Three tRNA Binding Sites in Rabbit Liver Ribosomes and Role of the Intrinsic ATPase in 80S Ribosomes from Higher Eukaryotes

El'skaya, A. V.; Ovcharenko, G. V.; Palchevskii, S. S.; Petrushenko, Zoya M.; Triana-Alonso, Francisco J.; Nierhaus, Knud H.

doi:10.1021/bi970631e

Cited by 39 publications

(22 citation statements)

References 22 publications

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“…As expected, on medium-containing glucose, i.e., no eEF3 overexpression, the strain harboring the GCN2 c allele showed reduced growth compared with the wild-type control ( Fig. 2A, left panel, rows 1-4 versus rows [5][6][7][8]. On galactose, however, the GCN2 c strain overexpressing eEF3 showed improved growth as compared with the GCN2 c strain overexpressing GST alone ( Fig.…”

Section: Eef3 Overexpression Elicits Sensitivity To 3-amino-24-triazsupporting

confidence: 74%

“…eEF3 is a fungal specific elongation factor, and it has been proposed that in nonfungal eukaryotes a factor permanently attached to the ribosome executes the function of eEF3 (5). We believe that our model may also be applicable to nonfungal eukaryotes.…”

Section: Discussionmentioning

confidence: 89%

“…However, the high conservation of the translational process implies that nonfungal organisms must have a factor with a function equivalent to that of eEF3. Supporting this idea, it was found that metazoan ribosomes have an intrinsic ATPase activity that is absent in yeast ribosomes (5), suggesting that in metazoans a ribosome bound factor executes the eEF3-like function rather than a soluble factor. Interestingly, in Escherichia coli the protein RbbA (ribosome-bound ATPase) was identified that exhibits ATPase activity and is tightly associated with ribosomes (6,7) and thus may have an eEF3-like function.…”

mentioning

confidence: 97%

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Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation

Visweswaraiah

Lee

Hinnebusch

et al. 2012

Journal of Biological Chemistry

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Section: Eef3 Overexpression Elicits Sensitivity To 3-amino-24-triazsupporting

confidence: 74%

Section: Discussionmentioning

confidence: 89%

mentioning

confidence: 97%

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Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation

Visweswaraiah

Lee

Hinnebusch

et al. 2012

Journal of Biological Chemistry

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“…The eEF1A preparation, containing approximately one molecule of the fluorescence reagent (FITC) per one protein molecule was obtained using an optimized labeling procedure. The functional activity of the FITC-modified eEF1A was verified by two independent techniques: the GDP/[ 3 H]GDP exchange and stimulation of poly(Phe) synthesis on poly(U)-programmed 80S ribosomes in reconstituted cell-free translation system [26]. The FITC-eEF1A activity was found to be 85-95% of the native protein activity in both tests (data not shown).…”

Section: Stability Of the [Eef1aaegdp/gmp-pnpaetrna] Complexesmentioning

confidence: 95%

Novel complexes of mammalian translation elongation factor eEF1A·GDP with uncharged tRNA and aminoacyl‐tRNA synthetase

Petrushenko

Budkevich

Shalak

et al. 2002

European Journal of Biochemistry

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Multimolecular complexes involving the eukaryotic elongation factor 1A (eEF1A) have been suggested to play an important role in the channeling (vectorial transfer) of tRNA during protein synthesis [Negrutskii, B.S. & El'skaya, A.V. (1998) Prog. Nucleic Acids Res. Mol. Biol. 60, 47–78]. Recently we have demonstrated that besides performing its canonical function of forming a ternary complex with GTP and aminoacyl‐tRNA, the mammalian eEF1A can produce a noncanonical ternary complex with GDP and uncharged tRNA [Petrushenko, Z.M., Negrutskii, B.S., Ladokhin, A.S., Budkevich, T.V., Shalak, V.F. & El'skaya, A.V. (1997) FEBS Lett. 407, 13–17]. The [eEF1A·GDP·tRNA] complex has been hypothesized to interact with aminoacyl‐tRNA synthetase (ARS) resulting in a quaternary complex where uncharged tRNA is transferred to the enzyme for aminoacylation. Here we present the data on association of the [eEF1A·GDP·tRNA] complex with phenylalanyl‐tRNA synthetase (PheRS), e.g. the formation of the above quaternary complex detected by the gel‐retardation and surface plasmon resonance techniques. To estimate the stability of the novel ternary and quaternary complexes of eEF1A the fluorescence method and BIAcore analysis were used. The dissociation constants for the [eEF1A·GDP·tRNA] and [eEF1A·GDP·tRNAPhe·PheRS] complexes were found to be 20 nm and 9 nm, respectively. We also revealed a direct interaction of PheRS with eEF1A in the absence of tRNAPhe (Kd = 21 nm). However, the addition of tRNAPhe accelerated eEF1A·GDP binding to the enzyme. A possible role of these stable novel ternary and quaternary complexes of eEF1A·GDP with tRNA and ARS in the channeled elongation cycle is discussed.

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“…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).…”

mentioning

confidence: 99%

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

Anand¹,

Balar²,

Ulloque³

et al. 2006

Journal of Biological Chemistry

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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, ...

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Three tRNA Binding Sites in Rabbit Liver Ribosomes and Role of the Intrinsic ATPase in 80S Ribosomes from Higher Eukaryotes

Cited by 39 publications

References 22 publications

Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation

Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation

Novel complexes of mammalian translation elongation factor eEF1A·GDP with uncharged tRNA and aminoacyl‐tRNA synthetase

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

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