Structure of eEF3 and the mechanism of transfer RNA release from the E-site

Abstract

Elongation factor eEF3 is an ATPase that, in addition to the two canonical factors eEF1A and eEF2, serves an essential function in the translation cycle of fungi. eEF3 is required for the binding of the aminoacyl-tRNA-eEF1A-GTP ternary complex to the ribosomal A-site and has been suggested to facilitate the clearance of deacyl-tRNA from the E-site. Here we present the crystal structure of Saccharomyces cerevisiae eEF3, showing that it consists of an amino-terminal HEAT repeat domain, followed by a four-helix bundle and two ABC-type ATPase domains, with a chromodomain inserted in ABC2. Moreover, we present the cryo-electron microscopy structure of the ATP-bound form of eEF3 in complex with the post-translocational-state 80S ribosome from yeast. eEF3 uses an entirely new factor binding site near the ribosomal E-site, with the chromodomain likely to stabilize the ribosomal L1 stalk in an open conformation, thus allowing tRNA release.Protein synthesis requires, in general, only two canonical GTPase elongation factors. eEF1A (known as EF-Tu in prokaryotes) recruits cognate aminoacyl-tRNAs to the A-site of the ribosome, and, after peptidyl transfer, eEF2 (EF-G in prokaryotes) catalyses translocation of the messenger RNA and the transfer RNAs from the A-and P-sites to the P-and E-sites. In contrast to the canonical factors, eEF3 has an ATPase activity that is stimulated by ribosomes. It interacts with both ribosomal subunits 1-3 , competes with eEF2 for binding to ribosomes, and stimulates eEF1A-dependent binding of cognate aminoacyltRNA to the ribosomal A-site 1,4 . Because, according to the allosteric three-site model of the ribosomal elongation cycle, E-site release is required for efficient A-site binding, it has been suggested that eEF3 functions as a so-called 'E-site factor' 4,5 . Moreover, ATP hydrolysis by eEF3 is required in every elongation cycle to allow chasing of deacyltRNA from the E-site 4 .eEF3 belongs to the family of ABC (ATP-binding cassette) proteins that includes proteins involved in transport across membranes, DNA repair, and translation. The membrane proteins of this class especially represent important targets for development of novel therapeutic strategies. The proteins contain ATP/ADP-binding ABC domains, which convert chemical energy derived from binding of ATP or its hydrolysis into a 'powerstroke' of mechanical energy 6 . ABC proteins function as either homodimers or as twin-cassette proteins with two ABC domains within the same polypeptide.The ribosome exhibits very dynamic behaviour, such as the ratchet movement 7 or the movement of the L1 and the L7/L12 stalks [8][9][10][11] . Hence, an intriguing question is how the interaction of eEF3 with the ribosome is correlated with its dynamic properties as an ABC protein, and how the energy derived from binding/hydrolysis of ATP is used for its function. Crystal structure of eEF3Three crystal structures of residues 1-980 of eEF3 in the apo state (2.7 Å ), in complex with ADP (2.4 Å ), or in complex with the nonhydrolysable ATP analogue ADPNP (3...

Eukaryote-specific extensions in ribosomal proteins of the small subunit: Structure and function

“…14,26,30,31,47,69,71 One of the key features differentiating eukaryotic from prokaryotic ribosomes is the extent of protein-protein interactions on the ribosome surface. It is believed that this structural complexity is directly related to the evolution of the translation apparatus, including appearance of new translation factors and multiple sophisticated mechanisms of translation control that are absent in prokaryotic cells.…”

“…70 eEF3 binds near the E-site and is necessary for clearance of the deacylated tRNA from the ribosome. 70,71 Cryo-EM analysis of the eEF3-80S complex (at 9.9A ) showed that the factor binds near the head of the 40S ribosomal subunit and makes contacts with both the 40S and 60S subunits. 71 The so-called HEAT domain of eEF3 was mapped to interact with eukaryote-specific extensions of uS13 and 2 protein-rich regions (unassigned due to low resolution) termed rpSX1 and rpSX2.…”

“…70,71 Cryo-EM analysis of the eEF3-80S complex (at 9.9A ) showed that the factor binds near the head of the 40S ribosomal subunit and makes contacts with both the 40S and 60S subunits. 71 The so-called HEAT domain of eEF3 was mapped to interact with eukaryote-specific extensions of uS13 and 2 protein-rich regions (unassigned due to low resolution) termed rpSX1 and rpSX2. 71 However, an X-ray structure of the yeast ribosome subsequently demonstrated that these regions are occupied by eukaryote-specific extensions of the conserved ribosomal proteins uS7, uS10 and uS19, respectively.…”

“…71 The so-called HEAT domain of eEF3 was mapped to interact with eukaryote-specific extensions of uS13 and 2 protein-rich regions (unassigned due to low resolution) termed rpSX1 and rpSX2. 71 However, an X-ray structure of the yeast ribosome subsequently demonstrated that these regions are occupied by eukaryote-specific extensions of the conserved ribosomal proteins uS7, uS10 and uS19, respectively. 6 In line with this observation, biochemical analyses showed that deletions/mutations of the uS7 yeast-specific Nterminus affects association of eEF3 with the 80S subunit and results in elongation defects.…”

Rustless translation

“…Research by many colleagues and ourselves extended this analysis to complete ABC proteins (e.g., Lamers et al , 2000 ;Obmolova et al , 2000 ;Locher et al , 2002 ;Andersen et al , 2006 ;Dawson and Locher , 2006 ;Lammens et al , 2011 ;Lim et al , 2011 ), and there is now a good understanding of the principal architecture and ATP-dependent structural dynamics of a variety of ABC proteins ( Figure 1A). A pair of NDBs undergoes conformational cycles by ATP driven engagement and disengagement, which impacts on the structure and substrate binding properties of the NBDs and its function-specific associated domains.…”