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The Mechanism of Eukaryotic Translation Initiation: New Insights and Challenges

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The Mechanism of Eukaryotic Translation Initiation: New Insights and Challenges Downloaded from http://cshperspectives.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press The Mechanism of Eukaryotic Translation Initiation: New Insights and Challenges Alan G. Hinnebusch1 and Jon R. Lorsch2 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 Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Correspondence: [email protected]; [email protected] Translation initiation in eukaryotes is a highly regulated and complex stage of gene expres- sion. It requires the action of at least 12 initiation factors, many of which are known to be the targets of regulatory pathways. Here we review our current understanding of the molecular mechanics of eukaryotic translation initiation, focusing on recent breakthroughs from in vitro and in vivo studies. We also identify important unanswered questions that will require new ideas and techniques to solve. his work aims to present the current state of current paradigm outlined below (Hinnebusch Tour knowledge of the molecular mechanics et al. 2007; Pestova et al. 2007; Lorsch and Dever of translation initiation in eukaryotes. Wefocus 2010; Hinnebusch 2011; Parsyan et al. 2011). on advances that have taken place over the last Identification of the initiation codon by the few years and, because of space limitations, as- eukaryotic translational machinery begins with sume readers will be able to find referencesto the binding of the ternary complex (TC) consisting foundational literature for the field (published of initiator methionyl-tRNA (Met-tRNAi) and before 2000) in the more recent works that are the GTP-bound form of eukaryotic initiation cited here. As always, we apologize for not hav- factor 2 (eIF2) to the small (40S) ribosomal ing the space to cite many important works. subunit to form the 43S preinitiation complex Please view this as merely an introduction to (PIC). Binding of the TC to the 40S subunit is the field rather than a complete summary. promoted by eIFs 1, 1A, 5, and the eIF3 complex (Fig. 1). A network of physical interactions links eIFs 1, 3, 5, and TC in a multifactor complex OVERVIEW OF THE INITIATION PATHWAY (MFC) in yeast (Asano et al. 2000), plants (Den- Figure 1 presents the basic outline of the eukary- nis et al. 2009), and mammals (Sokabe et al. otic cap-dependent initiation pathway, and the 2011). In budding yeast, the MFC enhances reader is referred to a number of recent reviews the formation or stability of the 43S PIC in that summarize the evidence supporting the vivo (reviewed in Hinnebusch et al. 2007). 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. Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011544 1 Downloaded from http://cshperspectives.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press A.G. Hinnebusch and J.R. Lorsch elFs 1,1A, 3 3 EPA 1 1A 80S 60S + 40S 5 1A Ternary complex (TC) 7 elF2 elF2 mRNA m G AUG (A) n 3 5 1 ATP elFs 4A, 4B, 4E, 4G ADP PABA elF2 PABP (A) AAAAAAAAAAAAAAAAAAAAAAAAAAA 3 5 n 1A 4G 1 4A 43S m7G 4B AUG mRNA activation PABP Met-tRNA Met elF2 i (A)nAAAAAAAAAAAAAAAAAAAAAAAAAA 4G 5 43S•mRNA 4A 4B 3 1A m7G 1 AUG ATP elF2 Scanning elF2•GTP ADP P PABP i + elF2 (A)nAAAAAAAAAAAAAAAAAAAAAAAAAAA A elF2B 4G 5 4A 3 1A m7G 4B 1 AUG ATP elF2•GDP elF2 elF2 elF2α ADP kinase P Pi PABP elF2 (A)nAAAAAAAAAAAAAAAAAAAAAAAAAAAA A 4G 3 5 48S 7 4A 4B 1A m G AUG 1 AUG recognition 5B elF5B•GTP elF2 PABP elF2•GDP (A)nAAAAAAAAAAAAAAAAAAAAAAAAAAA A 4G 4A 3 7 4B 1A m G AUG 5B 60S elFs Subunit joining PABP (A)nAAAAAAAAAAAAAAAAAAAAAAAAAA A 4G 4A m7G 4B AUG Elongation 80S Figure 1. Model of canonical eukaryotic translation initiation pathway. The pathway is shown as a series of discrete steps starting with dissociation of 80S ribosomes into subunits. Binding of factors is depicted both as a single step via the multifactor complex and as two separate steps, with eIFs 1, 1A, and 3 binding first followed by binding of ternary complex and eIF5. The resulting 43S preinitiation complex (PIC) is then loaded onto an activated mRNP near the 50 cap. (Legend continues on facing page.) 2 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011544 Downloaded from http://cshperspectives.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Mechanism of Eukaryotic Translation Initiation The 43S PIC binds to the messenger RNA initiation complex (IC) containing Met-tRNAi (mRNA) near the 50-7-methylguanosine cap in base-paired to AUG in the P site and ready to a process facilitated by eIF3, the poly(A)-bind- begin the elongation phase of protein synthesis ing protein (PABP), and eIFs 4B, 4H (in mam- (Fig. 1) (reviewed in Hinnebusch et al. 2007; mals), and 4F. The eIF4F complex is comprised Pestova et al. 2007; Hinnebusch 2011). of the cap-binding protein eIF4E, eIF4G, andthe RNA helicase eIF4A. eIF4G is a scaffold protein that harbors binding domains for PABP, eIF4E, RECRUITMENT OF Met-tRNAi TO eIF4A, and (in mammals) eIF3. Both yeast and THE 40S RIBOSOMAL SUBUNIT human eIF4G also bind RNA. The binding do- eIF2 Is a G-Protein Switch that Carries mains for eIF4E and PABP in eIF4G, along with Met-tRNA onto the Ribosome its RNA-binding activity, enable eIF4G to coor- i dinate independent interactions with mRNAvia The Met-tRNAi is delivered to the 40S subunit in the cap, poly(A) tail, and sequences in the theTCwitheIF2.GTP.TheaffinityofMet-tRNAi mRNA body to assemble a stable, circular mes- is greater for eIF2.GTP than for eIF2.GDP, and senger ribonucleoprotein (mRNP), referred to this affinity switch depends on the methionine as the “closed-loop” structure. The eIF4G–eIF3 moiety on the Met-tRNAi (Kapp and Lorsch interaction is expected to establish a protein 2004). This contribution of methionine, plus bridge between this “activated mRNP” and the the stimulatory role of the unique A1:U72 base 43S PIC to stimulate 43S attachment to the pair (bp) in the acceptor stem of tRNAi in bind- mRNA, and the helicase activity of eIF4A is ing eIF2 (Kapp and Lorsch 2004; Pestova et thought to generate a single-stranded landing al. 2007), presumably act to prevent binding of pad in the mRNA on which the 43S PIC can elongator tRNAs to the factor. This specificity, load (reviewed in Hinnebusch et al. 2007; Pes- coupled with the requirement for eIF2 to load tova et al. 2007; Lorsch and Dever 2010; Hinne- tRNA onto the 40S subunit, is thought to elim- busch 2011). inate the need for a mechanism to reject elonga- Once bound near the cap, the 43S PIC scans tor tRNAs during PIC assembly, a process in the mRNA leader for an AUG codon in a suit- bacteria that relies heavily on IF3 (Hershey and able sequence context. Base-pairing between Merrick 2000). (As described below, a structural the anticodon of Met-tRNAi and the AUG in homolog of IF3 is absent in eukaryotes, but eIF1 the peptidyl-tRNA (P) site of the 40S subunit acts similarly to ensure selection of AUG as a is the initial event in start codon recognition start codon.) Understanding the structural basis (Lomakin et al. 2006; Kolitz et al. 2009; Hinne- for the stimulatory effects of methionine, the busch 2011). AUG recognition causes arrest of A1:U72 bp, and GTP versus GDP on initiator the scanning PIC and triggers conversion of tRNA binding to eIF2 would be advanced by eIF2 in the TC to its GDP-bound state via gated high-resolution structural analysis of the com- phosphate (Pi) release and the action of the plete TC. Whereas the crystal structure of the GTPase-activating (GAP) factor eIF5. Following archaeal ortholog (aIF2) has been solved, as release of eIF2.GDP and several other eIFs pre- well as various aIF2 subcomplexes bound to sent in the PIC, joining of the large (60S) sub- GDP or GTP analogs (reviewed in Schmitt unit is catalyzed by eIF5B to produce an 80S et al. 2010), no crystal structures or cryo-EM Figure 1. (Continued) Subsequent scanning of the mRNA allows recognition of the start codon, which triggers downstream steps in the pathway including eIF1 release from the PIC, Pi release from eIF2, and conversion to the closed, scanning-arrested state of the complex. eIF2.GDP released after subunit joining is recycled back to eIF2.GTP by the exchange factor eIF2B. eIF5B in its GTP-bound form promotes joining of the 60S subunit to the preinitiation complex, which triggers release of eIF5B.GDP and eIF1A to form the final 80S initiation complex, which can begin the elongation phase of protein synthesis. Throughout, GTP is depicted as a green ball and GDP as a red ball. (Modified from Hinnebusch 2011; reproduced, with permission, from the author.) Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a011544 3 Downloaded from http://cshperspectives.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press A.G. Hinnebusch and J.R. Lorsch (electron microscopy) models of heterotrimeric whether they are important in solution and eIF2 have been described. conserved in eukaryotic TC. eIF2g binds directly to both GTP and Met- Importantly, the patterns of free radical-in- tRNAi and it appears that the a and b subunits duced cleavages of 18S rRNA observed in this each increase the affinity of the eIF2 complex last study suggested that eIF2g domain III inter- for Met-tRNAi by 100-fold (Naveau et al.
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