiotics than offered by lysine modification alone. Mutation of tRNAAla and tRNALys had little effect on either MprF activity, indicating that the aminoacyl moiety is the primary determinant for aminoacyl-tRNA recognition. The lack of discrimination of the tRNA is consistent with the role of MprF as a virulence factor, because species-specific differences in tRNA sequence would not present a barrier to horizontal gene transfer. Taken together, our findings reveal how the MprF proteins provide a potent virulence mechanism by which pathogens can readily acquire resistance to chemically diverse antibiotics.

Loss of Editing Activity during the Evolution of Mitochondrial Phenylalanyl-tRNA Synthetase

Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNAPhe. Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNAPhe, an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol.

When contemporary aminoacyl-tRNA synthetases invent their cognate amino acid metabolism

Faithful protein synthesis relies on a family of essential enzymes called aminoacyl-tRNA synthetases, assembled in a piecewise fashion. Analysis of the completed archaeal genomes reveals that all archaea that possess asparaginyl-tRNA synthetase (AsnRS) also display a second ORF encoding an AsnRS truncated from its anticodon binding-domain (AsnRS2). We show herein that Pyrococcus abyssi AsnRS2, in contrast to AsnRS, does not sustain asparaginyl-tRNAAsn synthesis but is instead capable of converting aspartic acid into asparagine. Functional analysis and complementation of an Escherichia coli asparagine auxotrophic strain show that AsnRS2 constitutes the archaeal homologue of the bacterial ammonia-dependent asparagine synthetase A (AS-A), therefore named archaeal asparagine synthetase A (AS-AR). Primary sequence- and 3D-based phylogeny shows that an archaeal AspRS ancestor originated AS-AR, which was subsequently transferred into bacteria by lateral gene transfer in which it underwent structural changes producing AS-A. This study provides evidence that a contemporary aminoacyl-tRNA synthetase can be recruited to sustain amino acid metabolism.

The tRNA synthetase paralog PoxA modifies elongation factor-P with (R)-β-lysine

The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with α-lysine at low efficiency. Cell-free extracts contained non-α-lysine substrates of PoxA that modified EF-P by a change in mass consistent with β–lysine, a substrate also predicted by genomic analyses. EF-P was efficiently, functionally, modified with (R)-β-lysine but not (S)-β-lysine or genetically encoded α-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases.

Resampling and Editing of Mischarged tRNA Prior to Translation Elongation

Faithful translation of the genetic code depends on the GTPase EF-Tu delivering correctly charged aminoacyl-tRNAs to the ribosome for pairing with cognate codons. The accurate coupling of cognate amino acids and tRNAs by the aminoacyl-tRNA synthetases is achieved through a combination of substrate specificity and product editing. Once released by aminoacyl-tRNA synthetases, both cognate and near-cognate aminoacyl-tRNAs were considered to be committed to ribosomal protein synthesis through their association with EF-Tu. Here we show instead that aminoacyl-tRNAs in ternary complex with EF-Tu*GTP can readily dissociate and rebind to aminoacyl-tRNA synthetases. For mischarged species, this allows resampling by the product editing pathway, leading to a reduction in the overall error rate of aminoacyl-tRNA synthesis. Resampling of mischarged tRNAs was shown to increase the accuracy of translation over ten fold during in vitro protein synthesis, supporting the presence of an additional quality control step prior to translation elongation.

Tuning the properties of the bacterial membrane with aminoacylated phosphatidylglycerol

The bacterial envelope is a semi‐permeable barrier that protects the cell from the hostilities of the environment. To survive the ever‐changing conditions of their surroundings, bacteria need to rapidly adjust the biochemical properties of their cellular envelope. Amino acid (aa) addition to phosphatidylglycerol (PG) of the membrane is one of the mechanisms used by bacteria to lower the net negative charge of their cellular envelope, thereby decreasing its affinity for several antibacterial agents such as the cationic antimicrobial peptides (CAMPs) produced by the innate immune response during host infection. This process requires the activity of an integral membrane protein, called aa‐PG synthase (aaPGS), to transfer the aa of aminoacyl‐tRNA (aa‐tRNA) onto the PG of the membrane. aaPGSs constitute a new family of virulence factors that are found in a wide range of microorganisms. aa‐PGs not only provide resistance to CAMPs but also to other classes of antibacterial agents and to environmental stresses such as those encountered during extreme osmotic or acidic conditions. This review will describe the known biochemical properties of aa‐PGSs, their specificity for aa‐tRNAs and phospholipids, and the growing repertoire of aa used as substrates by these enzymes. Their prevalence in bacteria and the phenotypes and modulations of membrane properties associated with these molecules will be addressed, as well as their regulation as a component of the envelope stress response system in certain bacteria.

Mechanism of tRNA-dependent editing in translational quality control

Protein synthesis requires the pairing of amino acids with tRNAs catalyzed by the aminoacyl-tRNA synthetases. The synthetases are highly specific, but errors in amino acid selection are occasionally made, opening the door to inaccurate translation of the genetic code. The fidelity of protein synthesis is maintained by the editing activities of synthetases, which remove noncognate amino acids from tRNAs before they are delivered to the ribosome. Although editing has been described in numerous synthetases, the reaction mechanism is unknown. To define the mechanism of editing, phenylalanyl-tRNA synthetase was used to investigate different models for hydrolysis of the noncognate product Tyr-tRNAPhe. Deprotonation of a water molecule by the highly conserved residue βHis-265, as proposed for threonyl-tRNA synthetase, was excluded because replacement of this and neighboring residues had little effect on editing activity. Model building suggested that, instead of directly catalyzing hydrolysis, the role of the editing site is to discriminate and properly position noncognate substrate for nucleophilic attack by water. In agreement with this model, replacement of certain editing site residues abolished substrate specificity but only reduced the catalytic efficiency of hydrolysis 2- to 10-fold. In contrast, substitution of the 3′-OH group of tRNAPhe severely impaired editing and revealed an essential function for this group in hydrolysis. The phenylalanyl-tRNA synthetase editing mechanism is also applicable to threonyl-tRNA synthetase and provides a paradigm for synthetase editing.

Cell-specific differences in the requirements for translation quality control

Protein synthesis has an overall error rate of approximately 10-4 for each mRNA codon translated. The fidelity of translation is mainly determined by two events: synthesis of cognate amino acid:tRNA pairs by aminoacyl-tRNA synthetases (aaRSs) and accurate selection of aminoacyl-tRNAs (aa-tRNAs) by the ribosome. To ensure faithful aa-tRNA synthesis, many aaRSs employ a proofreading (“editing”) activity, such as phenylalanyl-tRNA synthetases (PheRS) that hydrolyze mischarged Tyr-tRNAPhe. Eukaryotes maintain two distinct PheRS enzymes, a cytoplasmic (ctPheRS) and an organellar form. CtPheRS is similar to bacterial enzymes in that it consists of a heterotetramer in which the α-subunits contain the active site and the β-subunits harbor the editing site. In contrast, mitochondrial PheRS (mtPheRS) is an α-subunit monomer that does not edit Tyr-tRNAPhe, and a comparable transacting activity does not exist in organelles. Although mtPheRS does not edit, it is extremely specific as only one Tyr-tRNAPhe is synthesized for every ∼7,300 Phe-tRNAPhe, compatible with an error rate in translation of ∼10-4. When the error rate of mtPheRS was increased 17-fold, the corresponding strain could not grow on respiratory media and the mitochondrial genome was rapidly lost. In contrast, error-prone mtPheRS, editing-deficient ctPheRS, and their wild-type counterparts all supported cytoplasmic protein synthesis and cell growth. These striking differences reveal unexpectedly divergent requirements for quality control in different cell compartments and suggest that the limits of translational accuracy may be largely determined by cellular physiology.