Stimulation of the GTPase Activity of Translation Elongation Factor G by Ribosomal Protein L7/12

Savelsbergh, Andreas; Mohr, Dagmar; Wilden, Berthold; Wintermeyer, Wolfgang; Rodnina, Marina V.

doi:10.1074/jbc.275.2.890

Cited by 80 publications

(91 citation statements)

References 48 publications

(52 reference statements)

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“…Using mant-GTP as a fluorescent reporter, we observed that its fluorescence increased substantially when wild type EF-G was added (Fig. 3A), as reported previously (21). When we subsequently added a limiting amount of the ribosome, the fluorescence gradually dropped to a lower plateau.…”

Section: Figure 1 Locations Of Mutated Residues In the G Subdomain Osupporting

confidence: 64%

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Nechifor

Murataliev

Wilson

2007

Journal of Biological Chemistry

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Protein L7/L12 of the bacterial ribosome plays an important role in activating the GTP hydrolytic activity of elongation factor G (EF-G), which promotes ribosomal translocation during protein synthesis. Previously, we cross-linked L7/L12 from two residues (209 and 231) flanking ␣-helix A G in the G subdomain of Escherichia coli EF-G. Here we report kinetic studies on the functional effects of mutating three neighboring glutamic acid residues (224, 228, and 231) to lysine, either singly or in combination. Two single mutations (E224K and E228K), both within helix A G , caused large defects in GTP hydrolysis and smaller defects in ribosomal translocation. Removal of L7/L12 from the ribosome strongly reduced the activities of wild type EF-G but had no effect on the activities of the E224K and E228K mutants. Together, these results provide evidence for functionally important interactions between helix A G of EF-G and L7/L12 of the ribosome.

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Section: Figure 1 Locations Of Mutated Residues In the G Subdomain Osupporting

confidence: 64%

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Nechifor

Murataliev

Wilson

2007

Journal of Biological Chemistry

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“…The overall similarity in these determinations gives us greater confidence in the reported binding data. Taken together, our data suggest that GTP binding should predominate if nucleotide-free RF3 were simply incubated in the cellular milieu; however, given that RF3:GDP leaves the ribosome and must bind to GTP to function in the next cycle, there remains a kinetic problem (Savelsbergh et al 2000;Gromadski et al 2002). The off-rates of GDP from RF3 are slow enough that a GEF is likely needed to increase the rate of nucleotide exchange to physiologically relevant levels.…”

Section: Discussionmentioning

confidence: 94%

“…While the levels of GTP are eightfold higher than GDP in the cell (1660 μM vs. 233 μM, respectively) (Buckstein et al 2008), it is not clear how GDP for GTP exchange can happen on a physiologically relevant time scale in the cell in the absence of a GEF that promotes more rapid nucleotide exchange. Indeed, the off-rates of nucleotides from two other translational GTPases, EFTu and EFG, differ greatly from one another: in the case of EFG, the off-rates are relatively fast (k off,GDP = 23 sec −1 and k off,GTP = 7 sec −1 ) (Savelsbergh et al 2000), while in the case of EFTu, the off-rates are significantly slower (k off,GDP = 0.002 sec −1 , k off,GTP = 0.03 sec −1 ) (Gromadski et al 2002). It is, thus, not surprising that EFTu has a dedicated GEF in the cell, EFTs, while EFG functions as far as is known without any GEF.…”

Section: Discussionmentioning

confidence: 99%

RF3:GTP promotes rapid dissociation of the class 1 termination factor

Koutmou

McDonald

Brunelle

et al. 2014

RNA

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Translation termination is promoted by class 1 and class 2 release factors in all domains of life. While the role of the bacterial class 1 factors, RF1 and RF2, in translation termination is well understood, the precise contribution of the bacterial class 2 release factor, RF3, to this process remains less clear. Here, we use a combination of binding assays and pre-steady state kinetics to provide a kinetic and thermodynamic framework for understanding the role of the translational GTPase RF3 in bacterial translation termination. First, we find that GDP and GTP have similar affinities for RF3 and that, on average, the t 1/2 for nucleotide dissociation from the protein is 1-2 min. We further show that RF3:GDPNP, but not RF3:GDP, tightly associates with the ribosome pre-and post-termination complexes. Finally, we use stopped-flow fluorescence to demonstrate that RF3:GTP enhances RF1 dissociation rates by over 500-fold, providing the first direct observation of this step. Importantly, catalytically inactive variants of RF1 are not rapidly dissociated from the ribosome by RF3:GTP, arguing that a rotated state of the ribosome must be sampled for this step to efficiently occur. Together, these data define a more precise role for RF3 in translation termination and provide insights into the function of this family of translational GTPases.

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“…It was shown that isolated L7/L12 ribosomal proteins were able to stimulate GTP hydrolysis on EF-G to some extent. 52 However ribosomes lacking the L7/L12 were still able to hydrolyze GTP on EF-G (although at a reduced rate), 10,53 thus arguing for additional ribosomal components involved in GTPase activation. It is likely that the primary role of the L7/L12 stalk is factor recruitment rather than GTPase activation.…”

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confidence: 99%