P i Release from eif2, Not GTP Hydrolysis, Is the Step Controlled by Start-Site Selection during Eukaryotic Translation Initiation

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1 Molecular Cell, Vol. 20, , October 28, 2005, Copyright 2005 by Elsevier Inc. DOI /j.molcel P i Release from eif2, Not GTP Hydrolysis, Is the Step Controlled by Start-Site Selection during Eukaryotic Translation Initiation Mikkel A. Algire, David Maag, and Jon R. Lorsch* Department of Biophysics and Biophysical Chemistry Johns Hopkins University School of Medicine Baltimore, Maryland Summary Irreversible GTP hydrolysis by eif2 is a critical step in translation initiation in eukaryotes because it is thought to commit the translational machinery to assembling the ribosomal complex at the selected point in the mrna. Our quantitative analysis of the steps and interactions involved in activating GTP hydrolysis by eif2 during translation initiation in vitro indicates that a structural rearrangement in the 43S preinitiation complex activates it to become fully competent to hydrolyze GTP. Contrary to the prevailing model, release of inorganic phosphate after GTP hydrolysis by eif2, not hydrolysis itself, is controlled by recognition of the AUG codon. Release of P i, which makes GTP hydrolysis irreversible, appears to be controlled by the AUG-dependent dissociation of eif1 from the preinitiation complex. Introduction Translation initiation in eukaryotes requires at least 12 initiation factors (eifs) and the hydrolysis of ATP and GTP (Kapp and Lorsch, 2004b). In the current model, the first step is formation of the eif2 GTP Met-tRNA i ternary complex (hereafter TC). The TC binds the small (40S) ribosomal subunit to form the 43S complex in a process facilitated by eifs 1 and 1A. This complex can bind to an mrna near the 5# 7-methylguanosine cap structure with the help of the eif4f group of factors, poly(a) binding protein, and eif3. The 43S complex is thought to scan along the mrna in search of the AUG start codon. Recognition of the start codon by the 43S mrna complex is believed to result in activation of GTP hydrolysis by eif2. This requires eif5, a GTPase-activating protein (GAP) for eif2. After GTP hydrolysis, eif2 GDP dissociates, leaving the Met-tRNA i in the P site of the 40S subunit. At this point, the large (60S) ribosomal subunit can be joined to the 40S MettRNA i mrna complex with help from eif5b. Following subunit joining, eif5b hydrolyzes its bound GTP and dissociates, leaving an 80S initiation complex awaiting delivery of an aminoacyl-trna to the A site. Correct selection of the start codon in the 43S mrna complex is a critical point in gene expression because it sets the reading frame in which the mrna will be translated. GTP hydrolysis by eif2 is the first irreversible step in the pathway and is thought to be ultimately responsible for selecting the codon at which to begin translation. Previous studies have indicated that eif5 is intimately involved in start-site selection (Huang et al., *Correspondence: jlorsch@jhmi.edu 1997) and acts to increase the rate of GTP hydrolysis by eif2 (Chakrabarti and Maitra, 1991; Das et al., 2001; Odom et al., 1978; Paulin et al., 2001). In the current model, GTP hydrolysis only occurs upon location of the start codon, although previous studies have only reported a weak (w3-fold) dependence on the presence of mrna or an AUG codon (Chakrabarti and Maitra, 1991; Raychaudhuri et al., 1985; Unbehaun et al., 2004). It was proposed that eif1 is a negative regulator of GTP hydrolysis by eif2 (Valasek et al., 2004) and that a reduction in affinity of the 43S mrna complex for eif1 upon start codon identification that results in its release or movement is the trigger for GTP hydrolysis (Maag et al., 2005; Unbehaun et al., 2004; Valasek et al., 2004), although the molecular details of this process remain mysterious. We have conducted a quantitative analysis of the kinetic and thermodynamic parameters governing eif5- promoted GTP hydrolysis by eif2 using a reconstituted yeast translation initiation system (Algire et al., 2002). GTP hydrolysis by eif2 is cooperatively enhanced by incorporation of the TC into the 43S mrna complex and binding of eif5. We provide evidence that a structural rearrangement takes place within the 43S complex that activates it for rapid eif5-dependent GTP hydrolysis, even in the absence of mrna. We have found that the release of inorganic phosphate (P i ) from eif2 is highly dependent on the presence of an AUG codon in the mrna, while the actual GTP hydrolysis step is only slightly dependent. The data also indicate that eif1 release from the 43S mrna complex in response to start codon selection regulates the release of P i from eif2. Results Thermodynamics of the Interactions between eif5 and eif2 Complexes Proper formation of the complex between eif5 and eif2 is thought to be necessary for eif5 to act as a GAP (Asano et al., 1999; Chaudhuri et al., 1994). We have used a fluorescence-anisotropy-based binding assay for measuring the affinity between eif5 and eif2. A fluorescein-conjugated dipeptide (Cys-Lys- NH-fluorescein) was attached to eif5 at its C terminus (eif5-fl) using expressed protein ligation (Muir et al., 1998), as described previously for labeling of eifs 1 and 1A (Maag and Lorsch, 2003). A C-terminal FLAG-epitopetagged version of eif5 has been shown to fully complement an eif5 null yeast strain (Asano et al., 1999), indicating that eif5 can tolerate even moderate-length C-terminal extensions and suggesting that the CK-fluorescein dipeptide should not perturb its function. Consistent with this, eif5-fl behaves identically to the wt factor in our eif2 GTPase assays (data not shown), demonstrating that the fluorescein label does not interfere with the GAP function of the factor. We measured the binding affinity of eif5 for three eif2 complexes thought to be important during translation initiation: eif2 GDP, ternary complex (TC), and

2 Molecular Cell S mrna(aug) complex consisting of 40S eif1 eif1a mrna(aug) TC (in these binding experiments, the TC was made with GDPNP to prevent hydrolysis). The minimal model mrna used (mrna[aug]; 5#-GGA A[UC] 7 UAUG[CU] 10 C-3#) is unstructured and thus obviates the need for the 7-methylguanosine cap, poly(a) tail, and factors involved in mrna remodeling and loading, simplifying the system and allowing the steps studied in this work to be isolated (Algire et al., 2002; Lorsch and Herschlag, 1999). In this context, only eifs 1 and 1A are required for efficient formation of the 43S mrna complex (Algire et al., 2002; Maag et al., 2005). Increasing the concentrations of eif2 GDP, TC or 43S mrna(aug) complexes in the presence of limiting eif5-fl resulted in a saturable increase in fluorescence anisotropy (data not shown). The equilibrium dissociation constants (K d s) for eif5-fl binding to eif2 GDP or TC are identical (eif2 GDP K d =23±9 nm, TC K d = 23 ± 5 nm). The affinity of eif5-fl for the 43S mrna(aug) complex is increased by at least 20- fold (K d % 1 nm). We next carried out competitive binding experiments to examine the binding of unlabeled eif5 and two previously characterized eif5 mutants: a C-terminal alanine substitution mutant (eif5-7a: FI388,389AA, WL391,392AA, ESD AAA) known to negatively affect the interaction between eif5 and eif2 in vivo (Asano et al., 1999) and an N-terminal point mutation (R15M) shown to interact with eif2 but not stimulate its GTP hydrolysis activity (Das et al., 2001; Paulin et al., 2001). The unlabeled wt or mutant proteins were used to compete with eif5-fl for binding to both eif2 GDP and TC. These experiments yielded the same K d within error for the wt factor, as was measured in the direct binding assays for the labeled factor (36 ± 4 nm for eif2 GDP; 21 ± 2 nm for TC), indicating that the label does not interfere with binding between eif2 and eif5. We observed a R10-fold reduction in affinity for eif5-7a binding to both eif2 GDP and TC complexes, while the eif5-r15m point mutant bound to both eif2 GDP and TC with the same affinity as wt eif5 (30 ±5nMfor eif2 GDP; 18 ± 1 nm for TC), consistent with previous qualitative results. Activation of GTP Hydrolysis by eif5 Very little is known about how eif5 stimulates GTP hydrolysis by eif2 or how the activity of eif5 is regulated. To begin to elucidate the mechanism of this critical step, we examined activation of GTP hydrolysis by eif5 in a variety of complexes. The reconstituted system (Algire et al., 2002) has very low contaminating GTPase activity; the individual factors and 40S ribosomal subunits alone yield no hydrolysis activity above the experimental background. The background (and limit of detection of the assay) was determined by incubating free GTP in the buffer conditions used to form TC and measuring the rate of hydrolysis (Table 1). We first measured the rate constant for GTP hydrolysis by eif2 in isolated TC. In the absence of eif5, we could only obtain an upper limit on the rate constant for GTP hydrolysis because the observed rate constant is at the limit of detection for the assay (Figure 1A). When a saturating concentration of eif5 (2 M) is added, the rate constant for hydrolysis is increased at least 800-fold (Figure 1A). This is in contrast to previous reports that eif5 cannot stimulate GTP hydrolysis by the isolated TC (Chaudhuri et al., 1994; Das et al., 2001). This discrepancy is most likely explained by the increased sensitivity of our assay, which has allowed us to accurately measure even slow rates of GTP hydrolysis. The activation by eif5 is also specific, as the GAPdeficient R15M mutant that binds eif2 with wt affinity failed to give any stimulation over background. Addition of eif1 and eif1a (800 nm each) and mrna(aug) (1 M) also did not affect the observed rate constant for GTP hydrolysis by isolated TC eif5 (data not shown). We next measured the rate constant for GTP hydrolysis by eif2 in the 43S mrna(aug) complex. 43S mrna(aug) complexes were formed by mixing TC with 40S 1 1A mrna(aug) complexes. In the absence of eif5, the rate constant for GTP hydrolysis by the 43S mrna complex is at the limit of detection (Figure 1B). The addition of saturating eif5 to preformed 43S mrna(aug) complexes increased the rate constant for GTP hydrolysis fold (Figure 1B). The rate constant for TC eif5 is 2400-fold lower than that for the 43S mrna(aug) eif5 complex, indicating that the 40S ribosomal complex cooperates with eif5 to promote GTP hydrolysis. This cooperativity could result from the 43S mrna complex altering the conformations or positions of eif5 or the TC to allow optimal activating interactions. In all cases, increasing the concentrations of all the components at least 3-fold did not result in a change in the observed rate constant, indicating that GTP hydrolysis is first order under these conditions and not rate limited by a binding event. As described for activation of the isolated TC, eif5- R15M did not activate GTP hydrolysis much above the level seen in the absence of the factor (Figure 1B), demonstrating the activation of hydrolysis we observe is specific. Also, the 43S mrna complexes formed are able to assemble into active 80S initiation complexes that catalyze the formation of methionyl-puromycin, indicating the 43S mrna complexes are competent intermediates under these conditions (data not shown). In order to probe the nature of the rate-limiting step for GTP hydrolysis, we changed the order of addition of the components. In the previously described experiments, 43S mrna complex was preformed and GTP hydrolysis initiated by addition of eif5. With this protocol, there is a possibility that rearrangements could take place within the 43S mrna complex during the time it was incubated prior to addition of eif5 (R2 min; under these conditions, 43S mrna complex formation is very fast; see Experimental Procedures). If a rearrangement is required for full GTP hydrolysis activity, we reasoned that it might become rate limiting, and be revealed, if the reactions were initiated by addition of TC to preformed 40S eif1 eif1a mrna complexes in the presence of eif5. To test this possibility, TC was added to the 40S eif1 eif1a mrna complex in the presence of eif5, and GTP hydrolysis was monitored (Figure 1C). Under these conditions, the observed rate constant for hydrolysis is 100-fold lower than when 43S mrna complexes were preformed and the reactions initiated with eif5 (0.13 versus 13 s 1, respectively). Increasing the concentrations of the compo-

3 Kinetics of eif5-promoted GTP Hydrolysis 253 Table 1. Summary of Rate Constants for GTP Hydrolysis Rate Constant of GTP Hydrolysis Rate Constant of Structural Rate Enhancement d Complex (k obs )(s 1 ) Change (k obs )(s 1 ) (Fold Above TC Alone) Buffer only (background) (7 ± 3) 10 6 a NA TC (eif2 GTP Met-tRNA i ) (8.5 ± 2.3) 10 6 a NA TC eif ± R825 43S mrna(aug) (6 ± 0.2) 10 6 a 43S mrna(aug) eif5 13 ± 3 b 0.13 ± 0.02 c R e 43S eif5 (no mrna) 9 ± 2 b 43S mrna(cuc) eif5 5 ± 2 b Errors are the mean deviations of at least three measurements. a Limit of detection of assay. b Observed rate constant when the reaction is initiated with eif5. c Observed rate constant when the reaction is initiated with TC. d Rate enhancements were calculated using the observed rate constant of the TC-only case as an upper limit. e Calculated for postrearrangement hydrolysis rate constant. nents in the reaction at least 3-fold did not change the observed rate constant, indicating that the rate-limiting step of the reaction under these conditions is first order and not a binding event. In addition, when reactions were initiated by adding preformed TC eif5 complex to 40S eif1 eif1a complex plus mrna(aug), the observed rate constant was the same as that when eif5 was present with the 40S eif1 eif1a mrna(aug) complex (data not shown), suggesting that the 0.13 s 1 rate constant does not represent release of eif5 nonproductively bound to the ribosomal complex. The data indicate that, when the reaction is initiated by the addition of TC, the rate of GTP hydrolysis is limited by a step with a rate constant of 0.13 s 1 that is a first-order process and occurs before GTP hydrolysis but after TC binding. Such a step is likely a structural rearrangement of the 43S mrna complex that permits rapid and efficient GTP hydrolysis. To gain further evidence that this step is a structural rearrangement in the 43S complex, we performed the following experiment. 43S mrna(aug) complexes (without eif5) were formed and allowed to undergo the putative structural rearrangement for various times, after which the fraction of complexes that had completed the rearrangement, and thus could hydrolyze GTP very rapidly when eif5 was added, was measured. In this experiment, as the time increases, a greater fraction of the complexes are converted to the fast-reacting species (13 s 1 ), while the remaining species are slower reacting (0.13 s 1 ). The 100-fold difference in rate constants allowed us to estimate the fraction of complexes that went through the rearrangement by adding eif5 to the reaction and quenching after a short interval (2 s). A 2 s time point is sufficient for complete GTP hydrolysis by the fast-reacting complexes but is only long enough for hydrolysis by 23% of the slow-reacting complexes. If the 43S mrna(aug) complex can undergo a structural change in the absence of eif5 that activates it for GTP hydrolysis, we would expect to observe the same rate constant as that measured in the experiment described above, in which the reaction was initiated with TC in the presence of eif5. In fact, the rate constant obtained from this experiment was 0.14 ± 0.01 s 1, identical to the rate constant obtained when reactions were initiated with TC (Figure 1D). P i Release Is Accelerated by AUG Codon Recognition To test the dependence of the steps leading up to GTP hydrolysis on the presence of an mrna in the 43S complex and on the AUG codon in the mrna, experiments were performed in the absence of the model message or in the presence of a saturating concentration of a model mrna identical to mrna(aug), except that the AUG was replaced with CUC (mrna[cuc]). The concentration of 40S eif1 eif1a complexes and the concentration of mrna(cuc) (75 M) was saturating in these experiments (Maag et al., 2005). Increasing the concentrations of the components at least 2-fold did not change the results, indicating that a binding event was not rate limiting and that the 43S complexes were fully formed and mrna binding was complete. Initiating GTP hydrolysis by adding saturating eif5 to 43S complexes as in Figure 1B resulted in a very small reduction (1.5-fold) in the observed rate constant (Figure 2A, Table 1). The rate constant for GTP hydrolysis when mrna(cuc) was in the complex was also slightly reduced. In addition to the reduction in rate constants, there is a significant decrease in the apparent endpoints of the hydrolysis reaction (from 86% to 20% 30%) with no mrna or mrna(cuc). Using the experimental setup shown in Figure 1C, we observed a small reduction (w3-fold) in the rate constant for the putative structural rearrangement in the 43S complex when mrna was omitted from the reaction or mrna(cuc) was present in the complex (Figure 2B), indicating that the binding of mrna accelerates the conformational change in the 43S complex. Interestingly, eif3 increases mrna the dependence of the structural rearrangement from 2.6-fold to 7-fold, although it has no effect on the rate constant in the presence of mrna(aug) (Figure 2B). Although both the structural rearrangement and GTP hydrolysis steps described here are accelerated by mrna binding and by the presence of an AUG codon in the mrna, the stimulation is not nearly as large as what we would have expected for the first committed step in protein synthesis, nor as large as the effect of an AUG codon in the mrna on 43S complex formation itself (>1000-fold) (Maag et al., 2005). The low dependence is, however, consistent with previous studies of

4 Molecular Cell 254 Figure 1. GTP Hydrolysis by eif2 (A) GTP hydrolysis in isolated TC without ( ) or with saturating wt eif5 (C) or eif5-r15m (%). The rate constants observed with isolated TC ([8.5 ± 2.3] 10 6 s 1 ) and TC with eif5-r15m ( s 1 ) are at the background level of the assay. Wild-type eif5 stimulates GTP hydrolysis by the TC R 825-fold (k obs =[7±0.4] 10 3 s 1 ). (B) Time courses of GTP hydrolysis measured using the protocol shown. TC was mixed with 40S subunits, mrna, and eifs 1 and 1A. Following a short time, eif5 was added to initiate GTP hydrolysis. The observed rate constant for the 43S mrna(aug) complex, shown in the full panel, was 13 ± 3 s 1.k obs in the absence of eif5 (inset, C) was (6 ± 0.2) 10 6 s 1.k obs with eif5-r15m (inset, ) was (2.4 ± 0.08) 10 5 s 1. (C) 40S eif1 eif1a mrna(aug) complexes were preformed in the presence of eif5 and mixed with TC to initiate the GTP hydrolysis reaction. The observed rate constant was 0.13 ± 0.02 s 1. (D) Preformed 40S eif1 eif1a mrna(aug) complexes were mixed with TC to form 43S mrna(aug) complexes. The putative structural rearrangement proceeded for various times before eif5 was added to the 43S mrna(aug) complex and the reaction rapidly quenched. The observed rate constant was 0.14 ± 0.01 s 1. 43S mrna(aug) complex formation under these conditions is fast (k obs w3s 1 ; D.M. and J.R.L., unpublished data). GTP hydrolysis by eif2 in mammalian systems (Chakrabarti and Maitra, 1991; Raychaudhuri et al., 1985; Unbehaun et al., 2004). Because the fidelity of aminoacyl trna selection during elongation in bacterial systems is dependent on Mg 2+ concentration (Gromadski and Rodnina, 2004), we investigated whether decreasing the concentration of Mg +2 would improve the mrna and codon dependence in our system. Lowering the concentration of Mg 2+ from 3.2 to 1.2 mm eliminated the mrna dependence, whereas increasing the concentration to 5.2 mm lowered the rate constants for hydrolysis by both the 43S and 43S mrna complexes by 5-fold (data not shown). Another possible explanation for the lowerthan-expected dependence on binding of an AUG-containing mrna is that a component that helps set the

5 Kinetics of eif5-promoted GTP Hydrolysis 255 Figure 2. mrna and AUG Codon Dependence of GTP Hydrolysis and the Putative Structural Rearrangement (A) GTP hydrolysis from 43S complexes using the scheme in Figure 1B except with mrna(cuc) replacing mrna(aug) or without mrna. The observed rate constants were 5 ± 2 s 1 with mrna- (CUC) (%) and9±2s 1 with no mrna ( ) versus 13 s 1 with mrna(aug) (C). (B) Dependence of the putative structural rearrangement on mrna, an AUG codon, and eif3. GTP hydrolysis by eif2 was followed using the incubation scheme of Figure 1C. In the absence of mrna, eif3 decreases the rate constant for the structural rearrangement by 2.5-fold (0.05 ± 0.01 s 1 versus 0.02 ± 0.01 s 1 for no eif3 [C] and plus eif3 [%], respectively). eif3 does not change the rate constant in the presence of mrna(aug) (0.14 ± 0.02 s 1, :). The rate constant with mrna(cuc) is consistently between that without mrna and with mrna(aug) (0.06 ± 0.02 s 1, ). threshold for activation of GTP hydrolysis is missing from our system. The addition of the eif4f set of factors, eif4b and/or eif5b, however, had no effect on the dependence of the putative structural rearrangement on the presence of mrna (data not shown). Since eif5-promoted GTP hydrolysis by eif2 has only a small dependence on the presence of mrna and the nature of the start codon in the mrna, we looked at inorganic phosphate (P i ) release from eif2 after GTP hydrolysis. The quenches employed for the GTP hydrolysis assays (EDTA or formic acid) disrupt the complex, and thus we cannot distinguish irreversible hydrolysis in which P i has been released from eif2 and internal hydrolysis in which an equilibrium between GTP and GDP P i has been established on the enzyme but P i has not yet been released. In the latter case, release of P i from the eif2 GDP P i complex would make the hydrol- Figure 3. P i Release from eif2 Is Strongly Dependent on an AUG Codon in the mrna (A) P i release from eif2 after hydrolysis in a 43S mrna(aug) complex. Reactions were set up using the same scheme as in Figure 1B, and fluorescence from PBP-MDCC was followed. The observed rate constant for P i release from the 43S mrna(aug) complex with WT eif5 was 6.7 ± 0.3 s 1. When the GAP-deficient mutant eif5- R15M was used, no P i release was observed (lower trace). (B) P i release from 43S or 43S mrna(cuc) complexes. Reactions were performed as in (A), except the mrna was either mrna(cuc) ( ) or omitted (C). In the absence of mrna, P i is released with a rate constant of 0.04 ± 0.01 s 1. With mrna(cuc), the rate constant is 0.06 ± 0.02 s 1. ysis reaction irreversible and consequently could be a point of regulation in the system. To monitor phosphate release from eif2 in real time, we employed a well-characterized fluorescently labeled form of the phosphate binding protein from E. coli (PBP-MDCC) (Gilbert et al., 1995; Nixon et al., 1995; Webb, 2003). To measure P i release from various 43S complexes, the experimental approach used previously to measure GTP hydrolysis (Figure 1B) was employed, except that GTPγ 32 P was replaced with unlabeled GTP and PBP-MDCC was added to the 43S complexes prior to initiation with eif5. In all cases, the final amount of P i released was w85% of the GTP incorporated into 43S complexes (data not shown). The observed rate constant of P i release from 43S mrna(aug) complexes under these conditions was 6.7 ± 0.3 s 1 (Figure 3A, Table 2). This rate constant is 2-fold lower than that for GTP hydrolysis initiated in the same manner. To further demonstrate that the fluorescence signal was due

6 Molecular Cell 256 Table 2. Summary of Rate Constants for P i Release Complex k obs (s 1 ) 43S mrna(aug) 6.7 ± S 0.04 ± S mrna(cuc) 0.06 ± S mrna(aug) with WT eif1 a 0.54 ± S mrna(aug) with eif1-g107r a 0.09 ± 0.01 Errors are the mean deviations of at least three measurements. a Experiment initiated with mrna(aug) and eif5. to release of P i from eif2 after GTP hydrolysis, we used the eif5-r15m protein that is unable to promote hydrolysis to initiate the reaction and observed no P i release (Figure 3A). We also measured a rate constant of s 1 for P i release from isolated TC eif5 in solution, similar to the rate constant for GTP hydrolysis by this complex (0.007 s 1 ). Next, we examined the dependence of the P i release step on the presence of mrna by omitting the mrna and measuring P i release from the 43S complex. In the absence of mrna, P i is released 170-fold slower than in the presence of mrna(aug) (6.7 s 1 versus 0.04 s 1 ) (Figure 3B). A similar rate constant was measured when eif3 was added to the 43S complex in the absence of mrna (0.04 s 1 without eif3 versus 0.02 s 1 with eif3, see Figure S1 in the Supplemental Data available with this article online). We then examined the dependence on an AUG codon in the mrna by replacing mrna (AUG) in the complex with mrna(cuc). The measured rate constant for P i release was 0.06 ± 0.02 s 1, again more than 100-fold lower than when an AUG is in the mrna (Figure 3B). The rate constants for P i release from 43S and 43S mrna(cuc) are at least 100-fold lower than the rate constants for GTP hydrolysis by the same complexes, indicating that P i release is rate limiting for irreversible GTP hydrolysis. These data suggest that, on the time scale of GTP hydrolysis, an equilibrium between GTP and GDP P i might be established on eif2 in the 43S complex. If this is so, we can estimate this equilibrium constant because the time elapsing during the GTP hydrolysis experiments (1 s) with no mrna or mrna- (CUC) is only long enough to allow 5% of the P i to dissociate from eif2. Therefore, the amplitudes of these time courses serve as approximations of the ratio of GTP and GDP P i bound to eif2. The amplitudes of the GTP hydrolysis reactions with no mrna and mrna- (CUC) are 30% and 21%, respectively, whereas GTP hydrolysis with mrna(aug) results in 86% turnover (Figure 2A). Assuming that the maximal amount of GTP that can be productively bound and hydrolyzed is 86% of the total, we can estimate the internal equilibrium constant (K int ) between GTP and GDP P i on eif2 to be approximately 0.5 in the absence of mrna and in the presence of mrna(cuc) (K int = [GDP P i ]/[GTP]; in the no mrna case, K int z 0.30/( ) z 0.5). If an internal equilibrium is responsible for the decreased amplitudes of the reactions in the absence of an AUG codon, roughly one third of the GTP is hydrolyzed to GDP P i on eif2 in the 43S complex prior to AUG recognition. One alternate possibility to explain the decreased amplitudes is that the majority of the 43S complexes formed in the absence of mrna or in the presence of mrna(cuc) are unable to hydrolyze GTP or are unstable and dissociate. However, when GTP hydrolysis by 43S complexes formed without mrna or with mrna (CUC) is followed for longer times (300 s), a second, slower phase of the reaction is observed. The amplitudes of the slow phases approach the final endpoint observed in the reaction with mrna(aug) and have rate constants of w0.05 s 1 (Figure S2), identical to the rate constant for P i release from these complexes. This is what would be expected of a situation in which a rapid internal equilibrium is reached between GTP and GDP P i on eif2, which is then slowly driven to completion by release of P i. Recycling of dissociated eif2 into new complexes is unlikely to account for the slow second phase because a GDP chase was added along with eif5 to prevent multiple turnover (see Experimental Procedures). Furthermore, the rate constant for TC binding to 40S subunits under these conditions is significantly lower (w20-fold) than the observed slow phase (D.M. and J.R.L., unpublished data). We cannot rule out the possibility, however, that an active state of the 43S complex that hydrolyzes 100% of its GTP is in equilibrium with an inactive state (with a K eq w0.5) and this equilibrium is pulled along as P i is released. Evidence that P i Release from eif2 Is Controlled by Start Codon Recognition-Dependent eif1 Dissociation from the 43S mrna Complex Data from several labs have suggested that eif1 is a negative regulator of GTP hydrolysis by eif2 and its release or movement in the complex in response to AUG recognition is the trigger for irreversible GTP hydrolysis (Maag et al., 2005; Unbehaun et al., 2004; Valasek et al., 2004). To investigate whether eif1 dissociation controls P i release, we followed P i release from eif2, utilizing the experimental setup previously used to monitor dissociation of eif1 from a 43S mrna(aug) complex (Maag et al., 2005). In the experiments of Figure 3, eif1 had already dissociated before reactions were initiated with eif5 because the 43S mrna(aug) complex was preformed and AUG recognition triggers eif1 dissociation. The experiments to measure eif1 dissociation were done by mixing TC with 40S ribosomal subunits, eif1 labeled with TAMRA, and eif1a labeled with fluorescein to form 43S complex that does not contain mrna. In this complex, there is FRET between eif1a-fl and eif1-tamra. When the labeled complex is rapidly mixed with mrna in a stopped-flow device, the FRET between eif1a-fl and eif1-tamra decreases (fluorescein fluorescence increases) as the complex undergoes a conformational change (k obs = 10 s 1 ) and releases eif1 (k obs = 0.6 s 1 ) in response to AUG recognition (Maag et al., 2005). To measure P i release from eif2 under conditions where eif1 dissociation could be rate limiting, we used a similar experimental setup, except eif1 and eif1a were unlabeled, PBP-MDCC was included, and eif5 was added with mrna(aug) to initiate the reaction. The rate constant for P i release under these conditions was 0.54 ± 0.05 s 1 (Figure 4A), the same as the rate constant for eif1

7 Kinetics of eif5-promoted GTP Hydrolysis 257 Figure 4. Evidence that Dissociation of eif1 from the 43S mrna(aug) Complex after Start-Site Selection Limits Release of P i from eif2 (A) P i release from the 43S complex was measured after initiation of the reaction with eif5 and mrna(aug) (k obs = 0.54 ± 0.05 s 1 ). (B) The rate constants for dissociation of wt eif1 and eif1-g107r were measured using FRET. eif1-g107r (right curve) dissociates more slowly than wt eif1(left curve) (0.03 s 1 versus 0.6 s 1, respectively). (C) When P i release was followed as in (A) but with eif1-g107r instead of wt eif1, the rate constant was reduced 6-fold (0.09 s 1 versus 0.54 s 1 for G107R and wt, respectively). (D) GTP hydrolysis from the 43S complex after initiation with eif5 and mrna(aug). Data from Figure 2A are shown for comparison (43S complex, ; 43S mrna(aug) complex, C). When the rate constant for GTP hydrolysis was measured using the same experimental setup as in (A) (%), two kinetic phases were observed. The fast phase has a rate constant of 11 ± 2 s 1 and is followed by a slow phase with a rate constant of 0.64 ± 0.15 s 1. The endpoint of the fast phase is approximately the same as that with no mrna ( ), while the slower phase approaches the endpoint observed with 43S mrna(aug) complexes (C). (E) The experimental data were fit using the program Berkeley Madonna to simulate a reaction in which GTP hydrolysis rapidly reaches an internal equilibrium on eif2 followed by another rate-limiting step (P i release) that drives the reaction to completion. The data from (D) are shown with the best fit from the simulation (dotted line).

8 Molecular Cell 258 dissociation (0.6 s 1 ), consistent with eif1 dissociation being rate limiting for release of P i after start-site recognition. We reasoned that, if eif1 dissociation controlled P i release from eif2, then a decrease in the eif1 dissociation rate constant would be reflected by a corresponding change in the P i release rate constant. To this end, we utilized an eif1 mutant protein (G107R) that has a decreased dissociation rate from the 43S mrna(aug) complex. eif1-g107r was labeled with TAMRA and used in the FRET dissociation assay (Maag et al., 2005). The rate constant for dissociation of eif1-g107r from the 43S mrna(aug) complex after start-site selection is 18-fold lower than the rate constant for wt eif1(0.03 s 1 for eif1-g107r versus 0.6 s 1 for wt eif1; Figure 4B). Next, we measured the rate constant for P i release from 43S mrna(aug) complexes containing eif1- G107R instead of wt eif1 and observed a 6-fold decrease compared to the rate constant with wt eif1, supporting the idea that P i release is controlled by eif1 dissociation from the 43S mrna(aug) complex after start-site selection (Figure 4C). Further evidence that eif1 dissociation controls release of P i from eif2 comes from GTP hydrolysis experiments initiated in the same manner as the P i release experiments described above. When 43S complexes are mixed with eif5 and mrna(aug) and GTP hydrolysis is measured, we observed two kinetic phases (Figure 4D, diamonds). The first phase has a rate constant of 11 ± 2 s 1 and an amplitude of 0.21 ± 0.02, while the second phase is slower, with a rate constant of 0.64 ± 0.15 s 1 and an amplitude of 0.46 ± 0.03, as would be predicted if eif1 dissociation limits the release of P i from eif2. It would be expected that, when eif5 and mrna(aug) are added to 43S complexes, eif5 will bind the 43S complex before AUG recognition and promote the rapid hydrolysis of GTP within the complex with a rate constant of 9 s 1 (the rate constant of GTP hydrolysis in the absence of mrna), achieving an internal equilibrium between GTP and GDP P i of approximately 0.5. This is what is observed in the first kinetic phase of the hydrolysis experiment (k obs =11s 1 ). The endpoint for the 11 s 1 phase also matches the amplitude reached when mrna is absent (compare diamonds to squares). Following the first kinetic phase, the posthydrolysis 43S complex locates the start codon in the mrna, allowing dissociation of eif1 and subsequent release of P i from eif2, making the reaction irreversible and driving it to completion. If the rate-limiting step under these conditions is the dissociation of eif1, which occurs with a rate constant of 0.6 s 1,we would expect that P i release would occur with the same rate constant. This is, in fact, exactly what is seen in the second phase of the reaction (k obs = 0.64 s 1 ), further supporting the model. To confirm that these data are consistent with our kinetic model, we used the program Berkeley Madonna to simulate a reaction in which GTP hydrolysis reaches equilibrium rapidly within the 43S complex and is followed by a rate-limiting step (P i release) that drives the reaction to completion. We then fit the experimental data with the kinetic model by nonlinear regression (Figure 4E). The data are fit well with this kinetic model, and the rate constants obtained are in agreement with those obtained by fitting with the sum of two exponentials (13 and 0.7 s 1 and 11 and 0.64 s 1 for the simulation and direct two exponential fit, respectively). Discussion To begin to elucidate the molecular mechanisms underlying eif5-promoted irreversible hydrolysis of GTP by eif2, we have conducted a quantitative analysis of the steps and interactions surrounding this critical event in translation initiation. A Model for Irreversible GTP Hydrolysis in the 43S mrna Complex during Start-Site Selection Setting the reading frame of translation is a critical step because errors at this point will result in production of aberrant proteins. It has previously been assumed that GTP hydrolysis by eif2 is the critical step in selecting the start codon. The data presented here indicate that eif5 is able to promote GTP hydrolysis by eif2 in the absence of mrna and before start-site selection, suggesting that hydrolysis itself is not the key codondependent event. However, the release of P i from eif2 is highly dependent on selection of the AUG codon, and it is this dissociation step that causes the hydrolysis reaction to become irreversible, allowing it to proceed to completion, thereby committing the 43S mrna complex to beginning translation at the selected codon. Recently, our lab has provided data suggesting that a conformational change in the 43S mrna complex also accompanies start codon recognition and that this structural change reduces the affinity of the complex for eif1 (Maag et al., 2005). These results, along with other recent experiments in vivo and in vitro, suggested that eif1 might be a negative regulator of GTP hydrolysis by eif2 in the 43S complex (Unbehaun et al., 2004; Valasek et al., 2004). The data presented here indicate that eif1 does not inhibit GTP hydrolysis itself but instead regulates the release of P i from eif2 GDP P i. The combined data suggest the following model for the mechanism of 43S complex activation, start codon recognition, and irreversible GTP hydrolysis (Figure 5). Binding of the 43S complex to an mrna accelerates a structural rearrangement that makes the complex fully competent to hydrolyze GTP. The rearrangement may involve repositioning of eif5 within the complex to allow it to bind correctly to the GTPase activation site on eif2. The rearrangement might also alter the conformation of eif2, similar to the role some GAPs play in stabilizing the active conformation of other G proteins (Scheffzek et al., 1998). It is also conceivable that a group on the ribosome itself might be involved in directly stabilizing the transition state for GTP hydrolysis and that activation involves the proper positioning of this group. The rate constant for the activation step, 0.13 s 1, which is rate limiting for GTP hydrolysis when reactions are initiated with TC, is at least an order of magnitude larger than rate constants calculated from previous studies of GTP hydrolysis by eif2 in vitro (<0.01 s 1 )(Das et al., 2001; Huang et al., 1997; Paulin et al., 2001). This rate constant is also large enough to support the estimated rate of translation initiation in vivo (k obs w0.1 s 1 )(Palmiter, 1975).

9 Kinetics of eif5-promoted GTP Hydrolysis 259 Figure 5. Model of the Steps Leading Up to Irreversible Hydrolysis of GTP by the 43S Complex in Response to Start Codon Recognition The binding of mrna to the 43S complex increases the rate constant for a structural rearrangement that makes the complex fully competent to hydrolyze GTP in the presence of eif5. The nature of the rearrangement is not yet known but is depicted as an alteration of eif5 s position for simplicity. GTP is rapidly hydrolyzed by eif2 before start-site selection, resulting in an internal equilibrium between GTP and GDP P i on eif2. Although our data indicate that hydrolysis can also happen prior to mrna binding by the 43S complex, for simplicity we have only shown it happening after mrna binding. The complex then locates the start codon, triggering a second conformational change that results in a decrease in affinity for eif1 (Maag et al., 2005). This conformational change results in the C termini of eifs 1 and 1A moving away from one another and could be a movement of the 40S subunit, the factors themselves, or both (depicted in the model as a change in eif1). Dissociation of eif1 from the complex allows rapid release of P i from eif2, making GTP hydrolysis irreversible and allowing downstream events in the pathway, such as eif2 GDP dissociation and subunit joining, to proceed. eif2 GDP is shown bound to the 40S subunit after P i release because we do not yet know what steps must occur between P i release and eif2 GDP dissociation, if any. After the 43S complex switches into its active state, it scans the mrna in search of an AUG codon. At this stage, reversible GTP hydrolysis can occur rapidly within the complex in the absence of an AUG codon. This hydrolysis is reversible within the complex because P i release does not happen on a relevant time scale prior to AUG recognition. The 43S complex will continue to search the mrna for the AUG codon, with GTP in equilibrium with GDP P i in the active site of eif2. Recognition of the start codon triggers a rapid conformational change in the 43S complex and dissociation or movement of eif1, thereby allowing P i release from eif2. Following the release of P i, eif2 GDP is able to dissociate from the 40S complex, leaving the MettRNA i in the P site awaiting subunit joining. Control of product release as a mechanism for fidelity is not unprecedented. For example, in the Klenow fragment of DNA polymerase I, the release of pyrophosphate is accelerated by having the correctly paired dexoynucleotide in the active site (Eger and Benkovic, 1992). P i release has also been reported to be the rate-limiting step for neurofibromin-activated GTP hydrolysis by the small G protein Ras (Phillips et al., 2003), a relative of the γ subunit of eif2. Why does this system appear to gate the release of P i in response to start codon recognition rather than having fast P i release and controlling the rate of GTP hydrolysis? One possibility is that this is simply the way the system evolved and that there is no inherent benefit in controlling one step or the other. It should be noted, however, that the release of P i from Walker-type NTPases such as eif2 is the step at which switching from one functional state to another occurs. This is because the Switch I and Switch II regions of the active site, which transduce NTP hydrolysis into conformational changes in the enzymes, interact with the γ-phosphate and move upon P i release (Sprang, 1997). Thus, P i dissociation would be expected to induce the conformational changes in eif2 that trigger release of the initiator trna. The rate constant for dissociation of eif1 from a post-codon recognition 43S mrna(aug) complex is 0.6 s 1. This rate constant is similar to the rate constant for the rearrangement of the 43S mrna complex we have described that limits the rate of GTP hydrolysis (0.13 s 1 ), raising the possibility that these two steps are the same. However, observation of the conformational change that triggers the rapid release of eif1 is strictly dependent upon the binding of the 43S complex to an mrna, whereas the structural rearrangement we observe here can occur without mrna, albeit with a 3-fold lower rate constant. These data suggest that the structural rearrangement that activates the 43S complex for GTP hydrolysis is a separate step from the conformational change and eif1 release that accompany start codon recognition. Although the system in which we have performed these studies does not contain all of the factors or recapitulate every step thought to be involved in eukaryotic translation initiation, the strong dependencies we have measured for GTP hydrolysis on eif5 and for P i release on start codon recognition suggest that we have shed

10 Molecular Cell 260 light on key molecular events in the pathway. It will be important, however, to more extensively explore the role of eif3 in start codon recognition and related steps. Although genetic selections have indicated that eifs 1, 2, and 5 and the initiator trna are the central components of the start codon selection machinery (Cui et al., 1998; Hashimoto et al., 2002; Huang et al., 1997; Yoon and Donahue, 1992), recently, large-scale alanine-scanning mutagenesis studies have suggested that eif3 plays a role in the process as well (Valasek et al., 2004). In addition to the triggering of irreversible GTP hydrolysis by eif2 via the gating of P i release upon AUG recognition, other events may contribute to the fidelity of initiation site selection. For example, the scanning process, in which the 43S complex is thought to move along the mrna searching for the start codon, could influence start codon selection fidelity (Pestova and Kolupaeva, 2002). The length of time the complex stalls at each codon as it is scanning may help dictate whether that site is chosen for initiation; the longer the pause, the more chance the complex has of triggering P i release. It will be important to determine how the scanning machinery and the scanning process itself influence the steps we have studied here. Finally, events after P i release might further contribute to the fidelity of initiation codon selection. For example, a dissociation pathway might exist that allows post-p i release complexes stopped at non-aug codons to be discarded rather than continuing with the initiation process. This would provide a mechanism of proofreading analogous to what occurs after GTP hydrolysis by EF-Tu/EF1A during aminoacyl trna selection in the elongation phase of translation (Rodnina and Wintermeyer, 2001). The kinetic and thermodynamic analyses described here provide a necessary foundation for understanding how the fidelity of start codon selection is achieved during translation initiation in eukaryotes and will allow the roles of additional factors and steps in this critical process to be elucidated. Experimental Procedures Initiation Factors eif1 was purified as described (Algire et al., 2002). The eif1a gene and eif5 gene were cloned into the vectors ptyb1 and ptyb2, respectively (NEB), and purified as described for eif1. His-tagged eif2 was purified from yeast strain GP3511 as described (Algire et al., 2002) with minor modifications (Kapp and Lorsch, 2004a). A 1 hr dialysis was performed in storage buffer plus 1 mm EDTA before the overnight dialysis against storage buffer to produce apo-eif2. His-tagged eif3 was prepared as described previously (Algire et al., 2002). All protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad) microassay procedure as described by the manufacturer, using BSA as the standard. Purification of Yeast 40S Ribosomal Subunits 40S ribosomal subunits were isolated as described (Algire et al., 2002) with minor modifications. After clarification of the lysate, the ribosomes were pelleted through a sucrose cushion (20 mm HEPES-KOH [ph 7.6], 100 mm KOAc, 2.5 mm Mg(OAc) 2, 500 mm KCl, 1 M sucrose, 2 mm DTT). Following fractionation of the gradients, the sucrose gradient buffer was exchanged for ribosome storage buffer using 100,000 MWCO Amicon Ultra centrifugal filter devices (Millipore) by concentrating the 40S subunits to a volume of l and diluting up to 15 ml with ribosome storage buffer and reconcentrating the sample (three times). Model mrna Templates The model mrna templates were synthesized by T7 polymerase run-off transcription and purified by denaturing PAGE (Lorsch and Herschlag, 1999). The model mrna sequences are mrna(aug), 5#-GGAA[UC] 7 UAUG[CU] 10 C-3#; and mrna(cuc), 5#-GGAA[UC] 7 UCUC[UC] 10 U-3#. Methionyl-tRNA i Preparation The trna Met i was synthesized by T7 polymerase run-off of HH- IMTpUC plasmid. The HH-IMTpUC plasmid contains a T7 RNA polymerase promoter followed by a hammerhead ribozyme directly Met linked to the trna i sequence as described for the trna Tyr (Fechter et al., 1998). Cleavage of the hammerhead ribozyme to generate the free trna Met i was performed as described. The rationale for using the hammerhead-containing construct was to increase the transcription efficiency of the trna i.[ 35 S]Met-tRNA i and stoichiometric-charged Met-tRNA i were prepared as described (Kapp and Lorsch, 2004a). GTPase Assays Manually quenched GTPase experiments were conducted as follows. TC was formed at two times concentration by mixing one times reaction buffer (30 mm HEPES-KOH [ph 7.4], 100 mm KOAc, 3 mm Mg(OAc) 2, 2 mm DTT), 1.6 M eif2, 1.6 M Met-tRNA i, and 125 pm GTPγ[ 32 P] and incubating for 15 min at 26 C. After the incubation, a sample was removed for quantification of background GTP hydrolysis. Ribosomal complex was formed at two times concentration by mixing one times reaction buffer, 400 nm 40S ribosomal subunits, 1.6 M eif1 and eif1a, 2 M model mrna, 1.6 M eif5, and 2 mm GDP (to prevent multiple turnover). For each time point, 2 l of TC was mixed with 2 l of ribosomal complex (resulting in the final concentrations) for the desired time, after which 2 l was removed and quenched into 5 l of 100 mm EDTA (ph 8.0). To quantify the extent of GTP hydrolysis, 15% polyacrylamide TBE gels were run to separate GTPγ[ 32 P] from free 32 P i followed by PhosphorImager analysis. To obtain observed rate constants, data were fit with a single exponential equation, A(1 exp( kt)), in which A is the amplitude and k is the observed rate constant. All fits were performed using KaleidaGraph software (Synergy Software). For rapid quench GTPase experiments, TC was formed at four times concentration by mixing one times reaction buffer, 3.2 M eif2, 3.2 M Met-tRNA i, and 250 pm GTPγ[ 32 P]. Ribosomal complex was also formed at four times the final concentrations by mixing 800 nm 40S ribosomal subunits, 3.2 M eif1 and eif1a, and 4 M model mrna. After formation of the TC, equal volumes of the four times TC and four times ribosomal complex were mixed to form 43S mrna complex (at two times concentration). The 43S mrna complex was loaded into a sample port of the rapid quench apparatus (Kintek). The other sample port was loaded with one times reaction buffer, 1.6 M eif5, and 2 mm GDP (two times concentration). Reactions were quenched with 100 mm EDTA. Using 40% formic acid as a quench gave identical results. In the experiments without mrna or with mrna(cuc), the 43S or 43S mrna(cuc) complexes were allowed to form for 1 hr before initiation with eif5, which was found to be ample time for complex formation. In the experiment where mrna(aug) was present with eif5 to initiate the reaction, the concentration of mrna was 20 M. This is sufficient to insure binding of mrna is not rate limiting (Maag et al., 2005). All reported rate constants are the averages of at least three experiments, and the errors are the mean deviations. Fluorescence Anisotropy eif5 was labeled on its C terminus with a cysteine-lysine-fluorescein dipeptide conjugate by expressed protein ligation as described (Maag and Lorsch, 2003). Fluorescence anisotropy experiments were carried out as described (Maag and Lorsch, 2003). eif5-fluorescein (50 nm) was used in the direct titration experiments. In the experiments using eif2 GDP, a final concentration of 1 mm GDP was included in the cuvette. TC with GDPNP was formed as described (Maag et al., 2005). The cuvette also contained 500 M GDPNP and 80 nm Met-tRNA i in order to prevent net dissociation of ternary complex at the lower concentrations. The direct titration data were fit by nonlinear regression as previously described (Maag and Lorsch, 2003). Competitive binding