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

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.

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

Science ◽  
2012 ◽  
Vol 339 (6115) ◽  
pp. 85-88 ◽  
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.

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

2018 ◽  
Vol 115 (43) ◽  
pp. 11072-11077 ◽  
Author(s):  
Rodney Tollerson ◽  
Anne Witzky ◽  
Michael Ibba
Keyword(s):  
Growth Rate ◽  
Bond Formation ◽  
Elongation Factor ◽  
High Growth ◽  
Peptide Bond ◽  
Global Changes ◽  
Peptide Bond Formation ◽  
Translation Rate ◽  
Ribosome Pausing ◽  
Elongation Factor P

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.

scholarly journals pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA

2010 ◽  
Vol 108 (1) ◽  
pp. 79-84 ◽  
Author(s):  
Magnus Johansson ◽  
Ka-Weng Ieong ◽  
Stefan Trobro ◽  
Peter Strazewski ◽  
Johan Åqvist ◽  
...  
Keyword(s):  
Ph Value ◽  
Ph Dependence ◽  
Peptide Bond ◽  
Bulk Solution ◽  
Peptide Bond Formation ◽  
Transfer Rates ◽  
Peptidyl Transfer ◽  
A Site ◽  
Rate Limiting ◽  
Site Binding

We studied the pH-dependence of ribosome catalyzed peptidyl transfer from fMet-tRNAfMet to the aa-tRNAs Phe-tRNAPhe, Ala-tRNAAla, Gly-tRNAGly, Pro-tRNAPro, Asn-tRNAAsn, and Ile-tRNAIle, selected to cover a large range of intrinsic pKa-values for the α-amino group of their amino acids. The peptidyl transfer rates were different at pH 7.5 and displayed different pH-dependence, quantified as the pH-value, , at which the rate was half maximal. The -values were downshifted relative to the intrinsic pKa-value of aa-tRNAs in bulk solution. Gly-tRNAGly had the smallest downshift, while Ile-tRNAIle and Ala-tRNAAla had the largest downshifts. These downshifts correlate strongly with molecular dynamics (MD) estimates of the downshifts in pKa-values of these aa-tRNAs upon A-site binding. Our data show the chemistry of peptide bond formation to be rate limiting for peptidyl transfer at pH 7.5 in the Gly and Pro cases and indicate rate limiting chemistry for all six aa-tRNAs.

scholarly journals Irreversible chemical steps control intersubunit dynamics during translation

2008 ◽  
Vol 105 (40) ◽  
pp. 15364-15369 ◽  
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.

scholarly journals Elongation Factor P Interactions with the Ribosome Are Independent of Pausing

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
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.

scholarly journals Effect of modeccin on the steps of peptide-chain elongation

1978 ◽  
Vol 176 (2) ◽  
pp. 371-379 ◽  
Author(s):  
L Montanaro ◽  
S Sperti ◽  
M Zamboni ◽  
M Denaro ◽  
G Testoni ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Peptide Bond ◽  
Inhibitory Effect ◽  
Artemia Salina ◽  
Chain Elongation ◽  
Peptide Bond Formation ◽  
Experimental Conditions ◽  
Related Plant ◽  
The Individual

Modeccin inhibits polypeptide-chain elongation catalysed by Artemia salina (brine shrimp) ribosomes by inactivating the 60 S ribosomal subunit. Among the individual steps of elongation, peptide-bond formation, catalysed by 60 S peptidyltransferase, is unaffected by the toxin, whereas the binding of EF 2 (elongation factor 2) to ribosomes is strongly inhibited. Modeccin does not affect the poly(U)-dependent non-enzymic binding of either deacylated tRNAPhe or phenylalanyl-tRNA to ribosomes. The inhibitory effect of modeccin on the EF 1 (elongation factor 1)-dependent binding of phenylalanyl-tRNA is discussed, since it is decreased by tRNAPhe, which stimulates the binding reaction. The analysis of the distribution of ribosome-bound radioactivity during protein synthesis shows that modeccin consistently inhibits the radioactivity bound as long-chain peptides, but depending on the experimental conditions, can leave unchanged or even greatly stimulates the radioactivity bound as phenylalanyl-tRNA and/or short-chain peptides. It is concluded that, during the complete elongation cycle, modeccin does not affect the binding of the first aminoacyl-tRNA to ribosomes, but inhibits some step in the subsequent repetitive activity of either EF 1 or EF 2. The results obtained indicate that the mechanism of action of modeccin is very similar to that of ricin and related plant toxins such as abrin and crotin.

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

2020 ◽  
Vol 117 (7) ◽  
pp. 3610-3620 ◽  
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.

scholarly journals Essential Mechanisms in the Catalysis of Peptide Bond Formation on the Ribosome

2005 ◽  
Vol 280 (43) ◽  
pp. 36065-36072 ◽  
Author(s):  
Malte Beringer ◽  
Christian Bruell ◽  
Liqun Xiong ◽  
Peter Pfister ◽  
Peter Bieling ◽  
...  
Keyword(s):  
Ph Dependence ◽  
Bond Formation ◽  
Peptide Bond ◽  
Activation Entropy ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Chemical Catalysis ◽  
Catalytic Function ◽  
Peptidyl Transfer

Peptide bond formation is the main catalytic function of the ribo-some. The mechanism of catalysis is presumed to be highly conserved in all organisms. We tested the conservation by comparing mechanistic features of the peptidyl transfer reaction on ribosomes from Escherichia coli and the Gram-positive bacterium Mycobacterium smegmatis. In both cases, the major contribution to catalysis was the lowering of the activation entropy. The rate of peptide bond formation was pH independent with the natural substrate, amino-acyl-tRNA, but was slowed down 200-fold with decreasing pH when puromycin was used as a substrate analog. Mutation of the conserved base A2451 of 23 S rRNA to U did not abolish the pH dependence of the reaction with puromycin in M. smegmatis, suggesting that A2451 did not confer the pH dependence. However, the A2451U mutation alters the structure of the peptidyl transferase center and changes the pattern of pH-dependent rearrangements, as probed by chemical modification of 23 S rRNA. A2451 seems to function as a pivot point in ordering the structure of the peptidyl transferase center rather than taking part in chemical catalysis.

scholarly journals Translation elongation factor P (EF-P)

2020 ◽  
Vol 44 (2) ◽  
pp. 208-218 ◽  
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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