Binding of translation elongation factors to individual copies of the archaeal ribosomal stalk protein aP1 assembled onto aP0

https://doi.org/10.1016/j.bbrc.2016.12.175Get rights and content

Highlights

  • Each of the six archaeal aP1s in the stalk complex can bind tightly to aEF2.

  • C-terminal F107 residue of aP1 in the stalk complex is crucial for factor binding.

  • aEF1A binding to the stalk complex is stabilized in the presence of aEF2.

Abstract

Ribosomes in all organisms contain oligomeric and flexible proteins called stalks, which are responsible for the recruitment of translational GTPase factors to the ribosome. Archaeal ribosomes have three stalk homodimers (aP1)2 that constitute a heptameric complex with the anchor protein aP0. We investigated the factor binding ability of aP1 proteins assembled onto aP0, by gel-retardation assays. The isolated aP0(aP1)2(aP1)2(aP1)2 complex, as well as the form bound to the Escherichia coli 50S core, as a hybrid 50S particle, interacted strongly with elongation factor aEF2, but weakly with aEF1A. These interactions were disrupted by a point mutation, F107S, at the C-terminus of aP1. To examine the ability of each copy of aP0-associated aP1 to bind to elongation factors, we constructed aP0·aP1 variant trimers, composed of an aP0 mutant and a single (aP1)2 dimer. Biochemical and quantitative analyses revealed that the resultant three trimers, aP0(aP1)2I, aP0(aP1)2II, and aP0(aP1)2III, individually bound two molecules of aEF2, suggesting that each copy of the aP1 C-terminal region in the aP0·aP1 trimers can bind tightly to aEF2. Interestingly, the unstable binding of aEF1A to each of the three aP0·aP1 trimers was remarkably stabilized in the presence of aEF2. The stability of the aEF1A binding to the stalk complex may be affected by the presence of aEF2 bound to the complex, by an unknown mechanism.

Introduction

The ribosomal interactions with two elongation factors are pivotal events for translation elongation. These interactions strictly depend on the presence of multiple copies of a flexible ribosomal protein on the large subunit, the so-called “stalk” [1], [2], which is responsible for the recruitment of GTP-bound forms of elongation factors to the ribosome and the accompanying GTP hydrolysis at the sarcin/ricin loop (SRL) region of the large subunit [3], [4]. The stalk proteins are designated as L12 (or bL12) in bacteria, P1/P2 in eukaryotes [5], and aP1 in archaea [6]. In all cases, the stalk proteins form dimers through interactions between their N-terminal regions [7], and two or three stalk dimers bind to their respective stalk base proteins: bacterial L10 [2], [8], eukaryotic P0 [9], and archaeal aP0 [6]. These bindings occur through interactions between the N-terminal dimerization regions of the stalk and the C-terminal helical regions of the stalk base proteins [2], [10]. A comparison of the primary structures indicated a close evolutionary relationship between the archaeal aP1 and eukaryotic P1/P2 stalks [11]. In contrast, there is little structural similarity between the bacterial and archaeal/eukaryotic stalk proteins, despite their functional similarity [7], [11].

In the case of bacterial L12, the C-terminal 70 amino acid residues form a globular structure (CTD), which has long been implicated in factor binding [7], [12], [13], [14]. Our recent biochemical study demonstrated that the isolated archaeal stalk aP1 protein binds to the translation factors, aEF1A and aEF2, in a nucleotide (GTP/GDP)-independent manner, and that three hydrophobic amino acid residues at the C-terminal end of aP1 are responsible for the factor binding [6]. More recently, we determined the crystal structure of the complex between aEF1A and the C-terminal fragment including residues 83–108 of aP1 [15]. This structure revealed that the long extended α-helix of the C-terminal fragment bound to the cleft between domains 1 and 3 of aEF1A. In contrast, an NMR study of the human P1-P2 stalk dimer showed that the C-terminal halves of P1/P2 are unstructured and can extend up to 125 Å away from the dimerization domains [16]. These lines of evidence suggest that, unlike bacterial L12, the archaeal/eukaryotic stalk does not form a stable domain structure in the C-terminal region, although a helical C-terminal structure is induced by factor binding.

One of the common features shared by the stalk proteins of the three domains of life is their oligomeric state: the bacterial pentameric L10(L12)2(L12)2 [8] or heptameric L10(L12)2(L12)2(L12)2 [2], the archaeal aP0(aP1)2(aP1)2(aP1)2 heptamer [10], and the eukaryotic P0(P1·P2)(P1·P2) pentamer [17]. The functional significance of these oligomeric forms of the stalk has been suggested to be the enhancement of factor recruitment to the ribosome [2], [10]. However, it remains unknown whether all of the copies of the stalk in the complex participate equally in binding to translation factors. In the present study, we prepared Pyrococcus horikoshii aP1, aP0, and their mutants, and reconstituted various stalk complex variants including complexes composed of aP0 and one of the three aP1 dimers. The experimental results suggested that all copies of the C-terminal region of aP1 associated with aP0 have efficient ability to bind aEF2, and that aEF1A binding to the aP0·aP1complex is stabilized in the presence of aEF2.

Section snippets

Plasmid construction and protein expression

The gene encoding P. horikoshii ribosomal protein aP0 was cloned into the E. coli expression vector pET28c (Novagen), as described previously [18]. By site-directed mutagenesis, two (aP1)2 binding sites among the three sites, I, II, and III, in aP0 (see Fig. 2A) were disrupted in three combinations, as described previously [10]. The resultant aP0 mutant genes were used for the expression of aP0 mutants to constitute three aP0·aP1 variant trimers, aP0(aP1)2I, aP0(aP1)2II, and aP0(aP1)2III (see

Factor binding by the ribosomal aP1 stalk dimers associated with aP0

We reconstituted the aP0(aP1)2I(aP1)2II(aP1)2III stalk heptamer (see Fig. 2A, WT) [10], and analyzed it by polyacrylamide native gel electrophoresis (Fig. 1A, lane 1). By mixing the aP0·aP1 heptamer with aEF2, several shifted bands appeared (lane 3) with lower mobilities than that of free aEF2 (lane 5), suggesting that multiple molecules of aEF2 bound to the aP0·aP1 heptamer. Since the C-terminal F107 residue in aP1 was crucial for the binding of isolated aP1 to elongation factors aEF1A and

Acknowledgements

We thank Profs. Min Yao, and Takehito Tanzawa (Hokkaido University) for providing plasmids to express aP0 mutants lacking aP1 binding ability. This work was supported by Grant-in-Aid for Scientific Researches (16H04741 and 26242075), and a Grant-in-Aid for Challenging Exploratory Research (15K14472).

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