The hinge region of Escherichia coli ribosomal protein L7/L12 is required for factor binding and GTP hydrolysis

Dey, Debendranath; Oleinikov, Andrew V.; Traut, Robert R.

doi:10.1016/0300-9084(95)80003-4

Cited by 33 publications

(30 citation statements)

References 27 publications

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“…A recent crystal structure of L12 from the hyperthermophilic bacterium Thermotoga maritima shows two alternative conformations for the hinge region, an extended coil or a long ␣-helix that folds back on the N-terminal domain forming a compact overall protein structure (18). The flexible hinge conformation agrees better with the body of evidence that indicates that the E. coli L7/L12 protein has a great deal of overall flexibility (19). L7/L12 is easily and selectively dissociated from and reconstituted into the ribosome (20).…”

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confidence: 75%

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Cryo-electron Microscopic Localization of Protein L7/L12 within the Escherichia coli 70 S Ribosome by Difference Mapping and Nanogold Labeling

Montesano-Roditis¹,

Glitz²,

Traut³

et al. 2001

Journal of Biological Chemistry

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The Escherichia coli ribosomal protein L7/L12 is central to the translocation step of translation, and it is known to be flexible under some conditions. The assignment of electron density to L7/L12 was not possible in the recent 2.4 Å resolution x-ray crystallographic structure (Ban, N., Nissen, P., Hansen, J., Moore, P. B., and Steitz, T. A. (2000) Science 289, 905-920). We have localized the two dimers of L7/L12 within the structure of the 70 S ribosome using two reconstitution approaches together with cryo-electron microscopy and single particle reconstruction. First, the structures were determined for ribosomal cores from which protein L7/L12 had been removed by treatment with NH 4 Cl and ethanol and for reconstituted ribosomes in which purified L7/L12 had been restored to core particles. Difference mapping revealed that the reconstituted ribosomes had additional density within the L7/L12 shoulder next to protein L11. Second, ribosomes were reconstituted using an L7/L12 variant in which a single cysteine at position 89 in the C-terminal domain was modified with Nanogold (Nanoprobes, Inc.), a 14 Å gold derivative. The reconstruction from cryo-electron microscopy images and difference mapping placed the gold at four interfacial positions. The finding of multiple sites for the C-terminal domain of L7/L12 suggests that the conformation of this protein may change during the steps of elongation and translocation.The ribosome is the platform for all protein synthesis and the catalyst of peptide bond formation. The centrality of the ribosome is shown by its universality, its conservation throughout all forms of life, and its frequent targeting by toxins and antibiotics (1). The ribosome has been the subject of scores of studies that have linked the structure of the particle with its mechanism of action in protein synthesis. Traditional electron microscopy studies have played a central role in allowing visualization of the ribosome and its subunits and in the placement of component proteins, segments of its large RNA molecules, and functional sites (2, 3).Cryo-electron microscopy (EM) 1 adds new dimensions to these investigations while confirming the basic observations of earlier work; it allows a description of a native particle frozen in vitreous ice and undistorted by drying and staining, and it permits visualization of the interior of the particle rather than simply its stained or shadowed surfaces. Cryo-EM reconstructions of the ribosome have been generated through independent investigations in two different laboratories. This work has resulted in the attainment of greater resolution in the description of the overall structure of the ribosome (4, 5) and also has allowed the structural studies of ribosomes complexed with tRNAs and protein factors (6 -8). More recently, cryo-EM data have aided in the determination of phases for crystal structures of the 30 S subunit (9), the 50 S subunit (10), and the 70 S ribosome (11). Crystal structures of the 30 S (12, 13) and 50 S (14) subunits have been solved at 3.0 -3.3 an...

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confidence: 75%

“…The stalk and full activity are restored when L7/L12 is added back, and the ribosome nomenclature has equated L7/L12 and the stalk. A body of evidence suggests that L7/L12 exists in at least two conformations, and its mobility is essential for its function (19,21,22).…”

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confidence: 99%

Cryo-electron Microscopic Localization of Protein L7/L12 within the Escherichia coli 70 S Ribosome by Difference Mapping and Nanogold Labeling

Montesano-Roditis¹,

Glitz²,

Traut³

et al. 2001

Journal of Biological Chemistry

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“…It is clear that, in the cell, eEF-2 undergoes several other interactions with ribosomal proteins and rRNA [11,12]. The fact that we were unable to measure any interaction with eEF-2 when the P proteins were covalently linked to the sensor chip is possibly due to the fact that in the ribosome these proteins are flexible and at least partly mobile, as shown for their prokaryotic equivalents L7/L12 [13], whereas they were fixed on the sensor chip and could not move in our assay. Another explanation may come from the fact that interaction with eEF-2 would be possible only when P proteins are in a dimeric form, the formation of which could be hindered by covalent coupling.…”

Section: Discussionmentioning

confidence: 85%

Interaction of elongation factor eEF‐2 with ribosomal P proteins

Bargis-Surgey

Lavergne

Gonzalo

et al. 1999

European Journal of Biochemistry

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The eukaryotic P1 and P2 ribosomal proteins which constitute, with P0, a pentamer forming the lateral stalk of the 60 S ribosomal subunit, exhibit several differences from their prokaryotic equivalents L7 and L12; in particular, P1 does not have the same primary structure as P2 and both of them are phosphorylated, the significance of the latter remaining unclear. Rat liver P1 and P2 were overproduced in Escherichia coli cells and their interaction with elongation factor eEF-2 was studied. Both recombinant proteins were found to be required for the ribosomedependent GTPase activity of eEF-2, with P2 in the phosphorylated form. The surface plasmon resonance technique revealed that, in vitro, both proteins interact specifically with eEF-2, with a higher affinity for P1 (K d = 3.8 Â 10 28 m) than for P2 (K d = 2.2 Â 10 26 m). Phosphorylation resulted in a moderate increase (two-to four-fold) in these affinities. The interaction of both P1 and P2 (phosphorylated or not) with eEF-2 resulted in a conformational change in the factor, revealed by an increase in the accessibility of Glu554 to proteinase Glu-C. This increase was observed in both the presence and absence of GTP and GDP, which themselves produced marked opposite effects on the conformation of eEF-2. Our results suggest that the two proteins P1 and P2 both interact with eEF-2 inducing a conformational transition of the factor, but have acquired some specific properties during evolution.Keywords: acidic ribosomal proteins; ribosomal; elongation factor 2; phosphorylation.P proteins are present in the 60 S ribosomal subunits of all eukaryotic cells. In mammalian cells, there are three different proteins called P0, P1 and P2. P1 and P2 are 12-kDa acidic proteins which exhibit some similarities to and some differences from the prokaryotic ribosomal proteins L7 and L12. Among the similarities are their localization on the large ribosomal subunit forming a lateral stalk, their molecular mass and physicochemical properties, and their requirement for translational factor activity. Thus, the GTPase activity of the eukaryotic elongation factor eEF-2 which catalyses the translocation of peptidyl-tRNA from the A to the P site of the ribosome is dependent on the presence of P1 and P2 on the large ribosomal subunit [1], just as the GTPase activity of the corresponding prokaryotic factor EF-G is dependent on the presence of L7/L12 on this subunit. A direct interaction between L7/L12 and EF-G has been recently visualized directly by cryoelectron microscopy [2]. The differences between P1/P2 and L7/L12 are numerous and interesting to study in terms of evolution. First, sequence homologies between eukaryotic and prokaryotic proteins are not obvious [3]. Secondly, P1 and P2 do not have the same primary structure [3], whereas L7 and L12 do, with the only exception being an N-acetyl group. Thirdly, in contrast with L7 and L12, P1 and P2 are phosphorylated on serine residues when present on the ribosome [4], and this phosphorylation seems to be important for their function, althoug...

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“…Different studies indicated that the presence of one L7/L12 dimer on the body of the 50 S particle in an extended conformation directed toward the central protuberance (22, 23). Additional evidence that a C-terminal domain can occupy a location not only extended across the body of the ribosome but also near the base of the stalk came from cross-linking between a predetermined location in the C-terminal domain, 25) and also from hinge deletion studies (26,27). A different heterobifunctional reagent, APDP 1 showed a cross-link between Cys-89 and L11 and also with L10 in a lesser extent near the EF-G binding site near the base of the stalk (28).…”

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confidence: 99%

Cross-linking of Selected Residues in the N- and C-terminal Domains of Escherichia coli Protein L7/L12 to Other Ribosomal Proteins and the Effect of Elongation Factor Tu

Dey

Bochkariov²,

Jokhadze³

et al. 1998

Journal of Biological Chemistry

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The Escherichia coli ribosomal protein, L7/L12, is the most extensively investigated representative of the small, four-copy, dimeric acidic proteins that are found in large ribosomal subunits of all organisms and exist as a conserved quaternary structural element in which two dimers are integrated into the ribosome through binding to a common anchoring protein (1-3). One or both of the L7/L12 dimers form a conspicuous morphological feature on the ribosome known in E. coli as the L7/L12 stalk (4). The proteins can be simply and selectively removed from and reconstituted into the ribosome (5), a property that greatly facilitates experiments of the type reported here in which genetically and biochemically modified proteins replace the wild type.The L7/L12 polypeptide is composed of two distinct organized structural domains linked by a flexible hinge (6, 7), as summarized in Fig. 1. The elongated, helical N-terminal domain, residues 1-33, is responsible for dimer interaction (8), and the globular C-terminal domain, residues 53-120, is necessary for factor binding (9 -11). The C-terminal domains can be cross-linked to each other in different orientations yet retain full functional activity in supporting polypeptide synthesis (12). Flexibility of L7/L12 has been demonstrated in solution by fluorescence techniques (13) and in the ribosome by proton NMR (14, 15), by electron microscopy (16), and with fluorescence probes attached to the C-terminal domains (13,17,18).Immunoelectron microscopy with monoclonal antibodies (19) directly showed the presence of the C-terminal domain at the tip of the stalk and the N-terminal domain at the base of the stalk. This was consistent with earlier demonstrations that the N-terminal domain was responsible for binding of the fulllength L7/L12 to L10 and to the ribosome (9, 10). It was shown that one dimer per particle was sufficient to form a visible stalk (20), despite earlier studies with polyclonal antibodies that had suggested that both dimers were present in the stalk (21). Different studies indicated that the presence of one L7/L12 dimer on the body of the 50 S particle in an extended conformation directed toward the central protuberance (22, 23). Additional evidence that a C-terminal domain can occupy a location not only extended across the body of the ribosome but also near the base of the stalk came from cross-linking between a predetermined location in the C-terminal domain, 25) and also from hinge deletion studies (26,27). A different heterobifunctional reagent, APDP 1 showed a cross-link between Cys-89 and L11 and also with L10 in a lesser extent near the EF-G binding site near the base of the stalk (28). The site-specific cross-linking experiments led to the proposal of a possible "bent" conformation for one of the dimers in which the C-terminal domain could lie on the body of the subunit near the N-terminal domain at the base of the stalk. We asked the question whether there was a preferred surface of the C-terminal domain that made these contacts and designed cysteine prob...

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The hinge region of Escherichia coli ribosomal protein L7/L12 is required for factor binding and GTP hydrolysis

Cited by 33 publications

References 27 publications

Cryo-electron Microscopic Localization of Protein L7/L12 within the Escherichia coli 70 S Ribosome by Difference Mapping and Nanogold Labeling

Cryo-electron Microscopic Localization of Protein L7/L12 within the Escherichia coli 70 S Ribosome by Difference Mapping and Nanogold Labeling

Interaction of elongation factor eEF‐2 with ribosomal P proteins

Cross-linking of Selected Residues in the N- and C-terminal Domains of Escherichia coli Protein L7/L12 to Other Ribosomal Proteins and the Effect of Elongation Factor Tu

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