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The Elongation, Termination, and Recycling Phases of Translation in Eukaryotes

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The Elongation, Termination, and Recycling Phases of Translation in Eukaryotes Downloaded from http://cshperspectives.cshlp.org/ on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press The Elongation, Termination, and Recycling Phases of Translation in Eukaryotes Thomas E. Dever1 and Rachel Green2 1Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 2Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Correspondence: [email protected]; [email protected] This work summarizes our current understanding of the elongation and termination/recy- cling phases of eukaryotic protein synthesis. We focus here on recent advances in the field. In addition to an overview of translation elongation, we discuss unique aspects of eukaryotic translation elongation including eEF1 recycling, eEF2 modification, and eEF3 and eIF5A function. Likewise, we highlight the function of the eukaryotic release factors eRF1 and eRF3 in translation termination, and the functions of ABCE1/Rli1, the Dom34:Hbs1 complex, and Ligatin (eIF2D) in ribosome recycling. Finally, we present some of the key questions in translation elongation, termination, and recycling that remain to be answered. he mechanism of translation elongation is triggers GTP hydrolysis by eEF1A, releasing Twell conserved between eukaryotes and bac- the factor and enabling the aminoacyl-tRNA to teria (Rodnina and Wintermeyer 2009), and, in be accommodated into the A site. Recent high- general, studies on the mechanism of transla- resolution structures of the bacterial ribosome tion elongation have focused on bacterial systems. bound to EF-Tu and aminoacyl-tRNA revealed Following translation initiation, an 80S ribo- distortion of the anticodon stem and at the some is poised on a messenger RNA (mRNA) junction between the acceptor and D stems that with the anticodon of Met-tRNAi in the P site enables the aminoacyl-tRNA to interact with base-paired with the start codon. The second both the decoding site on the small subunit and codon of the open reading frame (ORF) is pre- with EF-Tu. It is thought that the energetic pen- sent in the A (acceptor) site of the ribosome alty for this distortion is paid for by the perfect awaiting binding of the cognate aminoacyl- codon–anticodon match and the attendant sta- tRNA. The eukaryotic elongation factor eEF1A, bilizing interactions that occur between the A the ortholog of bacterial EF-Tu, binds amino- site and cognate tRNA to promote high-fidelity acyl-tRNA in a GTP-dependent manner and decoding (Schmeing et al. 2009, 2011). These then directs the tRNA to the A site of the ribo- interactions might exceed those involving 16S some (Fig. 1). Codon recognition by the tRNA rRNA bases A1492, A1493, and G530 with the Editors: John W.B. Hershey, Nahum Sonenberg, and Michael B. Mathews Additional Perspectives on Protein Synthesis and Translational Control available at www.cshperspectives.org Copyright # 2012 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a013706 Cite this article as Cold Spring Harb Perspect Biol 2012;4:a013706 1 Downloaded from http://cshperspectives.cshlp.org/ on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press T.E. Dever and R. Green Aminoacyl-tRNA EF1A EF1A EPA Aminoacyl-tRNA binding eEF1B EF2 EF1A EF1A Deacyl-tRNA EPA Translocation EF2 A-site tRNA accommodation EPA EPA EF2 Peptide bond formation EPA Figure 1. Model of the eukaryotic translation elongation pathway. In this model the large ribosomal subunit is drawn transparent to visualize tRNAs, factors, and mRNA binding to the decoding center at the interface between the large and small subunits and tRNAs interacting with the peptidyl transferase center in the large subunit. Starting at the top, an eEF1A.GTP.aminoacyl-tRNA ternary complex binds the aminoacyl-tRNA to the 80S ribosome with the anticodon loop of the tRNA in contact with the mRNA in the A site of the small subunit. Following release of eEF1A.GDP,the aminoacyl-tRNA is accommodated into the A site, and the eEF1A.GDP is recycled to eEF1A.GTP by the exchange factor eEF1B. Peptide bond formation is accompanied by transition of the A- and P-site tRNAs into hybrid states with the acceptors ends of the tRNAs moving to the P and E sites, respectively. Binding of eEF2.GTP promotes translocation of the tRNAs into the canonical P and E sites, and is followed by release of eEF2.GDP,which unlike eEF1A does not require an exchange factor. The ribosome is now ready for the next cycle of elongation with release of the deacylated tRNA from the E site and binding of the appropriate eEF1A.GTP.aminoacyl-tRNA to the A site. Throughout, GTP is depicted as a green ball and GDPas a red ball; also, the positions of the mRNA, tRNAs, and factors are drawn for clarity and are not meant to specify their exact places on the ribosome. minor groove of the codon–anticodon helix coordinate and position the water molecule re- (Ogle et al. 2001) to include residues in riboso- quired forGTP hydrolysis(Voorheesetal. 2010). malproteins andother regionsof the tRNA (Jen- It is expected that these mechanisms of initial ner et al. 2010). The recent structures of the ri- aminoacyl-tRNA binding, codon recognition, bosome bound to EF-Tu and aminoacyl-tRNA and GTPase activation will be shared between alsorevealedthat theconservednucleotideA2662 bacteria and eukaryotes. (Thermus thermophilus numbering) in the sar- Following accommodation of the amino- cin–ricin loop of 23S rRNA in the large subunit acyl-tRNA into the A site, peptide bond for- interacts with the conserved catalytic Hisresidue mation with the P-site peptidyl-tRNA occurs in the G domain enabling the His residue to rapidly. The peptidyl transferase center (PTC), 2 Cite this article as Cold Spring Harb Perspect Biol 2012;4:a013706 Downloaded from http://cshperspectives.cshlp.org/ on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Translation in Eukaryotes consisting primarily of conserved ribosomal strictly coupled to binding of aminoacyl-tRNA RNA (rRNA) elements on the large ribosomal in the A site (Semenkov et al. 1996; Uemura subunit, positions the substrates for catalysis. et al. 2010; Chen et al. 2011). Recent crystal structures of the Saccharomyces Although these basic mechanisms of trans- cerevisiae 80S ribosome and the T. thermophila lation elongation and peptide bond formation 60S subunit revealed that the rRNA structure of are conserved between bacteria and eukaryotes, the PTC is nearly superimposable between the several features of translation elongation are eukaryotic and bacterial ribosomes (Ben-Shem unique in eukaryotes. Moreover, recent studies et al. 2010, 2011; Klinge et al. 2011), supporting have characterized additional factors that may the idea that the mechanism of peptide bond function in translation elongation. formation, the heart of protein synthesis, is uni- versally conserved. eEF1 RECYCLING Following peptide bond formation, ratchet- ing of the ribosomal subunits triggers move- Following GTP hydrolysis both EF-Tu and ment of the tRNAs into so-called hybrid P/E eEF1A are released from the ribosome in com- and A/P states with the acceptor ends of the plex with GDP. The spontaneous rate of GDP tRNAs in the E and P-sites and the anticodon dissociation from these factors is slow, and a loops remaining in the P and A sites, respective- guanine nucleotide exchange factor is required ly. Translocation of the tRNAs to the canonical E to recycle the inactive GDP-bound factor to its and P sites requires the elongation factor eEF2 in active GTP-bound state. In the case of EF-Tu,the eukaryotes, which is the ortholog of bacterial single polypeptide factor EF-Tspromotes nucle- EF-G. Binding of the GTPase eEF2 or EF-G in otideexchange. Incontrast,theeukaryoticfactor complex with GTP is thought to stabilize the eEF1B, composed of two to four subunits, cata- hybrid state and promote rapid hydrolysis of lyzes guanine nucleotide exchange on eEF1A. GTP. Conformational changes in eEF2/EF-G Interestingly, despite the strong homology be- accompanying GTP hydrolysis and Pi release tween eEF1A and EF-Tu, the catalytic eEF1Ba are thought to alternatively unlock the ribo- subunit does not resemble EF-Ts and the two some allowing tRNA and mRNA movement proteins promote guanine nucleotide exchange and then lock the subunits in the posttranslo- by distinct mechanisms (Rodnina and Winter- cation state. Pi release is also coupled to release meyer 2009). Whereas EF-Ts binds to the EF- of the factor from the ribosome. A structure of Tu G domain and indirectly destabilizes Mg2þ EF-G bound to a posttranslocation bacterial ri- binding leading to GDP release, eEF1Ba inserts bosome revealed the interaction of EF-G do- an essential Lys residue into the Mg2þ and g- main IV with the mRNA, P-site tRNA, and de- phosphate binding site to directly destabilize coding center on the small ribosomal subunit Mg2þ binding. It is unclear why eukaryotes use (Gao et al. 2009), consistent with the notion a more elaborate exchange factor, although it that EF-G and eEF2 function, at least in part, might provide a means to regulate translation to prevent backward movement of the tRNAs in elongation (Sivan et al. 2011). the unlocked state of the ribosome. In the posttranslocation state of the ribo- eEF2 MODIFICATION some, a deacylated tRNA occupies the E site and the peptidyl-tRNA is in the P site. The A As noted above, eEF2 and EF-G promote trans- site is vacant and available for binding of the location by binding to the ribosome and insert- next aminoacyl-tRNA in complex with eEF1A. ing domain IV of the factor into the decoding Although it has been proposed that E-site tRNA center of the small subunit. Interestingly, a con- release is coupled to binding of aminoacyl- served His residue (His699 in yeast eEF2) at the tRNA in the A site (Nierhaus 1990), recent sin- tip of domain IV of eEF2 is posttranslationally gle molecule and ensemble kinetic analyses modified to diphthamide.
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