Interaction Sites of Ribosome Bound Eukaryotic Elongation Factor 2 in 18S and 28S rRNA

Holmberg, Lovisa; Nygård, Odd

doi:10.1021/bi00254a027

Cited by 34 publications

(18 citation statements)

References 65 publications

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“…Despite its apparent lack of CaM responsiveness, Clk1 was able to phosphorylate EF-2. Studies by others have indicated that hyperphosphorylation of EF-2 inhibits translation (63,64). Two other observations are at least consistent with a potential role for Clk1 in regulation of protein synthesis.…”

Section: Ca 2ϩmentioning

confidence: 56%

Identification and Characterization of the CLK1 Gene Product, a Novel CaM Kinase-like Protein Kinase from the Yeast Saccharomyces cerevisiae

Melcher

Thorner

1996

Journal of Biological Chemistry

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The CLK1 gene of Saccharomyces cerevisiae encodes a 610-residue protein kinase that resembles known type II Ca 2؉ /calmodulin-dependent protein kinases (CaM kinases), including the CMK1 and CMK2 gene products from the same yeast. The Clk1 kinase domain is preceded by a 162-residue N-terminal extension, followed by a 132-residue C-terminal extension (which contains a basic segment resembling known calmodulin-binding sites) and is as similar to mammalian CaM kinase (38% identity to rat CaM kinase ␣) as it is to yeast CaM kinase (37% identity to Cmk2). However, Clk1 shares 52% identity with Rck1, another putative protein kinase encoded in the S. cerevisiae genome. Clk1 tagged with a c-myc epitope (expressed in yeast) and a GST-Clk1 fusion (expressed in bacteria) underwent autophosphorylation and phosphorylated an exogenous substrate (yeast protein synthesis elongation factor 2), primarily on Ser. Neither Clk1 activity was stimulated by purified yeast calmodulin (CMD1 gene product), with or without Ca 2؉ ; no association of Clk1 with Cmd1 was detectable by other methods. C-terminally truncated Clk1(⌬487-610) was growth-inhibitory when overexpressed, whereas catalytically inactive Clk1(K201R ⌬487-610) was not, suggesting that the C terminus is a negative regulatory domain. Using immunofluorescence, Clk1 was localized to the cytosol and excluded from the nucleus. A clk1⌬ mutant, a clk1⌬ rck1⌬ double mutant, a clk1⌬ cmk1⌬ cmk2⌬ triple mutant, and a clk1⌬ rck1⌬ cmk1⌬ cmk2⌬ quadruple mutant were all viable and manifested no other overt growth phenotype.

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Section: Ca 2ϩmentioning

confidence: 56%

Identification and Characterization of the CLK1 Gene Product, a Novel CaM Kinase-like Protein Kinase from the Yeast Saccharomyces cerevisiae

Melcher

Thorner

1996

Journal of Biological Chemistry

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“…This residue is also strictly conserved in other members of the translation-factor family (Bourne et al 1990(Bourne et al , 1991, which, together with the similarities in their structures and ribosome-binding modes (Dever et al 1987;Moazed et al 1988;Holmberg and Nygård 1994) suggests a common mechanism by which the ribosome activates GTP hydrolysis in all these proteins.…”

Section: Introductionmentioning

confidence: 77%

Mechanism of activation of elongation factor Tu by ribosome: Catalytic histidine activates GTP by protonation

Aleksandrov¹,

Field²

2013

RNA

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Elongation factor Tu (EF-Tu) is central to prokaryotic protein synthesis as it has the role of delivering amino-acylated tRNAs to the ribosome. Release of EF-Tu, after correct binding of the EF-Tu:aa-tRNA complex to the ribosome, is initiated by GTP hydrolysis. This reaction, whose mechanism is uncertain, is catalyzed by EF-Tu, but requires activation by the ribosome. There have been a number of mechanistic proposals, including those spurred by a recent X-ray crystallographic analysis of a ribosome:EF-Tu:aatRNA:GTP-analog complex. In this work, we have investigated these and alternative hypotheses, using high-level quantum chemical/molecular mechanical simulations for the wild-type protein and its His85Gln mutant. For both proteins, we find previously unsuggested mechanisms as being preferred, in which residue 85, either His or Gln, directly assists in the reaction. Analysis shows that the RNA has a minor catalytic effect in the wild-type reaction, but plays a significant role in the mutant by greatly stabilizing the reaction's transition state. Given the similarity between EF-Tu and other members of the translational Gprotein family, it is likely that these mechanisms of ribosome-activated GTP hydrolysis are pertinent to all of these proteins.

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“…Moreover the ionic conditions are not identical in the two situations. 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.…”

Section: Discussionmentioning

confidence: 99%

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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Interaction Sites of Ribosome Bound Eukaryotic Elongation Factor 2 in 18S and 28S rRNA

Cited by 34 publications

References 65 publications

Identification and Characterization of the CLK1 Gene Product, a Novel CaM Kinase-like Protein Kinase from the Yeast Saccharomyces cerevisiae

Identification and Characterization of the CLK1 Gene Product, a Novel CaM Kinase-like Protein Kinase from the Yeast Saccharomyces cerevisiae

Mechanism of activation of elongation factor Tu by ribosome: Catalytic histidine activates GTP by protonation

Interaction of elongation factor eEF‐2 with ribosomal P proteins

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