Ingo Wohlgemuth

EF-P Is Essential for Rapid Synthesis of Proteins Containing Consecutive Proline Residues

Translating Polyproline Translation of messenger RNA into protein is carried out by the ribosome, together with a variety of accessory factors, which offer the potential for regulation of this critical step in gene expression (see the Perspective by Buskirk and Green). Ude et al. (p. 82, published online 13 December), using bacterial genetics and an in vitro reconstituted translation system, and Doerfel et al. (p. 85, published online 13 December), using a model assay for peptide bond formation, find that the universally conserved bacterial elongation factor P (EF-P) (which is orthologous to the archaeal and eukaryotic initiation factor 5A) is required for the efficient translation of polyproline-containing polypeptides. Such short polyproline stretches (with runs of two, three, or more proline residues) would otherwise cause ribosomal stalling. A universally conserved translation factor facilitates synthesis of peptides that would otherwise cause ribosome stalling. [Also see Perspective by Buskirk and Green] Elongation factor P (EF-P) is a translation factor of unknown function that has been implicated in a great variety of cellular processes. Here, we show that EF-P prevents ribosome from stalling during synthesis of proteins containing consecutive prolines, such as PPG, PPP, or longer proline strings, in natural and engineered model proteins. EF-P promotes peptide-bond formation and stabilizes the peptidyl–transfer RNA in the catalytic center of the ribosome. EF-P is posttranslationally modified by a hydroxylated β-lysine attached to a lysine residue. The modification enhances the catalytic proficiency of the factor mainly by increasing its affinity to the ribosome. We propose that EF-P and its eukaryotic homolog, eIF5A, are essential for the synthesis of a subset of proteins containing proline stretches in all cells.

Modulation of the rate of peptidyl transfer on the ribosome by the nature of substrates.

The ribosome catalyzes peptide bond formation between peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Here, we show that the nature of the C-terminal amino acid residue in the P-site peptidyl-tRNA strongly affects the rate of peptidyl transfer. Depending on the C-terminal amino acid of the peptidyl-tRNA, the rate of reaction with the small A-site substrate puromycin varied between 100 and 0.14 s–1, regardless of the tRNA identity. The reactivity decreased in the order Lys = Arg > Ala > Ser > Phe = Val > Asp ≫ Pro, with Pro being by far the slowest. However, when Phe-tRNAPhe was used as A-site substrate, the rate of peptide bond formation with any peptidyl-tRNA was ∼7 s–1, which corresponds to the rate of binding of Phe-tRNAPhe to the A site (accommodation). Because accommodation is rate-limiting for peptide bond formation, the reaction rate is uniform for all peptidyl-tRNAs, regardless of the variations of the intrinsic chemical reactivities. On the other hand, the 50-fold increase in the reaction rate for peptidyl-tRNA ending with Pro suggests that full-length aminoacyl-tRNA in the A site greatly accelerates peptide bond formation.

Evolutionary optimization of speed and accuracy of decoding on the ribosome

Speed and accuracy of protein synthesis are fundamental parameters for the fitness of living cells, the quality control of translation, and the evolution of ribosomes. The ribosome developed complex mechanisms that allow for a uniform recognition and selection of any cognate aminoacyl-tRNA (aa-tRNA) and discrimination against any near-cognate aa-tRNA, regardless of the nature or position of the mismatch. This review describes the principles of the selection—kinetic partitioning and induced fit—and discusses the relationship between speed and accuracy of decoding, with a focus on bacterial translation. The translational machinery apparently has evolved towards high speed of translation at the cost of fidelity.

Optimization of speed and accuracy of decoding in translation

The speed and accuracy of protein synthesis are fundamental parameters for understanding the fitness of living cells, the quality control of translation, and the evolution of ribosomes. In this study, we analyse the speed and accuracy of the decoding step under conditions reproducing the high speed of translation in vivo. We show that error frequency is close to 10−3, consistent with the values measured in vivo. Selectivity is predominantly due to the differences in kcat values for cognate and near‐cognate reactions, whereas the intrinsic affinity differences are not used for tRNA discrimination. Thus, the ribosome seems to be optimized towards high speed of translation at the cost of fidelity. Competition with near‐ and non‐cognate ternary complexes reduces the rate of GTP hydrolysis in the cognate ternary complex, but does not appreciably affect the rate‐limiting tRNA accommodation step. The GTP hydrolysis step is crucial for the optimization of both the speed and accuracy, which explains the necessity for the trade‐off between the two fundamental parameters of translation.

Entropic Contribution of Elongation Factor P to Proline Positioning at the Catalytic Center of the Ribosome

The peptide bond formation with the amino acid proline (Pro) on the ribosome is slow, resulting in translational stalling when several Pro have to be incorporated into the peptide. Stalling at poly-Pro motifs is alleviated by the elongation factor P (EF-P). Here we investigate why Pro is a poor substrate and how EF-P catalyzes the reaction. Linear free energy relationships of the reaction on the ribosome and in solution using 12 different Pro analogues suggest that the positioning of Pro-tRNA in the peptidyl transferase center is the major determinant for the slow reaction. With any Pro analogue tested, EF-P decreases the activation energy of the reaction by an almost uniform value of 2.5 kcal/mol. The main source of catalysis is the favorable entropy change brought about by EF-P. Thus, EF-P acts by entropic steering of Pro-tRNA toward a catalytically productive orientation in the peptidyl transferase center of the ribosome.

Rapid peptide bond formation on isolated 50S ribosomal subunits.

The catalytic site of the ribosome, the peptidyl transferase centre, is located on the large (50S in bacteria) ribosomal subunit. On the basis of results obtained with small substrate analogues, isolated 50S subunits seem to be less active in peptide bond formation than 70S ribosomes by several orders of magnitude, suggesting that the reaction mechanisms on 50S subunits and 70S ribosomes may be different. Here we show that with full‐size fMet‐tRNAfMet and puromycin or C‐puromycin as peptide donor and acceptor substrates, respectively, the reaction proceeds as rapidly on 50S subunits as on 70S ribosomes, indicating that the intrinsic activity of 50S subunits is not different from that of 70S ribosomes. The faster reaction on 50S subunits with fMet‐tRNAfMet, compared with oligonucleotide substrate analogues, suggests that full‐size transfer RNA in the P site is important for maintaining the active conformation of the peptidyl transferase centre.

An Uncharged Amine in the Transition State of the Ribosomal Peptidyl Transfer Reaction

The ribosome has an active site comprised of RNA that catalyzes peptide bond formation. To understand how RNA promotes this reaction requires a detailed understanding of the chemical transition state. Here, we report the Brønsted coefficient of the alpha-amino nucleophile with a series of puromycin derivatives. Both 50S subunit- and 70S ribosome-catalyzed reactions displayed linear free-energy relationships with slopes close to zero under conditions where chemistry is rate limiting. These results indicate that, at the transition state, the nucleophile is neutral in the ribosome-catalyzed reaction, in contrast to the substantial positive charge reported for typical uncatalyzed aminolysis reactions. This suggests that the ribosomal transition state involves deprotonation to a degree commensurate with nitrogen-carbon bond formation. Such a transition state is significantly different from that of uncatalyzed aminolysis reactions in solution.

Evolution of the protein stoichiometry in the L12 stalk of bacterial and organellar ribosomes

The emergence of ribosomes and translation factors is central for understanding the origin of life. Recruitment of translation factors to bacterial ribosomes is mediated by the L12 stalk composed of protein L10 and several copies of protein L12, the only multi-copy protein of the ribosome. Here we predict stoichiometries of L12 stalk for >1,200 bacteria, mitochondria and chloroplasts by a computational analysis, and validate the predictions by quantitative mass spectrometry. The majority of bacteria have L12 stalks allowing for binding of four or six copies of L12, largely independent of the taxonomic group or living conditions of the bacteria, whereas some cyanobacteria have eight copies. Mitochondrial and chloroplast ribosomes can accommodate six copies of L12. The last universal common ancestor probably had six molecules of L12 molecules bound to L10. Changes of the stalk composition provide a unique possibility to trace the evolution of protein components of the ribosome.

Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.

Essential structural elements in tRNAPro for EF-P-mediated alleviation of translation stalling.

The ribosome stalls on translation of polyproline sequences due to inefficient peptide bond formation between consecutive prolines. The translation factor EF-P is able to alleviate this stalling by accelerating Pro-Pro formation. However, the mechanism by which EF-P recognizes the stalled complexes and accelerates peptide bond formation is not known. Here, we use genetic code reprogramming through a flexible in-vitro translation (FIT) system to investigate how mutations in tRNAPro affect EF-P function. We show that the 9-nt D-loop closed by the stable D-stem sequence in tRNAPro is a crucial recognition determinant for EF-P. Such D-arm structures are shared only among the tRNAPro isoacceptors and tRNAfMet in Escherichia coli, and the D-arm of tRNAfMet is essential for EF-P-induced acceleration of fMet–puromycin formation. Thus, the activity of EF-P is controlled by recognition elements in the tRNA D-arm.