The Elongation Factor 3 Unique in Higher Fungi and Essential for Protein Biosynthesis Is an E Site Factor

Triana-Alonso, Francisco J.; Chakraburtty, Kalpana; Nierhaus, Knud H.

doi:10.1074/jbc.270.35.20473

Cited by 147 publications

(149 citation statements)

References 34 publications

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“…5) showed weaker binding. As suggested before, this indicates that eEF3 binds to post-state ribosomes, and that ATP hydrolysis is required for eEF3 dissociation from the ribosome 4 .…”

Section: Saxs Analysis Of Eef3mentioning

confidence: 93%

“…It interacts with both ribosomal subunits [1][2][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 .…”

mentioning

confidence: 99%

“…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 .…”

mentioning

confidence: 99%

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

Andersen

Becker

Blau

et al. 2006

Nature

152

166

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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...

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“…5) showed weaker binding. As suggested before, this indicates that eEF3 binds to post-state ribosomes, and that ATP hydrolysis is required for eEF3 dissociation from the ribosome 4 .…”

Section: Saxs Analysis Of Eef3mentioning

confidence: 93%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Andersen

Becker

Blau

et al. 2006

Nature

152

166

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“…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.…”

Section: Interaction Of 40s Ribosomal Proteins With Eef3mentioning

confidence: 99%

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

Ghosh

Komar

2015

Translation

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Keywords: conserved ribosomal proteins, eukaryotic/yeast ribosome, eukaryote-specific extensions, eukaryotic translation initiation factors, protein synthesis, small ribosomal subunitHigh-resolution structures of yeast ribosomes have improved our understanding of the architecture and organization of eukaryotic rRNA and proteins, as well as eukaryote-specific extensions present in some conserved ribosomal proteins. Despite this progress, assignment of specific functions to individual proteins and/or eukaryotespecific protein extensions remains challenging. It has been suggested that eukaryote-specific extensions of conserved proteins from the small ribosomal subunit may facilitate eukaryote-specific reactions in the initiation phase of protein synthesis. This review summarizes emerging data describing the structural and functional significance of eukaryotespecific extensions of conserved small ribosomal subunit proteins, particularly their possible roles in recruitment and spatial organization of eukaryote-specific initiation factors.

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“…The in vitro-synthesized tRNA f Met showed clear mRNAdependent P site binding, comparing well with the in vivogenerated tRNA (data not shown). Templates for the MFmRNA were generated by linearizing plasmid pTZ-MF [kindly provided by K. Nierhaus, (17)] with SspI.…”

Section: Generation Of Phosphorothioate-containing Trnas Andmentioning

confidence: 99%

Identification of specific Rp-phosphate oxygens in the tRNA anticodon loop required for ribosomal P-site binding

Schnitzer

Ahsen

1997

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACTtRNA binding to the ribosomal P site is dependent not only on correct codon-anticodon interaction but also involves identification of structural elements of tRNA by the ribosome. By using a phosphorothioate substitutioninterference approach, we identified specific nonbridging Rp-phosphate oxygens in the anticodon loop of tRNA Phe from Escherichia coli which are required for P-site binding. Stereospecific involvement of phosphate oxygens at these positions was confirmed by using synthetic anticodon arm analogues at which single Rp-or Sp-phosphorothioates were incorporated. Identical interference results with yeast tRNA Phe and E. coli tRNA f Met indicate a common backbone conformation or common recognition elements in the anticodon loop of tRNAs. N-ethyl-N-nitrosourea modification-interference experiments with natural tRNAs point to the importance of the same phosphates in the loop. Guided by the crystal structure of tRNA Phe , we propose that specific Rp-phosphate oxygens are required for anticodon loop (''U-turn'') stabilization or are involved in interactions with the ribosome on correct tRNAmRNA complex formation.

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The Elongation Factor 3 Unique in Higher Fungi and Essential for Protein Biosynthesis Is an E Site Factor

Cited by 147 publications

References 34 publications

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

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

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

Identification of specific Rp-phosphate oxygens in the tRNA anticodon loop required for ribosomal P-site binding

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