Elongation Factor P and the Control of Translation Elongation.

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Annu. Rev. Microbiol. 2017.71. Downloaded from www.annualreviews.org Access provided by Columbia University on 08/29/17. For personal use only.

Elongation Factor P and the Control of Translation Elongation Andrei Rajkovic1 and Michael Ibba1,2 1

Molecular, Cellular and Developmental Biology Program and Center for RNA Biology, Ohio State University, Columbus, Ohio 43210; email: [email protected]

2

Department of Microbiology, Ohio State University, Columbus, Ohio 43210

Annu. Rev. Microbiol. 2017. 71:117–31

Keywords

The Annual Review of Microbiology is online at micro.annualreviews.org

posttranslational modification, proline, protein synthesis, ribosome

https://doi.org/10.1146/annurev-micro-090816093629

Abstract

c 2017 by Annual Reviews. Copyright All rights reserved

Elongation factor P (EF-P) binds to ribosomes requiring assistance with the formation of oligo-prolines. In order for EF-P to associate with paused ribosomes, certain tRNAs with specific D-arm residues must be present in the peptidyl site, e.g., tRNAPro . Once EF-P is accommodated into the ribosome and bound to Pro-tRNAPro , productive synthesis of the peptide bond occurs. The underlying mechanism by which EF-P facilitates this reaction seems to have entropic origins. Maximal activity of EF-P requires a posttranslational modification in Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis. Each of these modifications is distinct and ligated onto its respective EF-P through entirely convergent means. Here we review the facets of translation elongation that are controlled by EF-P, with a particular focus on the purpose behind the many different modifications of EF-P.

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Contents


INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Controlling Translation Elongation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THE FUNCTION OF ELONGATION FACTOR P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Mechanism Behind EF-P–Assisted Catalysis of Prolyl Peptide Bond Synthesis Roles of EF-P Beyond Polyproline Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . POSTTRANSLATIONAL MODIFICATION OF EF-P . . . . . . . . . . . . . . . . . . . . . . . . . . . (R)-β-Lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L-Rhamnose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-Aminopentanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONCLUSIONS AND FUTURE OBJECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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INTRODUCTION Protein synthesis is an essential proce that creates the cellular machinery necessary for metabolism, motility, replication, and the cell’s architecture. At the nexus of protein synthesis lies the ribosome, a complex macromolecular machine composed of RNA and proteins (21). In bacteria, ribosomes are assembled on actively transcribed messenger RNA (mRNA). The sequences of mRNA contain the necessary features for initiating and concluding the production of a specific protein. Each protein is constructed from individual amino acids that are delivered by transfer RNAs (tRNAs), which recognize specific nucleotide triplets on the mRNA. Amino acids are connected together to produce a growing peptide chain during the phase of translation known as elongation. The average rate of elongation is about 20 amino acids per second in bacteria, but a closer examination into the factors influencing the velocity of protein synthesis reveals more complex control of translation elongation (38, 48).

Controlling Translation Elongation The rate of translation elongation can vary and depends on numerous factors. For instance, nutrient availability and quality, tRNA abundance, structural properties of the message being translated and the amino acids being incorporated all affect the rate of elongation to different degrees (7, 42, 46, 61). Changes in any of these parameters can have significant impacts with respect to the protein’s quantity, folding, and regulation. The result of a protein synthesis system that is dynamic provides the cell a means for sensing stress and, ultimately, generating responses that allow adaptation at the proteomic level. However, there are features intrinsic to translation that remain static, such as the comparatively slow rate of proline peptide bond formation (13, 58). When compared to other amino acids, proline has far fewer structural configurations it can sample because of the spatial arrangement of its pyrrolidine ring, which prevents flexibility across the phi/psi torsion angles (26). By its rigid nature, proline endows proteins with a variety of important structural features, but this comes at a kinetic cost during protein synthesis (62). Proline is the slowest of the proteogenic amino acids to form a peptide bond, especially when consecutive prolines are encountered during peptide bond formation (8, 13, 16, 22, 25, 46, 58, 59). However, the barrier to translate polyproline at a normal speed is overcome in the cell by translation elongation factor P (EF-P). 118

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tRNAPhe


70 Å

Elongation factor P

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Figure 1 Elongation factor P structurally mimics tRNA. The crystal structures of EF-P from Thermus thermophilus and tRNAPhe from the yeast Saccharomyces cerevisiae are represented as a surface and superimposed on each other. This figure was inspired by Reference 23.

Resembling tRNA in structure (Figure 1), EF-P binds between the peptidyl and exit sites of the ribosome, where it presumably guides proline into a favorable conformation, one conducive to forming peptide bonds. Optimal interaction between EF-P and the ribosome requires EF-P to be posttranslationally modified on a conserved basic residue that protrudes into the region of the ribosome known as the peptidyl-transferase center (PTC) (36, 51–53). Surprisingly, recent studies have revealed the existence of a variety of EF-P modification pathways that are distinct to the taxonomic classes within Proteobacteria and Firmicutes. Discovery of these modification pathways accounts for only 31% of all sequenced bacterial genomes, prompting questions, for example, as to whether the diversity of EF-P modifications extends beyond the three identified so far, and how these modifications achieve the same function of enhancing EF-P’s activity. The purpose of this review is to address the broad and nuanced impact of different EF-P modification systems within the confines of protein synthesis, to ultimately provide grounds for future investigations into the biology of EF-P.

THE FUNCTION OF ELONGATION FACTOR P Elongation factor P was discovered in the 1970s by the classic biochemical technique of fractionating cellular components and assaying these fractions for their ability to stimulate peptide bond formation (19). Specifically, the researchers sought an increase in the reaction speed between initiator-tRNA and the aminoacyl-tRNA analog, puromycin. Subsequent experiments tested a variety of other aminoacyl acceptors and determined smaller amino acids (leucine and glycine) depended more on EF-P to form the first peptide bond (2). From these biochemical data, EF-P was suggested to catalyze the start of translation initiation. Although a crystal structure of EF-P in complex with the Thermus thermophilus ribosome and initiator-tRNA was obtained, there www.annualreviews.org • Translation Elongation Factor P


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Diprolyl peptide bond formation EF-G Paused ribosomes

EF-Tu Vacant E-site Translocation


Actively elongating ribosomes Amino acid

Accelerated formation Elongation factor P

Figure 2 The role of elongation factor P (EF-P) in translation elongation. (a) The basic components required for translation elongation: tRNA, EF-Tu, amino acids, and the aminoacyl tRNA synthetases (aaRS). Cognate components are presented in light and dark shades of the same color. (b) General steps in translation elongation: EF-Tu delivers cognate tRNA to the acceptor site of the ribosome, where the ribosome decodes for the correct tRNA. After the tRNA is accommodated and forms a peptide bond, EF-G facilitates translocation of A-site tRNA into the P-site, to begin the next round of peptide bond formation. (c) EF-P (red ) rescuing translation elongation during an encounter of consecutive prolines (light orange).

remained a paucity of in vivo data to support the role of EF-P as a bona fide translation factor (5). The lack of in vivo data may in part have been due to efp having been assigned as an essential gene in Escherichia coli; this was later shown to be incorrect (2). More recently, several groups discovered a different role for EF-P and also demonstrated that efp is dispensable in a variety of organisms (4, 13, 58). There is now overwhelming evidence to suggest EF-P facilitates peptide bond formation when the ribosome encounters consecutive prolines, rather than EF-P only participating in the formation of the first peptide bond (Figure 2). Such evidence comes from kinetic experiments detailing the 100-fold rate increase EF-P provides during the synthesis of diprolyl peptide bonds, which can take 90 s to form without EF-P (13). Other compelling support comes from ribosome profiling data, where the in vivo impact EF-P has on protein synthesis was globally measured. Ribosome profiling identified regions where ribosomes failed to translate efficiently in efp strains; thus, the transcripts requiring EF-P for translation were revealed and a hierarchy of pausing motifs (PPX) delineated (16, 59). The culmination of these experiments ultimately explained the pleiotropy of phenotypes observed in E. coli efp strains, an organism with nearly 25% of genes containing diprolyl motifs (63). So far, there appears to be no clear indication as to why certain combinations of amino acids, when forming peptide bonds with diprolyl, exacerbate or enhance reactivity. For instance, 120

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ribosomes were found to accumulate at diprolyl motifs (PPX) to the greatest degree when attempting to form peptide bonds with tryptophan, asparagine, proline (Pro), lysine (Lys), glycine, and aspartate and to the least degree with leucine, methionine, tyrosine, and phenylalanine. There are no chemical or physical attributes shared between amino acids that strengthen the pause or that diminish it. However, taking tRNA copy number or, more accurately, intracellular tRNA abundance and decoding rates into account may provide a more complete picture as to why certain amino acid combinations take longer to form peptide bonds with proline (11). For example, E. coli has only one copy of the tryptophan tRNA gene, whereas it has eight copies of the methionine gene. By changing the tRNA copy number or varying the time it takes the ribosome to decode, the dependence of certain EF-P motifs can be tested to determine the rate-limiting determinants in the reaction, whether it be the tRNA abundance, decoding time, chemical and physical properties of the amino acid itself, or a combination of these features. The rate at which translation initiates also determines the extent to which synthesis of proteins containing a PPX motif depends on EF-P (24). Not all bacterial transcripts initiate translation with the canonical AUG codon. Instead, about 14% of E. coli K-12 genes use alternative start codons, namely GUG and, to a lesser degree, UUG (6). These less frequent sites of initiation result in substantially lower translation efficiency, potentially because of their weaker thermodynamic stability when binding to initiator tRNA (33). In ribosome profiling studies, the pause at PPX motifs had less of an effect in transcripts with slow initiation rates than in those with faster initiation rates. The observation was followed by in vivo studies using fluorescent reporters with PPX motifs, which confirmed that as the rate of initiation, and ribosome loading, decreased (because of a different start codon), the requirement for EF-P to alleviate pauses also decreased (24). The net effect is that for messages where initiation rates are low, ribosome binding is sufficiently rare that most stalled ribosomes will resume elongation before the next ribosome reaches the stall site (Figure 3). A bioinformatics search revealed 37% of genes initiating translation at GTG and TTG contain PPX motifs, whereas only 30% of genes with ATG for a start site contain PPX motifs.

The Mechanism Behind EF-P–Assisted Catalysis of Prolyl Peptide Bond Synthesis Ribosomes likely evolved without an intrinsic mechanism to rapidly incorporate oligo-prolines, or lost the means to robustly catalyze the reaction prior to the last common ancestor (17). Elongation factor P enhances the synthesis of polyproline in bacteria, possibly by helping peptidyl-tRNAPro navigate the terminal proline on the nascent chain into a favorable confirmation to form a peptide bond. The conditions required for EF-P to help rescue paused ribosomes are not completely understood, but it seems likely the ribosome must have both an unoccupied exit site and a peptidyl tRNA recognized by EF-P. The mechanism by which the ribosome is unlocked from this less active state involves EF-P increasing the entropy of the system (paused ribosome complex) to enable repositioning of the P-site Pro-tRNAPro (14). This is consistent with the physical properties of proline that prohibit adoption of certain configurations that most other amino acids can sample. Hence, EF-P provides the stalled complex with favorable entropy to compensate for prolines’ lack of flexibility. The reactions from which the thermodynamic terms were calculated used fluorinated analogs of proline that had different chirality. Therefore, the entropic contribution measured in the presence of EF-P could potentially also be due to differences in chirality. Chirality was shown to be important for the monofluorinated analogs; however, as the authors pointed out, such S and R configurations are not relevant to the proteogenic form of proline. Atomic resolution structures of ribosomes with a triproline peptide bound to a nonhydrolyzable Pro-tRNAPro analog, with and www.annualreviews.org • Translation Elongation Factor P


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High initiation rate

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Polyproline stretch Third round of initiation


Second round of initiation

Pause persists in absence of EF-P

EF-P enables more initiation events

EF-P rescue Pausing is rate limiting

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Rapid completion of the first translation event

Figure 3 The importance of EF-P when the initiation rate is varied. (a) A transcript with fewer translation initiation events is depicted when EF-P is absent (orange arrow) or present ( pink arrow). The white circle underneath the 70S ribosome indicates the same complex translating along the mRNA at different times. (b) A transcript with a higher frequency of translation initiation is represented with EF-P absent (red arrow) or present ( green arrow). Pink signifies a greater consequence with regards to the lack of EF-P when translation is at a higher initiation rate, compared to one with a lower initiation rate (orange).

without EF-P, will ultimately be required to advance this notion of favorable catalysis activated through entropic contributions.

Roles of EF-P Beyond Polyproline Synthesis The biochemical role of EF-P has been redefined through recent studies, and this in turn warrants a closer examination into whether EF-P actually stimulates the formation of the first peptide bond in vivo. One striking piece of evidence in favor of EF-P functioning at the start of translation is the crystal structure with EF-P bound to the ribosome and tRNAfMet . The structure clearly shows that the D-arm and CCA end of tRNAfMet make extensive contacts with EF-P; interestingly, tRNAfMet and tRNAPro from E. coli have identical D-arms. That observation was, in part, the motivation for a recent biochemical study that identified nucleotides C13, G12, G22, and C23 of tRNAPro and tRNAfMet as those required for EF-P activity (30). This study also reports that when tRNAfMet is charged with proline, it receives the same degree of assistance from EF-P to form a peptide bond with proline as Pro-tRNAPro does, but if tRNAfMet is charged with formyl-methionine, EF-P provides only a small enhancement when fMet-tRNAfMet reacts with puromycin. The results suggest EF-P discriminates paused ribosomes based on the identity of the tRNA in the P-site and could, under certain circumstances, participate in the formation of the first peptide bond.

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The conservation and distribution of tRNA EF-P recognition elements (tEREs) are unknown; however, the information to determine this could be retrieved from databases with curated tRNA sequences (9, 27). Knowing which tRNAs contain tEREs and which tRNAPro and tRNAfMet do not enables us to predict how EF-P operates in these organisms. For example, when the sequences of tRNAPro and tRNAfMet from Bacillus subtilis are contrasted with those from E. coli, the critical nucleotides for EF-P recognition are absent in B. subtilis tRNAPro but still present in tRNAfMet (34). Such observations might be misleading, since these differences in tRNAfMet and tRNAPro may have coevolved with EF-P in B. subtilis to achieve the same function that was characterized for E. coli EF-P. Therefore, it is also critical to compare the region of EF-P that interacts with the D-arm (residues 126 and 127 T. thermophilus numbering according to the crystal structure) in order to determine whether organisms with similar tERE profiles have similar EF-Ps (31). Such findings might give insight into the origin of EF-P’s function as having evolved to enhance the formation of the first peptide bond and later gained preference for tRNAPro , or vice versa. EF-P has been purported to prevent aberrant frameshifting during the first two rounds of peptide bond formation (18). In vivo frameshifting was monitored by using a lacZ construct modified with an in-frame homopolymeric cytosine sequence inserted after the start codon. In the absence of efp, a threefold decrease in lacZ activity compared to wild type was measured. However, the in vivo readout for frameshifting did not account for the decrease in translation capacity of efp strains, as two- to three fold reductions have been observed for non-EF-P targets when efp is absent (25). To mechanistically address the role of EF-P with regard to frameshifting, the authors then used an in vitro system and defined the kinetic parameters of the reaction. The in vitro experiment showed that in the presence of EF-P, the tripeptide MPV that resulted from a frameshift was produced very slowly. When EF-P was removed from the reaction, the formation of the frameshifting product occurred about 30-fold more readily. These kinetic rates conflict with prior studies, which showed EF-P to be required for rapid peptide bond formation only when certain proline amino acid combinations follow the second codon (MetProProGly, for example). Further evidence is now required to substantiate the physiological relevance of the in vitro data to determine whether EF-P also prevents frameshifting in vivo. EF-P has also been implicated in maintaining the coupling between transcription and translation in E. coli (15). By preventing translation pausing at polyproline stretches, EF-P allows the ribosome to closely follow the active RNA polymerase on a nascent mRNA, thus inhibiting the formation of elements that can lead to premature transcription termination such as intrinsic terminator hairpins or available Rho utilization (rut) sites. To assess uncoupling of translation from transcription in a efp E. coli strain, fluorescent reporters containing a polyproline motif placed upstream of either a Rho-dependent terminator or the intrinsic pyrL terminator hairpin were used. In both cases, a more than tenfold decrease in fluorescence was observed in the efp strain when the polyproline motif was present, whereas a 1.6-fold decrease was measured in the wild-type strain. These results support a role for EF-P in maintaining transcription-translation coupling. To further investigate the physiological relevance of this function for EF-P, two separate genes (narY and rsxC) that encode predicted intrinsic terminators within 500 base pairs downstream of PPX motifs were selected from among those whose transcript levels changed at least twofold between wild-type and efp strains. Premature termination of rsxC transcription was monitored in the efp strain using two hybridization probes complementary to sequences flanking the PPX and the predicted terminator, and a 1.8-fold increase in the before/after probe ratio was observed in the absence of EF-P. However, no significant differences were seen for narY. The results indicate that in certain cases, PPX pauses decrease the coupling efficiency, though it is also possible that pauses favor the formation of antiterminators. While these experiments support that EF-P

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EF-P

Hydroxylase

Lys34 (R)-β-Lysine Aminomutase

LysRS paralog

EF-P Arg32 L-Rhamnose

rmlABCD EarP


Lys32 Hypothetical pathway

5-Aminopentanol

Figure 4 Detailed EF-P posttranslational modification systems. (a) EF-P modification pathways with each of the components are represented on the left. Next to the pathway lies the respective chemical modification on EF-P (orange spheres, oxygen; gray spheres, carbon; purple spheres, nitrogen).

maintains coupling of translation and transcription, the number of genes dependent on EF-P to prevent decoupling seems to be fairly low.

POSTTRANSLATIONAL MODIFICATION OF EF-P The activity of EF-P is dependent on an augmentative posttranslational modification (PTM). At least three convergent EF-P posttranslational modification systems (EPMSs) have evolved across Gammaproteobacteria, Betaproteobacteria, and Firmicutes (Figures 4, 5) (36, 43, 47, 51–53, 60). Each EPMS constitutes the gene products involved in the synthesis and attachment of the modification. In all cases, EF-P is posttranslationally modified on a conserved basic residue that extends toward the peptidyl-transfer center of the ribosome, with the modification predicted to make contact with the CCA end of the tRNA. Two of the modifications have considerable structural similarity, while the third one deviates geometrically and chemically. The functional implications of these differences are not well understood, nor is it clear why so many EPMSs evolved in bacteria whereas in eukaryotes and archaea there is only one known modification pathway, and one variation on it, for the EF-P homologs EF5a (previously termed eIF5a) and aIF5a (12, 49). The following section describes in greater detail each modification and the potential reason for such diversity.

(R)-β-Lysine The first indication of a posttranslational modification on EF-P was from a partial amino acid sequencing experiment in the early 1990s (1). DNA sequencing data from E. coli EF-P predicted a lysine at position 31; however, the Edman degradation of EF-P did not identify a lysine at that position, a disparity that could have been explained by a posttranslational modification. The modification remained elusive for two more decades, possibly because of inaccuracies known to accumulate in Edman sequencing past 30 amino acids. 124

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≥ Pro3 per 1,000 amino acids

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Figure 5 Distribution of EF-P posttranslational modification systems across the bacterial taxonomic tree. Modification of EF-P convergently evolved in organisms with little correlation to polyproline abundance. The outermost circle represents the distribution of the poxA gene (aqua), the earP gene ( yellow), and Bacillus subtilis ( green). The innermost circle plots the number of three or more consecutive prolines found in a given genome divided by the total genomically encoded amino acids.



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Evidence to support a modified form of EF-P eventually came from a genetic screen that implicated three adjacent genes, efp, poxA (also referred to as epmA), and yjeK (also referred to as epmB) from Salmonella enterica, as responsible for nitric oxide resistance (43). The protein sequence of poxA matched that of a truncated lysyl-tRNA synthetase (LysRS), while the sequence of yjeK appeared to have homology to a 2,3-lysine aminomutase (3). Because structural analyses found that EF-P resembles tRNA (Figure 1) and that the poxA gene has strong homology with LysRS, aminoacylation reactions were performed with purified EF-P and PoxA. Initially, radiolabeled α-lysine was used as a substrate to modify EF-P on Lys34 (E. coli numbering), but the kinetics of the reactions appeared to be too slow to be physiologically relevant. However, sequence homology and phylogenetic studies predicted YjeK to convert α-lysine into β-lysine. Both chiral forms of β-lysine were tested in aminoacylation reactions, and (R)-β-lysine had the greatest efficiency by about 100-fold, compared to α-lysine and (S)-β-lysine (53). After addition of (R)-β-lysine, a subsequent hydroxylation event at position Lys34 is carried out by the hydroxylase yfcM (also referred to as epmC) (32). The role of this last reaction remains unclear, as there are no obvious consequences associated with removing the hydroxylation (47), although the formation of fMetpuromycin is stimulated by 1.5-fold (8). Hydroxylated EF-P’s enhanced reactivity for tRNAfMet toward puromycin may indicate either that an important hydrogen bond forms with the tRNA, or that the electronic configuration of lysine is slightly changed to stabilize the modification. The addition of (R)-β-lysine increases the observed KM of EF-P for paused ribosomes by 30-fold, possibly acting as an anchor to stabilize EF-P bound to P-site tRNA, although a more active role in controlling the electrostatic environment to enhance reactivity of prolyl-tRNA cannot be excluded.


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The posttranslational β-lysylation of Lys34 by PoxA is critical for EF-P activity in E. coli and S. enterica; however, the poxA gene is absent in 78% of sequenced bacteria. Based on the narrow distribution of poxA, discovery of an alternative modification pathway seemed inevitable, but it was somewhat surprising that three independent research groups ended up identifying the same alternative pathway, albeit in different organisms. Each group set out to investigate a particular efp encoding a conserved arginine (Arg) residue at a position equivalent to where β-Lys is attached to EF-P in E. coli (10, 36, 51). The most promising indication that efp was modified came from genomic neighborhood analysis. For the 9% of organisms with efp encoding Arg32, nearly all have a conserved predicted glycosyltransferase (earP) adjacent to efp (57). The metabolite substrate for EarP was not obvious, owing to a lack of homology with a specific glycosyltransferase. Therefore, mass spectrometry was necessary to identify the molecule attached on EF-P. The studies measured a single cyclic hexose moiety on the Arg32 residue of EF-P, when EF-P was isolated from Pseudomonas aeruginosa, Neisseria meningitidis, or Shewanella oneidensis. Since the mass spectrometry results were unable to distinguish between a fucose or a rhamnose molecule, two separate approaches were taken to clarify the ambiguity. When disrupted, a wellcharacterized dTDP-L-rhamnose modification pathway in P. aeruginosa resulted in the absence of modified EF-P. An additional pathway forms GDP-L-rhamnose, though EF-P was not affected by the inability to synthesize this molecule. Another study exploited the endogenous fucose sugar nucleotide biosynthesis pathway in E. coli, but S. oneidensis EF-P was only active when the dTDP-L-rhamnose biosynthesis pathway was introduced into E. coli. In vitro rhamnosylation assays established dTDP-L-rhamnose as the primary substrate for the posttranslational glycosylation of EF-P. The speed at which EF-P is rhamnosylated was not determined, since each study only performed endpoint reactions. If the kinetic parameters for EF-P 126

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rhamnosylation were to be measured, a comparison with the rate of β-lysylation could be made to provide a context for the relationship between metabolite selection and EF-P function. For example, (R)-β-lysine so far appears to be unique to EF-P, which may enable PoxA to be less kinetically efficient compared to LysRS, where there is more competition for α-lysine. Similar to α-lysine, the pool of dTDP-L-rhamnose is used by different enzymes to produce rhamnolipids, glycosylated flagellin, and posttranslationally modified EF-P (35, 39, 50, 54). Rhamnosylation of EF-P is, in contrast to β-lysylation, competing with a variety of other cellular processes that demand dTDP-L-rhamnose as a substrate. How the flow of metabolites is controlled by each of these processes could impart regulatory restrictions in order to effectively appropriate dTDP-L-rhamnose. When the function of efp was disrupted in the opportunistic pathogen P. aeruginosa, by deletion of either efp or earP, antibiotics targeting cell wall synthesis were observed in antibiograms to have a stronger potency. In addition to P. aeruginosa having greater susceptibility toward certain antibiotics, their ability to infect human cells was severely impeded, possibly because of the decrease in motility and rhamnolipid production. In contrast to the severe efp mutant phenotypes of P. aeruginosa, N. meningitidis is unable to survive without efp but is viable without earP. Therefore, posttranslational rhamnosylation of EF-P plays a key role in P. aeruginosa and N. meningitidis pathogenesis and could serve as a target for narrow-spectrum antibiotics (36, 51, 60).

5-Aminopentanol Discovery of EPMSs for P. aeruginosa and E. coli was simplified by the fact that genes neighboring efp in those organisms exhibited remarkable conservation. Furthermore, the genes to which they had homology made them promising targets to test for modifying EF-P. However, this cannot be taken as a generalized feature, since the majority of bacteria do not share such serendipity with respect to having clear targets for investigating new EPMSs. As an additional observation, the putative presence of rhamnosylation or β-lysylation EPMSs in a given genome was correlated with a relative enrichment in protein-coding genes containing three or more consecutive prolines (56). Based on this analysis, a dichotomy emerged whereby organisms encoding high numbers of polyprolines were predicted to require a modification, whereas those encoding few were not (37). For example, in Lactobacillus jensenii a modification that enhances EF-P function may not be required, as it has only one protein with three or more consecutive prolines (28). Instead, it is possible L. jensenii and other closely related species evolved an alternative strategy to deal with pausing. L. jensenii harbors two EF-Ps that share 47% sequence identity, suggesting that the role of a modification may perhaps have been replaced by having duplicate EF-Ps or that the two isoforms have different functions. Given that other modifications enhance EF-P’s affinity for the ribosome, increasing the intracellular concentration of EF-P could achieve a net effect similar to addition of a posttranslational modification. Organisms with genomes containing few polyprolines are provided with an opportunity to modulate the expression of a subset of proteins, whereas organisms that have a high abundance of polyprolines would presumably require EF-P to be fully functional under most conditions. This form of regulation could be controlled at the level of EF-P expression, such as in the case of L. jensenii, or by turning the modification pathway on under conditions that require proteins with polyprolines to be expressed, and turning the pathway off when the conditions no longer require their presence. B. subtilis has fourfold less polyproline motifs than E. coli, and tenfold less than P. aeruginosa. The only phenotype observed in the absence of efp is a defect in swarming motility, although multiple studies have also reported B. subtilis failing to sporulate when efp was disrupted by transposon www.annualreviews.org • Translation Elongation Factor P


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mutagenesis (41, 44). Further investigation into those studies revealed that reconstructed deletions of efp were unable to recapitulate the deficiency in sporulation, suggesting the sporulation genes surrounding efp of B. subtilis may have been inadvertently targeted during transposon mutagenesis. The lack of phenotypes observed when efp is deleted correlates well with the low abundance of consecutive prolines found in the B. subtilis genome; therefore, it was unexpected when structural mass spectrometry identified a 5-aminopentanol moiety attached to Lys32 of EF-P. Nevertheless, 5-aminopentanolylated EF-P is required for the efficient translation of a polyproline reporter and a few genes essential to swarming motility, thus challenging the widely held notion that polyproline abundance dictates the modification status of EF-P (52). Rather, the requirement for EF-P to be modified may have evolved from as yet unknown selective pressures alternative to the requirement for efficient polyproline synthesis.


CONCLUSIONS AND FUTURE OBJECTIVES A total of three chemically distinct, though functionally analogous modifications of EF-P have been discovered in separate bacteria. For two of the three modifications, the genes responsible for modifying EF-P have been well characterized, and phylogenetic distribution of these genes has been shown to be nonoverlapping, with the exception of Pseudohongiella spirulinae, Thalassolituus oleivorans MIL-1, and Nitrincola nitratireducens, which encode both rhamnosylation and β-lysylation modification pathways in their respective genomes (20, 45, 55). How and for what purpose multiple modification pathways exist in a single organism has yet to be explored, though previous studies have demonstrated the capacity for heterologous function of modification pathways (36). The putative distribution of known EF-P modification pathways is conserved in less than 31% of sequenced bacteria and is mostly confined to the Proteobacteria. It is tempting to speculate that within the other 69% of known bacteria more distinct EPMSs evolved, providing an even greater menagerie of EF-P posttranslational modifications. Future experiments will hopefully detail the rate of diprolyl synthesis with chimerically modified EF-Ps (e.g., rhamnosylated E. coli EF-P) to define the differences between each modification. One conclusion could be that each modification has the same impact on the velocity of diprolyl peptide bond formation, or the kinetic contributions made by the modifications are ranked in accordance with the demands of the proline pausing burden. Such a hypothesis has yet to be tested but could have important implications for the field of synthetic biology, which as of now requires new tools to be developed for rapid incorporation of nonstandard amino acids (29, 40). In the future customized EPMSs, and yet to be discovered modifications of EF-P, may provide new ways to modulate the chemistry of translation elongation.

DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS We thank R. Tollerson and A. Witzky for comments and helpful suggestions. Work in the authors’ lab on this topic was supported by the National Institutes of Health GM65183 (to M.I.) and an OSU Center for RNA Biology Fellowship (to A.R.). 128

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Maintenance of Transcription-Translation Coupling by Elongation Factor P.

Elongation factor P restricts Salmonella's growth by controlling translation of a Mg2+ transporter gene during infection.

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

Arginine-rhamnosylation as new strategy to activate translation elongation factor P.

Corrigendum: Arginine-rhamnosylation as new strategy to activate translation elongation factor P.

The translation elongation factor eEF1A1 couples transcription to translation during heat shock response.

Decoding the biosynthesis and function of diphthamide, an enigmatic modification of translation elongation factor 2 (EF2)

Effect of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 on translocation.

Neisseria meningitidis Translation Elongation Factor P and Its Active-Site Arginine Residue Are Essential for Cell Viability.

Isolation and sequence analysis of the gene for translation elongation factor 3 from Candida albicans.

Isolation and sequence analysis of the gene encoding translation elongation factor 3 from Candida albicans.

Independent overexpression of the subunits of translation elongation factor complex eEF1H in human lung cancer.

Sequence of a cDNA encoding the alpha-subunit of wheat translation elongation factor 1.

Cracking the control of RNA polymerase II elongation by 7SK snRNP and P-TEFb.

A conserved proline triplet in Val-tRNA synthetase and the origin of elongation factor P.

A novel mechanism for the control of translation of specific mRNAs by tumor suppressor protein Pdcd4: inhibition of translation elongation.

A new concept of the function of elongation factor 1 in peptid chain elongation.

Elongation factor-P at the crossroads of the host-endosymbiont interface.

Translation Elongation Factor eEF1A2 is a Novel Anticancer Target for the Marine Natural Product Plitidepsin.

Translation elongation factor 1-α gene as a potential taxonomic and identification marker in dermatophytes.

Translation Elongation Rate Measurement of Epstein-Barr Virus Strain GD1.

The RNA polymerase II elongation complex. Factor-dependent transcription elongation involves nascent RNA cleavage.

Correction: Translation Elongation Factor Tuf of Acinetobacter baumannii Is a Plasminogen-Binding Protein.

Elongation factor P and modifying enzyme PoxA are necessary for virulence of Shigella flexneri.