Ribosome-induced tuning of GTP hydrolysis by a translational GTPase

Maracci, Cristina; Peske, Frank; Dannies, Ev; Pohl, Corinna; Rodnina, Marina V.

doi:10.1073/pnas.1412676111

Cited by 45 publications

(92 citation statements)

References 63 publications

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“…3A). Consistent with previous observations (27), ribosomes are required to stimulate EF-Tu-mediated GTP hydrolysis, and virtually no hydrolysis of GTP was detected in their absence. EF-Tu hydrolyzed GTP in the presence of ribosomes in a time-dependent manner, and by 30 min ∼60% of the GTP was converted into GDP.…”

Section: Phosphorylation Of Ef-tu Inhibits Gtp Hydrolysis and Translasupporting

confidence: 92%

“…EF-Tu hydrolyzed GTP in the presence of ribosomes in a time-dependent manner, and by 30 min ∼60% of the GTP was converted into GDP. The relatively modest levels of GTP hydrolysis observed likely are caused by the absence of mRNA and aa-tRNA in the reactions, which are required for full activation of EF-Tu GTPase activity (27). However, under the same conditions, GTP hydrolysis was reduced significantly in the presence of P∼EF-Tu to levels similar to those observed in the absence of ribosomes.…”

Section: Phosphorylation Of Ef-tu Inhibits Gtp Hydrolysis and Translamentioning

confidence: 81%

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Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor

Pereira

Gonzalez

Dworkin

2015

Proc. Natl. Acad. Sci. U.S.A.

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In nature, most organisms experience conditions that are suboptimal for growth. To survive, cells must fine-tune energy-demanding metabolic processes in response to nutrient availability. Here, we describe a novel mechanism by which protein synthesis in starved cells is down-regulated by phosphorylation of the universally conserved elongation factor Tu (EF-Tu). Phosphorylation impairs the essential GTPase activity of EF-Tu, thereby preventing its release from the ribosome. As a consequence, phosphorylated EF-Tu has a dominant-negative effect in elongation, resulting in the overall inhibition of protein synthesis. Importantly, this mechanism allows a quick and robust regulation of one of the most abundant cellular proteins. Given that the threonine that serves as the primary site of phosphorylation is conserved in all translational GTPases from bacteria to humans, this mechanism may have important implications for growth-rate control in phylogenetically diverse organisms.A daptation to nutrient availability is a fundamental cellular process. From unicellular prokaryotes to complex multicellular organisms, cells sense and adjust their metabolism to respond to variations in nutrient levels. Protein synthesis is one of the most energy-intensive cellular processes, and both the initiation and elongation stages of translation are down-regulated in response to nutrient limitation (1). Proteins that mediate translation, such as the essential and universally conserved GTPase Elongation factor Tu (EF-Tu), are observed in the phosphoproteomes of diverse organisms (2), suggesting that they are subject to regulatory phosphorylation. However, EF-Tu is the most abundant protein in bacteria, present at ∼100,000 copies per cell of growing Escherichia coli (3), but less than 10% of the EF-Tu molecules are phosphorylated (4). Thus, a key question is how this relatively small fraction of phosphorylated EF-Tu can cause a down-regulation of protein synthesis.GTP-bound EF-Tu binds and delivers an aminoacyl-tRNA (aa-tRNA) molecule to the translating ribosome (3). Upon formation of a correctly base-paired mRNA codon-aa-tRNA anticodon interaction, the ribosome activates the GTPase activity of EF-Tu, followed by accommodation of the aa-tRNA into the ribosomal aa-tRNA binding (A) site, and release of the inactive GDP-bound EF-Tu from the ribosome. EF-Tu belongs to the GTPase superfamily which comprises molecular switches that share a core catalytic domain and mechanism but have evolved to perform diverse roles in many cellular processes (5). Central to their function is the hydrolysis of GTP, which controls the switch between the ON, GTP-bound, and the OFF, GDP-bound, states. Hydrolysis of GTP is followed by large conformational changes in two flexible regions known as "switch I" and "switch II." These regions are composed of highly conserved motifs that surround and contact the nucleotide and are involved in interactions with both exchange factors and effectors that regulate GTPase function.GTPases also can be regulated by direct inhibiti...

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Section: Phosphorylation Of Ef-tu Inhibits Gtp Hydrolysis and Translasupporting

confidence: 92%

Section: Phosphorylation Of Ef-tu Inhibits Gtp Hydrolysis and Translamentioning

confidence: 81%

Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor

Pereira

Gonzalez

Dworkin

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Nevertheless, the proposed general base mechanism continues to be intensively discussed and the role of the SRL, and the conserved histidine remains unclear (22,23). Recent studies favor a model where GTP hydrolysis is activated via substrate-assisted catalysis, thereby highlighting the importance of functional groups on the GTP substrate for the phosphoryl transfer mechanism (41,42). However, the contribution of the nonbridging phosphate oxygen at position 2662 of rRNA to GTP hydrolysis is still unclear.…”

Section: A2662 Thiophosphate Diastereomers Both Have Wt Activities Inmentioning

confidence: 99%

“…Very recently another residue, namely an aspartate residue (Asp21 of EF-Tu), has been shown to be crucial for stimulation of GTP hydrolysis by EF-Tu on cognate decoding, whereas it is dispensable for intrinsic GTPase activity (42). It was suggested that basal GTP hydrolysis of trGTPases does not involve side chains of Asp21 and His84.…”

Section: A2662 Thiophosphate Diastereomers Both Have Wt Activities Inmentioning

confidence: 99%

Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

Koch

Flür

Kreutz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

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“…His84 in the switch II is conserved in all trGTPases. Mutation of His84 to alanine (His84Ala) completely abolishes the acceleration of GTP hydrolysis by the ribosome . Due to the neutral pK a of histidine in solution and its position close to the catalytic water molecule, as seen from the structure of the ribosome‐bound EF‐Tu in the pre‐hydrolysis state (Figure b), His84 was proposed to act as a general base .…”

Section: Multiple Pathways Of Gtp Hydrolysismentioning

confidence: 99%

Review: Translational GTPases

Maracci

Rodnina

2016

Biopolymers

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Translational GTPases (trGTPases) play key roles in facilitating protein synthesis on the ribosome. Despite the high degree of evolutionary conservation in the sequences of their GTP‐binding domains, the rates of GTP hydrolysis and nucleotide exchange vary broadly between different trGTPases. EF‐Tu, one of the best‐characterized model G proteins, evolved an exceptionally rapid and tightly regulated GTPase activity, which ensures rapid and accurate incorporation of amino acids into the nascent chain. Other trGTPases instead use the energy of GTP hydrolysis to promote movement or to ensure the forward commitment of translation reactions. Recent data suggest the GTPase mechanism of EF‐Tu and provide an insight in the catalysis of GTP hydrolysis by its unusual activator, the ribosome. Here we summarize these advances in understanding the functional cycle and the regulation of trGTPases, stimulated by the elucidation of their structures on the ribosome and the progress in dissecting the reaction mechanism of GTPases. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 463–475, 2016.

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Ribosome-induced tuning of GTP hydrolysis by a translational GTPase

Cited by 45 publications

References 63 publications

Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor

Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor

Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

Review: Translational GTPases

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