Decrypting the functional design of unmodified translation elongation factor P.

Bacteria overcome ribosome stalling by employing translation elongation factor P (EF-P), which requires post-translational modification (PTM) for its full activity. However, EF-Ps of the PGKGP subfamily are unmodified. The mechanism behind the ability to avoid PTM while retaining active EF-P requires further examination. Here, we investigate the design principles governing the functionality of unmodified EF-Ps in Escherichia coli. We screen for naturally unmodified EF-Ps with activity in E. coli and discover that the EF-P from Rhodomicrobium vannielii rescues growth defects of a mutant lacking the modification enzyme EF-P-(R)-ß-lysine ligase. We identify amino acids in unmodified EF-P that modulate its activity. Ultimately, we find that substitution of these amino acids in other marginally active EF-Ps of the PGKGP subfamily leads to fully functional variants in E. coli. These results provide strategies to improve heterologous expression of proteins with polyproline motifs in E. coli and give insights into cellular adaptations to optimize protein synthesis.

Proline codon pair selection determines ribosome pausing strength and translation efficiency in bacteria.

The speed of mRNA translation depends in part on the amino acid to be incorporated into the nascent chain. Peptide bond formation is especially slow with proline and two adjacent prolines can even cause ribosome stalling. While previous studies focused on how the amino acid context of a Pro-Pro motif determines the stalling strength, we extend this question to the mRNA level. Bioinformatics analysis of the Escherichia coli genome revealed significantly differing codon usage between single and consecutive prolines. We therefore developed a luminescence reporter to detect ribosome pausing in living cells, enabling us to dissect the roles of codon choice and tRNA selection as well as to explain the genome scale observations. Specifically, we found a strong selective pressure against CCC/U-C, a sequon causing ribosomal frameshifting even under wild-type conditions. On the other hand, translation efficiency as positive evolutionary driving force led to an overrepresentation of CCG. This codon is not only translated the fastest, but the corresponding prolyl-tRNA reaches almost saturating levels. By contrast, CCA, for which the cognate prolyl-tRNA amounts are limiting, is used to regulate pausing strength. Thus, codon selection both in discrete positions but especially in proline codon pairs can tune protein copy numbers.

Synthetic post-translational modifications of elongation factor P using the ligase EpmA.

Canonically, tRNA synthetases charge tRNA. However, the lysyl-tRNA synthetase paralog EpmA catalyzes the attachment of (R)-ß-lysine to the ε-amino group of lysine 34 of the translation elongation factor P (EF-P) in Escherichia coli. This modification is essential for EF-P-mediated translational rescue of ribosomes stalled at consecutive prolines. In this study, we determined the kinetics of EpmA and its variant EpmA_A298G to catalyze the post-translational modification of K34 in EF-P with eight noncanonical substrates. In addition, acetylated EF-P was generated using an amber suppression system. The impact of these synthetically modified EF-P variants on in vitro translation of a polyproline-containing NanoLuc luciferase reporter was analyzed. Our results show that natural (R)-ß-lysylation was more effective in rescuing stalled ribosomes than any other synthetic modification tested. Thus, our work not only provides new biochemical insights into the function of EF-P, but also opens a new route to post-translationally modify proteins using EpmA.

A set of rhamnosylation-specific antibodies enables detection of novel protein glycosylations in bacteria.

Despite its potential importance for bacterial virulence, protein rhamnosylation has not yet been sufficiently studied. Specific anti-SerRha, anti-ThrRha and anti-AsnRha antibodies allowed the identification of previously unknown monorhamnosylated proteins in cytosol and membrane fractions of bacterial cell lysates. Mapping of the complete rhamnoproteome in pathogens should facilitate development of targeted therapies against bacterial infections.

Switching the Post-translational Modification of Translation Elongation Factor EF-P.

Tripeptides with two consecutive prolines are the shortest and most frequent sequences causing ribosome stalling. The bacterial translation elongation factor P (EF-P) relieves this arrest, allowing protein biosynthesis to continue. A seven amino acids long loop between beta-strands ß3/ß4 is crucial for EF-P function and modified at its tip by lysylation of lysine or rhamnosylation of arginine. Phylogenetic analyses unveiled an invariant proline in the -2 position of the modification site in EF-Ps that utilize lysine modifications such as Escherichia coli. Bacteria with the arginine modification like Pseudomonas putida on the contrary have selected against it. Focusing on the EF-Ps from these two model organisms we demonstrate the importance of the ß3/ß4 loop composition for functionalization by chemically distinct modifications. Ultimately, we show that only two amino acid changes in E. coli EF-P are needed for switching the modification strategy from lysylation to rhamnosylation.

Exceptionally versatile - arginine in bacterial post-translational protein modifications.

Post-translational modifications (PTM) are the evolutionary solution to challenge and extend the boundaries of genetically predetermined proteomic diversity. As PTMs are highly dynamic, they also hold an enormous regulatory potential. It is therefore not surprising that out of the 20 proteinogenic amino acids, 15 can be post-translationally modified. Even the relatively inert guanidino group of arginine is subject to a multitude of mostly enzyme mediated chemical changes. The resulting alterations can have a major influence on protein function. In this review, we will discuss how bacteria control their cellular processes and develop pathogenicity based on post-translational protein-arginine modifications.

Comparison of the functional properties of trimeric and monomeric CaiT of Escherichia coli.

Secondary transporters exist as monomers, dimers or higher state oligomers. The significance of the oligomeric state is only partially understood. Here, the significance of the trimeric state of the L-carnitine/γ-butyrobetaine antiporter CaiT of Escherichia coli was investigated. Amino acids important for trimer stability were identified and experimentally verified. Among others, CaiT-D288A and -D288R proved to be mostly monomeric in detergent solution and after reconstitution into proteoliposomes, as shown by blue native gel electrophoresis, gel filtration, and determination of intermolecular distances. CaiT-D288A was fully functional with kinetic parameters similar to the trimeric wild-type. Significant differences in amount and stability in the cell membrane between monomeric and trimeric CaiT were not observed. Contrary to trimeric CaiT, addition of substrate had no or only a minor effect on the tryptophan fluorescence of monomeric CaiT. The results suggest that physical contacts between protomers are important for the substrate-induced changes in protein fluorescence and the underlying conformational alterations.

Structural Basis for EarP-Mediated Arginine Glycosylation of Translation Elongation Factor EF-P.

Glycosylation is a universal strategy to posttranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida The enzyme is composed of two opposing domains with Rossmann folds, thus constituting a B pattern-type glycosyltransferase (GT-B). While dTDP-ß-l-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the KOW-like N-domain of EF-P. Based on our data, we propose a structural model for arginine glycosylation by EarP. As EarP is essential for pathogenicity in P. aeruginosa, our study provides the basis for targeted inhibitor design.IMPORTANCE The structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP not only provides the first molecular insights into arginine glycosylation but also lays the basis for targeted-inhibitor design against Pseudomonas aeruginosa infection.

A Versatile Toolbox for the Control of Protein Levels Using Nε-Acetyl-l-lysine Dependent Amber Suppression.

The analysis of the function of essential genes in vivo depends on the ability to experimentally modulate levels of their protein products. Current methods to address this are based on transcriptional or post-transcriptional regulation of mRNAs, but approaches based on the exploitation of translation regulation have so far been neglected. Here we describe a toolbox, based on amber suppression in the presence of Nε-acetyl-l-lysine (AcK), for translational tuning of protein output. We chose the highly sensitive luminescence system LuxCDABE as a reporter and incorporated a UAG stop codon into the gene for the reductase subunit LuxC. The system was used to measure and compare the effects of AcK- and Nε-(tert-butoxycarbonyl)-l-lysine (BocK) dependent amber suppression in Escherichia coli. We also demonstrate here that, in combination with transcriptional regulation, the system allows protein production to be either totally repressed or gradually adjusted. To identify sequence motifs that provide improved translational regulation, we varied the sequence context of the amber codon and found that insertion of two preceding prolines drastically decreases luminescence. In addition, using LacZ as a reporter, we demonstrated that a strain encoding a variant with a Pro-Pro amber motif can only grow on lactose when AcK is supplied, thus confirming the tight translational regulation of protein output. In parallel, we constructed an E. coli strain that carries an isopropyl ß-d-1-thiogalactopyranoside (IPTG)-inducible version of the AcK-tRNA synthetase (AcKRS) gene on the chromosome, thus preventing mischarging of noncognate substrates. Subsequently, a diaminopimelic acid auxotrophic mutant (ΔdapA) was generated demonstrating the potential of this strain in regulating essential gene products. Furthermore, we assembled a set of vectors based on the broad-host-range pBBR ori that enable the AcK-dependent amber suppression system to control protein output not only in E. coli, but also in Salmonella enterica and Vibrio cholerae.

Resolving the α-glycosidic linkage of arginine-rhamnosylated translation elongation factor P triggers generation of the first ArgRha specific antibody.

A previously discovered posttranslational modification strategy - arginine rhamnosylation - is essential for elongation factor P (EF-P) dependent rescue of polyproline stalled ribosomes in clinically relevant species such as Pseudomonas aeruginosa and Neisseria meningitidis. However, almost nothing is known about this new type of N-linked glycosylation. In the present study we used NMR spectroscopy to show for the first time that the α anomer of rhamnose is attached to Arg32 of EF-P, demonstrating that the corresponding glycosyltransferase EarP inverts the sugar of its cognate substrate dTDP-ß-l-rhamnose. Based on this finding we describe the synthesis of an α-rhamnosylated arginine containing peptide antigen in order to raise the first anti-rhamnosyl arginine specific antibody (anti-ArgRha). Using ELISA and Western Blot analyses we demonstrated both its high affinity and specificity without any cross-reactivity to other N-glycosylated proteins. Having the anti-ArgRha at hand we were able to visualize endogenously produced rhamnosylated EF-P. Thus, we expect the antibody to be not only important to monitor EF-P rhamnosylation in diverse bacteria but also to identify further rhamnosyl arginine containing proteins. As EF-P rhamnosylation is essential for pathogenicity, our antibody might also be a powerful tool in drug discovery.