Elongation factor P is required to maintain proteome homeostasis at high growth rate

Elongation factor P (EF-P) is a universally conserved translation factor that alleviates ribosome pausing at polyproline (PPX) motifs by facilitating peptide bond formation. In the absence of EF-P, PPX peptide bond formation can limit translation rate, leading to pleotropic phenotypes including slowed growth, increased antibiotic sensitivity, and loss of virulence. In this study, we observe that many of these phenotypes are dependent on growth rate. Limiting growth rate suppresses a variety of detrimental phenotypes associated with ribosome pausing at PPX motifs in the absence of EF-P. Polysome levels are also similar to wild-type under slow growth conditions, consistent with global changes in ribosome queuing in cells without EF-P when growth rate is decreased. Inversely, under high protein synthesis demands, we observe that Escherichia coli lacking EF-P have reduced fitness. Our data demonstrate that EF-P-mediated relief of ribosome queuing is required to maintain proteome homeostasis under conditions of high translational demands.

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Elongation Factor P Interactions with the Ribosome Are Independent of Pausing

mBio ◽

10.1128/mbio.01056-17 ◽

2017 ◽

Vol 8 (4) ◽

Cited By ~ 2

Author(s):

Rodney Tollerson ◽

Anne Witzky ◽

Michael Ibba

Keyword(s):

Single Molecule ◽

Bond Formation ◽

Elongation Factor ◽

Peptide Bond ◽

Cellular Distribution ◽

Peptide Bond Formation ◽

Translation Elongation ◽

Single Molecule Tracking ◽

Localization Pattern ◽

Elongation Factor P

ABSTRACT Bacterial elongation factor P (EF-P) plays a pivotal role in the translation of polyproline motifs. To stimulate peptide bond formation, EF-P must enter the ribosome via an empty E-site. Using fluorescence-based single-molecule tracking, Mohapatra et al. (S. Mohapatra, H. Choi, X. Ge, S. Sanyal, and J. C. Weisshaar, mBio 8:e00300-17, 2017, https://doi.org/10.1128/mBio.00300-17 !) monitored the cellular distribution of EF-P and quantified the frequency of association between EF-P and the ribosome under various conditions. Findings from the study showed that EF-P has a localization pattern that is strikingly similar to that of ribosomes. Intriguingly, EF-P was seen to bind ribosomes more frequently than the estimated number of pausing events, indicating that E-site vacancies occur even when ribosomes are not paused. The study provides new insights into the mechanism of EF-P-dependent peptide bond formation and the intricacies of translation elongation.

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Irreversible chemical steps control intersubunit dynamics during translation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0805299105 ◽

2008 ◽

Vol 105 (40) ◽

pp. 15364-15369 ◽

Cited By ~ 82

Author(s):

R. Andrew Marshall ◽

Magdalena Dorywalska ◽

Joseph D. Puglisi

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Large Scale ◽

Bond Formation ◽

Elongation Factor ◽

Resonance Energy ◽

Peptide Bond ◽

Peptide Bond Formation ◽

Gtp Hydrolysis ◽

30S Subunit

The ribosome, a two-subunit macromolecular machine, deciphers the genetic code and catalyzes peptide bond formation. Dynamic rotational movement between ribosomal subunits is likely required for efficient and accurate protein synthesis, but direct observation of intersubunit dynamics has been obscured by the repetitive, multistep nature of translation. Here, we report a collection of single-molecule fluorescence resonance energy transfer assays that reveal a ribosomal intersubunit conformational cycle in real time during initiation and the first round of elongation. After subunit joining and delivery of correct aminoacyl-tRNA to the ribosome, peptide bond formation results in a rapid conformational change, consistent with the counterclockwise rotation of the 30S subunit with respect to the 50S subunit implied by prior structural and biochemical studies. Subsequent binding of elongation factor G and GTP hydrolysis results in a clockwise rotation of the 30S subunit relative to the 50S subunit, preparing the ribosome for the next round of tRNA selection and peptide bond formation. The ribosome thus harnesses the free energy of irreversible peptidyl transfer and GTP hydrolysis to surmount activation barriers to large-scale conformational changes during translation. Intersubunit rotation is likely a requirement for the concerted movement of tRNA and mRNA substrates during translocation.

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

Science ◽

10.1126/science.1229017 ◽

2012 ◽

Vol 339 (6115) ◽

pp. 85-88 ◽

Cited By ~ 283

Author(s):

Lili K. Doerfel ◽

Ingo Wohlgemuth ◽

Christina Kothe ◽

Frank Peske ◽

Henning Urlaub ◽

...

Keyword(s):

Unknown Function ◽

Bond Formation ◽

Elongation Factor ◽

Peptide Bond ◽

Transfer Rna ◽

Peptide Bond Formation ◽

Rapid Synthesis ◽

Catalytic Center ◽

Cellular Processes ◽

Peptidyl Transfer

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.

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Molecular Functions of Conserved Developmentally-Regulated GTP-Binding Protein Drg1 in Translation

10.1101/2020.07.06.190082 ◽

2020 ◽

Author(s):

Fuxing Zeng ◽

Melissa Pires-Alves ◽

Christopher W. Hawk ◽

Xin Chen ◽

Hong Jin

Keyword(s):

Elongation Factor ◽

Peptide Bond ◽

Peptide Bond Formation ◽

Developmentally Regulated ◽

Single Particle Reconstruction ◽

Peptidyl Transfer ◽

Gtp Binding ◽

Processing Pathways ◽

Trna Translocation ◽

Ribosome Pausing

SUMMARYDevelopmentally-regulated GTP-binding (Drg) proteins are important for embryonic development, cell growth, proliferation, and differentiation. Despite their highly conserved nature, the functions of Drg proteins in translation are unknown. Here, we demonstrate the yeast Drg ortholog, Rbg1, alleviates ribosome pausing at Arginine/Lysine-rich regions in mRNAs, and mainly targets genes related to ribonucleoprotein complex biogenesis and non-coding RNA processing pathways. Furthermore, we reveal the global architecture of the ribosome and the molecular interactions involved when Rbg1 and its binding partner, Tma46, associate with the ribosome using biochemistry and single particle reconstruction using cryoEM. Our data show that Rbg1/Tma46 associate with the larger subunit of ribosome via the N-terminal zinc finger domain in Tma46, and that the protein complex helps to enrich translating ribosomes in the post-peptidyl transfer state, after peptide-bond formation, but before elongation factor binding and tRNA translocation. Based on our results and the conserved nature of Drg proteins, broader functions of the Drg proteins in the protein synthesis and quality control pathways of eukaryotic cells are proposed.

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Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904469117 ◽

2020 ◽

Vol 117 (7) ◽

pp. 3610-3620 ◽

Cited By ~ 4

Author(s):

Justin C. Morse ◽

Dylan Girodat ◽

Benjamin J. Burnett ◽

Mikael Holm ◽

Roger B. Altman ◽

...

Keyword(s):

Protein Synthesis ◽

Ternary Complex ◽

Bond Formation ◽

Critical Role ◽

Ribosomal Subunit ◽

Elongation Factor ◽

Peptide Bond ◽

Elongation Factor Tu ◽

Peptide Bond Formation ◽

Gtp Hydrolysis

The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu⋅GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tu⋅GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.

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Translation elongation factor P (EF-P)

FEMS Microbiology Reviews ◽

10.1093/femsre/fuaa003 ◽

2020 ◽

Vol 44 (2) ◽

pp. 208-218 ◽

Cited By ~ 1

Author(s):

Katherine R Hummels ◽

Daniel B Kearns

Keyword(s):

Elongation Factor ◽

Peptide Bond ◽

Post Translational Modification ◽

Translation Elongation Factor ◽

Translation Elongation ◽

Isolation And Characterization ◽

Domains Of Life ◽

Ribosome Pausing ◽

Elongation Factor P

ABSTRACT Translation elongation factor P (EF-P) is conserved in all three domains of life (called eIF5A and aIF5A in eukaryotes and archaea, respectively) and functions to alleviate ribosome pausing during the translation of specific sequences, including consecutive proline residues. EF-P was identified in 1975 as a factor that stimulated the peptidyltransferase reaction in vitro but its involvement in the translation of tandem proline residues was not uncovered until 2013. Throughout the four decades of EF-P research, perceptions of EF-P function have changed dramatically. In particular, while EF-P was thought to potentiate the formation of the first peptide bond in a protein, it is now broadly accepted to act throughout translation elongation. Further, EF-P was initially reported to be essential, but recent work has shown that the requirement of EF-P for growth is conditional. Finally, it is thought that post-translational modification of EF-P is strictly required for its function but recent studies suggest that EF-P modification may play a more nuanced role in EF-P activity. Here, we review the history of EF-P research, with an emphasis on its initial isolation and characterization as well as the discoveries that altered our perceptions of its function.

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

Biochemical Journal ◽

10.1042/bj1560015 ◽

1976 ◽

Vol 156 (1) ◽

pp. 15-23 ◽

Cited By ~ 10

Author(s):

L Montanaro ◽

S Sperti ◽

G Testoni ◽

A Mattioli

Keyword(s):

Rat Liver ◽

Bond Formation ◽

Elongation Factor ◽

Adenosine Diphosphate ◽

Peptide Bond ◽

Native Enzyme ◽

Peptide Bond Formation ◽

Elongation Factor 2 ◽

Hydrolysis Of ◽

Liver Ribosomes

1. The effect of elongation factor 2 (EF 2) and of adenosine diphosphate-ribosylated elongation factor 2 (ADP-ribosyl-EF 2) on the shift of endogenous peptidyl-tRNA from the A to the P site of rat liver ribosomes (measured by the peptidyl-puromycin reaction) and on the release of deacylated tRNA (measured by aminoacylation) was investigated. 2. Limiting amounts of EF2, pre-bound or added to ribosomes, catalyse the shift of peptidyl-tRNA in the presence of GPT; when the enzyme is added in substrate amounts GMP-P(CH2)P [guanosine (beta, gamma-methylene)triphosphate] can partially replace GTP. ADP-ribosyl-EF 2 has no effect on the shift of peptidyl-tRNA when present in catalytic amounts, but becomes almost as effective as EF 2 when added in substrate amounts together with GTP; GMP-P(CH2)P cannot replace GTP. 3. The release of deacylated tRNA is induced only by substrate amounts of added EF 2 and also occurs in the absence of guanine nucleotides. In this reaction ADP-ribosyl-EF 2 is only 25% as effective as EF 2 in the absence of added nucleotide, but becomes 60-80% as effective in the presence of GTP or GMP-P(CH2)P. 4. The results obtained on protein-synthesizing systems are consistent with the hypothesis that ADP-ribosyl-EF 2 can operate a single round of translocation followed by binding of aminoacyl-tRNA and peptide-bond formation. 5. From the data obtained with the native enzyme it is concluded that the two moments of translocation require different conditions of interaction of EF 2 with ribosomes; it is suggested that the shift of peptidyl-tRNA is catalysed by EF 2 pre-bound to ribosomes, and that the release of tRNA is induced by a second molecule of interacting EF 2. The hydrolysis of GTP would be required for the release of pre-bound EF 2 from ribosomes. 5. The inhibition of the utilization of limiting amounts of EF 2 on ADP-ribosylation is very likely the consequence of a concomitant decrease in the rate of association and dissociation of the enzyme from ribosomes.

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