Domain and Nucleotide Dependence of the Interaction between Saccharomyces cerevisiae Translation Elongation Factors 3 and 1A

Abstract

Eukaryotic translation elongation factor 3 (eEF3) is a fungalspecific ATPase proposed to catalyze the release of deacylatedtRNA from the ribosomal E-site. In addition, it has been shown to interact with the aminoacyl-tRNA binding GTPase elongation factor 1A (eEF1A), perhaps linking the E and A sites. Domain mapping demonstrates that amino acids 775-980 contain the eEF1A binding sites. Domain III of eEF1A, which is also involved in actin-related functions, is the site of eEF3 binding. The binding of eEF3 to eEF1A is enhanced by ADP, indicating the interaction is favored post-ATP hydrolysis but is not dependent on the eEF1A-bound nucleotide. A temperaturesensitive P915L mutant in the eEF1A binding site of eEF3 has reduced ATPase activity and affinity for eEF1A. These results support the model that upon ATP hydrolysis, eEF3 interacts with eEF1A to help catalyze the delivery of aminoacyl-tRNA at the A-site of the ribosome. The dynamics of when eEF3 interacts with eEF1A may be part of the signal for transition of the post to pre-translocational ribosomal state in yeast.The protein synthetic machinery is characterized by the interplay of different soluble factors in conjunction with ribosomes to translate the mRNA into the correct sequence of amino acids. The three phases of translation, initiation, elongation, and termination, are driven by factors that are highly conserved between yeast and metazoans (1). However, a major difference in elongation is the indispensability of eukaryotic elongation factor 3 (eEF3) 3 with yeast ribosomes (2, 3). eEF3 catalyzes an essential step in each elongation cycle by virtue of its ATPase activity. It has been proposed to act as an Exit-site (E-site) factor, facilitating the release of deacylated-tRNA and simultaneously impacting on the delivery of aminoacyl-tRNA (aa-tRNA) at the aminoacyl site (A-site) (4). Metazoan ribosomes have been reported to possess a compensatory intrinsic ATPase activity, although they differ kinetically from the fungal eEF3 (5). Escherichia coli, on the other hand, expresses the 911 amino acid RbbA protein that exhibits ATPase activity and is tightly associated with ribosomes (6, 7). Both pathogenic and non-pathogenic fungi have been reported to contain eEF3 (8 -10). In Saccharomyces cerevisiae, eEF3 is encoded by a single copy essential YEF3 gene. A paralog of the YEF3 gene, designated HEF3 or YEF3B, encodes an 84% identical protein but is not expressed during vegetative growth (11). However, expression of the HEF3 coding sequence under the YEF3 promoter produces a protein that has similar ATPase activity and ribosome binding properties to YEF3-encoded eEF3.eEF3 is a class 1 member of the ATP binding cassette (ABC) family of proteins. eEF3 possesses distinct motifs including the HEAT repeats on the N terminus, two nucleotide binding domains with tandemly arranged bipartite (ABC) cassettes in the middle, a conserved insertion in the intervening region of the Walker A and B motifs of ABC2, and a highly basic C terminus. HEAT (Huntington elongation factor 3, ...

Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation

“…The resulting transformants were subjected to semiquantitative growth assays using solid medium containing galactose as carbon source, and 3AT or no 3AT as control. As reported before (27), under replete conditions eEF3 overexpression did not lead to a significant growth defect ( Fig. 1, left panel), indicating that eEF3 overexpression does not severely affect general protein synthesis or other essential processes in the cell.…”

“…However, these scenarios may be less likely considering that overexpression of eEF3 did not mitigate the toxicity of overexpressing the eIF2␣ kinase PKR when expressed in yeast instead of Gcn2. Taken together, our data provide strong evidence that eEF3 negatively regulates Gcn2 activation in a manner requiring the HEAT domain and/or requiring an accessory ribosome-binding domain at the eEF3 C terminus that is dispensable for eEF3 essential function in translation elongation (1,27). These findings provide added support for the model that Gcn2 is activated by uncharged tRNAs on elongating ribosomes and reveal an unanticipated role for a general elongation factor, in addition to eEF1A, in controlling this process.…”

“…An attractive model would be that eEF3 does not bind with high affinity to an elongating ribosome containing tRNA deacyl in the A-site, which is the relevant sub-strate for the Gcn1/20-Gcn2 complex. In fact, there is evidence that eEF3 interacts with eEF1A to help catalyze delivery of the eEF1A-GTP-amino acyl tRNA ternary complex (TC) to the ribosomal A-site (1,27). Thus, eEF3 and Gcn1/20-Gcn2 would not normally occupy the same ribosomal complexes, whereas overexpressing eEF3 would force it to bind to the stalled complexes containing tRNA deacyl in the A-site, by mass action, and thereby interfere with Gcn2 activation.…”

Mutations in the Chromodomain-like Insertion of Translation Elongation Factor 3 Compromise Protein Synthesis through Reduced ATPase Activity

“…eEF3 has been shown to promote the release of deacylated tRNA from the post-translocation ribosomal E-site in an ATPdependent manner and consequently stimulate A-site aa-tRNA binding mediated by eEF1A, supporting the allosteric three-site model of translating ribosomes (15). eEF3 has also been shown to bind directly to eEF1A in the presence of ADP, suggesting that upon ATP hydrolysis, eEF3 interacts with eEF1A to promote aa-tRNA delivery to the ribosome A-site (8).…”

A Structural Domain Mediates Attachment of Ethanolamine Phosphoglycerol to Eukaryotic Elongation Factor 1A in Trypanosoma brucei

“…Domain I harbors the GTP/GDP binding site, while binding of eEF1Bα occurs to both domains I and II [2], [4]. In addition, domain II also contains the binding site for aminoacyl-tRNAs [5]–[7], whereas in yeast domain III represents the binding site for elongation factor 3 (eEF3) [8].…”