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

Huter, Paul; Arenz, Stefan; Bock, Lars V.; Graf, Michael; Frister, Jan Ole; Heuer, André; Peil, Lauri; Starosta, Agata L.; Wohlgemuth, Ingo; Peske, Frank; Nováček, Jiří; Berninghausen, Otto; Grubmüller, Helmut; Tenson, Tanel; Beckmann, Roland; Rodnina, Marina V.; Vaiana, Andrea C.; Wilson, Daniel N.

doi:10.1016/j.molcel.2017.10.014

Cited by 149 publications

(191 citation statements)

References 74 publications

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“…Sequences that were moderately expressed had a more or less random distribution of GC nucleotides, with a slight increase in C nucleotides for low expressed sequences (1≤GFP score<2). This could potentially be explained by the Crich codons for proline and previously described proline stalling during translation [36][37][38] . Taken together, these analyses indicate that local mRNA sequence and potentially base-pairing stability of nucleotides +7 to +15 influence expression of the protein.…”

Section: Mainmentioning

confidence: 96%

Short translational ramp determines efficiency of protein synthesis

Verma¹,

Choi

Cottrell³

et al. 2019

Preprint

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It is generally assumed that translation efficiency is governed by translation initiation. However, the efficiency of protein synthesis is regulated by multiple factors including tRNA abundance, codon composition, mRNA motifs and amino-acid sequence 1-4 . These factors influence the rate of protein synthesis beyond the initiation phase of translation, typically by modulating the rate of peptide-bond formation and to a lesser extent that of translocation. The slowdown in translation during the early elongation phase, known as the 5' translational ramp, likely contributes to the efficiency of protein synthesis 5-9 . Multiple mechanisms, which could explain the molecular basis for this translational ramp, have been proposed that include tRNA abundance bias 6,9 , the rate of translation initiation 10-15 , mRNA and ribosome structure 11,12,14,[16][17][18] , or retention of initiation factors during early elongation events 19 .Here, we show that the amount of synthesized protein (translation efficiency) depends on a short translational ramp that comprises the first 5 codons in mRNA. Using a library of more than 250,000 reporter sequences combined with in vitro and in vivo protein expression assays, we show that differences in the short ramp can lead to 3 to 4 orders of magnitude changes in protein abundance. The observed difference is not dependent on tRNA abundance, efficiency of translation initiation, or overall mRNA structure. Instead, we show that translation is regulated by amino-acid-sequence composition and local mRNA sequence. Single-molecule measurements of translation kinetics indicate substantial pausing of ribosome and abortion of protein synthesis on the 4 th or 5 th codon for distinct amino acid or nucleotide compositions. Introduction of preferred sequence motifs, only at the exact positions within the mRNA, improves protein synthesis for recombinant proteins, indicating an evolutionarily conserved mechanism for controlling translational efficiency.

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Section: Mainmentioning

confidence: 96%

Short translational ramp determines efficiency of protein synthesis

Verma¹,

Choi

Cottrell³

et al. 2019

Preprint

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“…In all of these cases, the acceptor amino acid is either lysine or arginine and located at the tip of domain I. When bound to the ribosome, the modification protrudes toward peptidyl transferase center, where it stabilizes the CCA sequence end of the P-site tRNA to promote an optimal geometry for peptide bond formation (24)(25)(26). Whereas lysyl lysine, 5-aminopentolyl lysine, and (deoxy)hypusine resemble chemically related structures, arginine rhamnosylation is exceptionally distinct.…”

mentioning

confidence: 99%

Complex Structure of Pseudomonas aeruginosa Arginine Rhamnosyltransferase EarP with Its Acceptor Elongation Factor P

Liu

et al. 2019

J Bacteriol

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A bacterial inverting glycosyltransferase EarP transfers rhamnose from dTDP-β-l-rhamnose (TDP-Rha) to Arg32 of translation elongation factor P (EF-P) to activate its function. We report here the structural and biochemical characterization of Pseudomonas aeruginosa EarP. In contrast to recently reported Neisseria meningitidis EarP, P. aeruginosa EarP exhibits differential conformational changes upon TDP-Rha and EF-P binding. Sugar donor binding enhances acceptor binding to EarP, as revealed by structural comparison between the apo-, TDP-Rha-, and TDP/EF-P-bound forms and isothermal titration calorimetry experiments. In vitro EF-P rhamnosylation combined with active-site geometry indicates that Asp16 corresponding to Asp20 of N. meningitidis EarP is the catalytic base, whereas Glu272 is another putative catalytic residue. Our study should provide the basis for EarP-targeted inhibitor design against infections from P. aeruginosa and other clinically relevant species. IMPORTANCE Posttranslational rhamnosylation of EF-P plays a key role in Pseudomonas aeruginosa, establishing virulence and antibiotic resistance, as well as survival. The detailed structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP from P. aeruginosa not only demonstrates that sugar donor TDP-Rha binding enhances acceptor EF-P binding to EarP but also should provide valuable information for the structure-guided development of its inhibitors against infections from P. aeruginosa and other EarP-containing pathogens.

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“…Protein sequence of CPLX1 is highly conserved in Rattus norvegicua, Mus musculus and Homo sapiens, and they all had the same PPG sequences which induce ribosomes become stalled resulting no full-length product produced ( Fig.6A). However, previously reports demonstrated that eukaryotic eIF5A1 can rescue the stalled ribosomes (Doerfel et al, 2013;Huter et al, 2017). To verify whether eIF5A1 was the main reason for down-regulated CPLX1 in GV treatment, we firstly used the primary cultured cortical neurons to confirm the translated and functional regulation of eIF5A1 to CPLX1.…”

Section: Gv Improves the Neurological Deficits Via Eif5a1 Regulating mentioning

confidence: 99%

Electroacupuncture at Governor Vessel improves neurobehavioral function via reduction of complexin1

Liu

Zhao

et al. 2019

Preprint

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Electroacupuncture at Governor Vessel (GV), as a traditional chinese medicine, has been proved that it can reduce scar and promote axon regeneration. However, the underlying mechanism remains unclear. Herein, complexin1 (CPLX1), as a candidate protein, was found using protein chip. Therefore, using a CRISPR/Cas9 knockout approach, we deleted CPLX1 specifically in the SD rats to assess the role of CPLX1 in GV treatment. Additionally, eIF5A1 stimulate the translation of CPLX1 with PPG sequence, we attempt to uncover whether eIF5A1 play a role in the GV treatment. In fact, GV can reduce scar and promote axon regeneration after SCC. CPLX1 -/+ SCC rats demonstrated that decreased CPLX1 improved the microenvironment of injured area via reducing the components of fibrotic scar and further enhanced the synaptic plasticity, which benefit the regeneration of axons. And eIF5A1 could regulate the expression of CPLX1 in the process of GV treatment. Therefore, GV contributes to axon regeneration and synapse plasticity via eIF5A1 regulating CPLX1 following SCC, providing a convincible mechanism for improving the therapeutic efficacy of GV for SCC.

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

Cited by 149 publications

References 74 publications

Short translational ramp determines efficiency of protein synthesis

Short translational ramp determines efficiency of protein synthesis

Complex Structure of Pseudomonas aeruginosa Arginine Rhamnosyltransferase EarP with Its Acceptor Elongation Factor P

Electroacupuncture at Governor Vessel improves neurobehavioral function via reduction of complexin1

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