doi: 10.1093/nar/gks660. Epub 2012 Jul 5.
Structural basis for translation termination by archaeal RF1 and GTP-bound EF1α complex
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- PMID:22772989
- PMCID: PMC3467058
- DOI: 10.1093/nar/gks660
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Structural basis for translation termination by archaeal RF1 and GTP-bound EF1α complex
Kan Kobayashi et al. Nucleic Acids Res.2012 Oct.
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Abstract
When a stop codon appears at the ribosomal A site, the class I and II release factors (RFs) terminate translation. In eukaryotes and archaea, the class I and II RFs form a heterodimeric complex, and complete the overall translation termination process in a GTP-dependent manner. However, the structural mechanism of the translation termination by the class I and II RF complex remains unresolved. In archaea, archaeal elongation factor 1 alpha (aEF1α), a carrier GTPase for tRNA, acts as a class II RF by forming a heterodimeric complex with archaeal RF1 (aRF1). We report the crystal structure of the aRF1·aEF1α complex, the first active class I and II RF complex. This structure remarkably resembles the tRNA·EF-Tu complex, suggesting that aRF1 is efficiently delivered to the ribosomal A site, by mimicking tRNA. It provides insights into the mechanism that couples GTP hydrolysis by the class II RF to stop codon recognition and peptidyl-tRNA hydrolysis by the class I RF. We discuss the different mechanisms by which aEF1α recognizes aRF1 and aPelota, another aRF1-related protein and molecular evolution of the three functions of aEF1α.
Figures
Overall structure of the aRF1·aEF1α·GTP complex, viewed from two perpendicular directions. Domains 1, 2 and 3 of aEF1α are colored red, brown and yellow, respectively; Domains A, B and C of aRF1 are colored turquoise, light blue and purple, respectively. The bound GTP is shown by a ball-and-stick model. The disordered loop of the residues Lys176–Tyr187 in aEF1α domain B is represented as the dashed line.
Interaction sites between aRF1 and aEF1α. Proteins are depicted by ribbon models, with the domains color-coded as in Figure 1. Hydrogen bonds and salt bridges are indicated by dashed blue lines. (A) The interaction interface between aRF1 and aEF1α, which is divided into three sites, sites 1, 2 and 3. The switch I and II regions of aEF1α are highlighted in red. (B) The interactions between aRF1 domain C and aEF1α domain 3 (Site 1). (C) The interactions between aRF1 domain B and aEF1α domain 2 (Site 2). (D) The interactions between aRF1 domain B and aEF1α domains 1 and 3 (Site 3). Water molecules and magnesium ions are depicted by red and grey ball-and-stick models, respectively.
Yeast two-hybrid analysis of aRF1 binding to aEF1α at Sites 2 and 3. (A) Two-hybrid analysis (3-d growth) of mutants of residues in Sites 2 and 3 in aRF1 (activation domain, AD) against aEF1α wild-type (binding domain, BD). (B) Two-hybrid analysis (3-d growth) of mutants of residues in Sites 2 and 3 in aEF1α (BD) against aRF1 wild-type (AD).
Comparison of the aRF1·aEF1α·GTP complex structure with related structures. (A) Superposition of domains 2 and 3 of aEF1α in the present complex (red) with those of the GDPNP-bound form of eRF3 (blue) (PDB ID: 1R5O). (B) Superposition of domains 2 and 3 of aEF1α in the present complex (red) with those of the GDPNP-bound form of EF–Tu (yellow) bound to tRNA (PDB ID: 1TTT). (C) The complex structure of aRF1·aEF1α·GTP (this work). Domains are color-coded as in Figure 1. (D) The complex structure of tRNA·EF–Tu·GDPNP (PDB ID: 1TTT). The domains of EF–Tu are color-coded as in aEF1α. The tRNA is colored turquoise (anticodon arm), light blue (acceptor stem) and purple (T stem). The bound GDPNP is depicted by a ball-and-stick model. (E) The complex structure of eRF1 and eRF3 lacking the GTP-binding domain (PDB ID: 3E1Y). Domains are color-coded as in aRF1·aEF1α.
Comparison of the complex structure of aRF1·aEF1α·GTP with that of aPelota·aEF1α·GTP. Domains are color-coded as in Figure 1. (A) aPelota·aEF1α·GTP complex structure (PDB ID: 3AGJ). (B) aRF1·aEF1α·GTP complex structure (this work). (C) The interaction interface between aPelota and aEF1α. Lys99 and Arg309 of aEF1α, and Asp135 and Asp137 of aPelota are depicted by ball-and-stick models. (D) The interaction interface between aRF1 and aEF1α. Lys99 and Arg309 of aEF1α, and Glu149 and Asp151 of aRF1 are depicted by ball-and-stick models. (E)In vitro binding assay of wild-type (WT) and mutant (K99A/R309A) aEF1α to aRF1 and aPelota. His-tagged aEF1α (His-aEF1α) was mixed with aRF1 or aPelota, immobilized by MagneHis™ Ni particles and then eluted. Eluted fractions were analyzed by SDS–PAGE.
Structural comparison of tRNA, aRF1 and aPelota. (A) Comparison of EF–Tu-bound tRNA in the isolated (PDB ID: 1TTT) and the ribosome-bound A/T state (PDB ID: 2XQD) forms, colored purple and yellow, respectively. (B) Comparison of aRF1 in the isolated (PDB ID: 3AGK) and aEF1α-bound (this work) forms, colored red and turquoise, respectively. (C) The structure of aPelota bound to aEF1α (PDB ID: 3AGJ).
Inter-domain interaction between domains 1 and 2 of aEF1α and EF–Tu through conserved arginine and glutamate residues (Glu134 and Arg413 for aEF1α, and Glu118 and Arg389 for EF–Tu). Domains are color-coded as in Figure 1. (A) aEF1α in the aRF1·aEF1α·GTP complex structure (this work). (B) EF–Tu in the tRNA·EF–Tu·GDPNP complex structure (PDB ID: 1TTT). (C) EF–Tu in the tRNA·EF–Tu·GDPCP complex structure on the ribosome (PDB ID: 2XQD).
Comparison of the arrangements of domains B and C in (A) aRF1 bound to aEF1α (this work), (B) eRF1 (PDB ID: 1DT9) and (C) eRF1 bound to eRF3 lacking the GTP-binding domain (PDB ID: 3E1Y). Domains are color-coded as in Figure 1.
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