Author response: The β-hairpin of 40S exit channel protein Rps5/uS7 promotes efficient and accurate translation initiation in vivo

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract The eukaryotic 43S pre-initiation complex bearing tRNAiMet scans the mRNA leader for an AUG start codon in favorable context. Structural analyses revealed that the β-hairpin of 40S protein Rps5/uS7 protrudes into the 40S mRNA exit-channel, contacting the eIF2∙GTP∙Met-tRNAi ternary complex (TC) and mRNA context nucleotides; but its importance in AUG selection was unknown. We identified substitutions in β-strand-1 and C-terminal residues of yeast Rps5 that reduced bulk initiation, conferred ‘leaky-scanning’ of AUGs; and lowered initiation fidelity by exacerbating the effect of poor context of the eIF1 AUG codon to reduce eIF1 abundance. Consistently, the β-strand-1 substitution greatly destabilized the ‘PIN’ conformation of TC binding to reconstituted 43S·mRNA complexes in vitro. Other substitutions in β-hairpin loop residues increased initiation fidelity and destabilized PIN at UUG, but not AUG start codons. We conclude that the Rps5 β-hairpin is as crucial as soluble initiation factors for efficient and accurate start codon recognition. https://doi.org/10.7554/eLife.07939.001 eLife digest To make a protein, the DNA sequence of a gene is first copied to make an mRNA molecule, which is then translated into a protein by a molecular machine called the ribosome. The first step of translation is known as initiation. Several proteins referred to as initiation factors can bind to the small subunit of the ribosome, which itself is composed of an RNA molecule and numerous proteins, and form a pre-initiation complex (or PIC for short). This complex contains a molecule called initiator tRNA that is specialized for initiation. The PIC then attaches to the mRNA and starts scanning it, searching the sequence for an AUG triplet to serve as the start codon. The sequence immediately surrounding an AUG triplet, known as the context, influences the likelihood of its selection as the start site. When the start codon is recognized by the initiator tRNA in the PIC, the complete ribosome assembles and begins to build the protein. Choosing the correct start codon is crucial to ensure that the correct protein is made from every mRNA in the cell. The PIC can adopt an ‘open’ state, which makes it easier to scan the mRNA for the correct start codon and ignore triplets similar in sequence to AUG (like UUG) or AUG triplets in a poor context. Once the correct AUG start codon has been recognized, the PIC changes to a ‘closed’ state, ceases to scan, and assembles the complete ribosome. One of the proteins that make up the small ribosomal subunit (called Rps5 in budding yeast) is located near the channel where the mRNA molecule exits the PIC during scanning, and is also thought to be involved in translation initiation. However, the role of Rps5 in recognizing the start codon is poorly understood. Visweswaraiah et al. have now studied Rps5—in particular, a region of this protein that adopts a hairpin structure that dips into the exit channel—using genetic and biochemical methods. In mutant yeast cells in which the hairpin structure was mutated, translation initiation was diminished at suboptimal start codons—including a UUG start codon and an AUG codon in poor context—thus making translation initiation more accurate. Visweswaraiah et al. then performed experiments on PICs built from purified components to determine how the Rps5 mutations might affect the assembly and stability of the PIC. The results revealed that mutating the upper region of the Rps5 hairpin destabilized the closed state of the PIC when either an AUG or UUG start codon was present in the mRNA. However, other mutations of the hairpin structure destabilized the closed state of the PIC only at a UUG start codon. In both cases, the mutations made the PIC more likely to remain in the open conformation and continue scanning at incorrect or suboptimal start codons, making it more likely that translation begins at the correct AUG start codon. These results indicate that the Rps5 hairpin is crucial for both efficiently and accurately recognizing the start codon to begin translation. This suggests that ribosomal proteins not only contribute to ribosome structure but can actively participate with other initiation factors in choosing the correct start sites for protein synthesis on all mRNAs in the cell. https://doi.org/10.7554/eLife.07939.002 Introduction Accurate identification of the translation initiation codon is critical to ensure synthesis of the correct cellular proteins. In eukaryotic cells this process generally occurs by a scanning mechanism, wherein the small (40S) ribosomal subunit first recruits initiator tRNA (Met-tRNAi) in a ternary complex (TC) with eIF2-GTP in a reaction stimulated by eIFs 1, 1A, and 3. The resulting 43S pre-initiation complex (PIC) attaches to the mRNA 5′ end and scans the 5′ UTR for an AUG, using complementarity with the anticodon of Met-tRNAi to identify the start codon and assemble a 48S PIC. Nucleotides immediately surrounding the AUG, particularly the −3 and +4 positions (referred to below as context nucleotides), also influence start codon selection. During scanning, the GTP bound to eIF2 in the TC is hydrolyzed in the 43S PIC in a manner dependent on the GTPase activating protein eIF5, but Pi release is blocked by eIF1, which also impedes stable binding of Met-tRNAi in the P site. Start codon recognition triggers dissociation of eIF1 from the 40S subunit, which allows interaction between eIF5 and the C-terminal tail (CTT) of eIF1A, Pi release from eIF2-GDP·Pi, and more stable TC binding in the P site (Figure 1). Subsequent dissociation of eIF2-GDP and other eIFs from the 48S PIC enables eIF5B-catalyzed subunit joining and formation of an 80S initiation complex with Met-tRNAi base-paired to AUG in the P site (reviewed in Hinnebusch, 2014). Figure 1 Download asset Open asset Model describing conformational rearrangements of the PIC during scanning and start codon recognition. Assembly of the PIC, scanning and start codon selection in WT cells. (i) eIF1 and the scanning enhancer (SEs) elements in the CTT of eIF1A stabilize an open conformation of the 40S subunit to which the TC loads rapidly. (ii) The 43S PIC in the open conformation scans the mRNA for the start codon with Met-tRNAi bound in the POUT state. The GAP domain in the N-terminal domain of eIF5 (5N) stimulates GTP hydrolysis by the TC to produce GDP•Pi, but release of Pi is blocked. The unstructured NTT of eIF2β interacts with eIF1 to stabilize eIF1•40S association and the open conformation. (iii) On AUG recognition, Met-tRNAi moves from the POUT to PIN state, clashing with eIF1 and the CTT of eIF1A. Movement of eIF1 and the eIF1A CTT away from the P site disrupts eIF1's interaction with eIF2β-NTT, and the latter interacts with the eIF5-CTD. eIF1 dissociates from the 40S subunit, and the eIF5-NTD disengages from eIF2 and interacts with the eIF1A CTT instead, dependent on the SE elements, thereby facilitating Pi release from eIF2. The eIF5-CTD moves into the position on the 40S subunit previously occupied by eIF1 and blocks reassociation of eIF1. (Below) Arrows summarize that eIF1 and the eIF1A SE elements promote POUT and block transition to the PIN state, whereas the scanning inhibitor (SI) element in the NTT of eIF1A stabilizes the PIN state. (Adapted from Hinnebusch and Lorsch, 2012; Nanda et al., 2013). https://doi.org/10.7554/eLife.07939.003 eIF1 plays a dual role in the scanning mechanism. It promotes an open, scanning-conducive conformation of the PIC (Pestova and Kolupaeva, 2002) to which TC rapidly loads, bound in a state capable of inspecting successive triplets entering the P site (dubbed POUT) (Passmore et al., 2007; Saini et al., 2010); and it also blocks recognition of near-cognate start codons (e.g., UUG) (Yoon and Donahue, 1992) and AUG codons in poor sequence context (Pestova and Kolupaeva, 2002). Hence, eIF1 must dissociate from the 40S subunit (Maag et al., 2005; Cheung et al., 2007) to allow Pi release (Algire et al., 2005) and rearrangement to a scanning-incompatible state with Met-tRNAi base paired with AUG and more tightly bound in the PIN conformation (Passmore et al., 2007; Saini et al., 2010). Consistent with this, structural analyses of different PICs reveal that eIF1 and eIF1A promote rotation of the 40S head relative to the body (Lomakin and Steitz, 2013) (Hussain et al., 2014), which is likely instrumental in TC binding in the POUT conformation, but that eIF1 physically obstructs Met-tRNAi binding in the PIN state (Rabl et al., 2011; Lomakin and Steitz, 2013). Accordingly, eIF1 is deformed and displaced from its 40S location in the open complex during the POUT to PIN transition (Hussain et al., 2014). Consequently, mutations that weaken eIF1 binding to the 40S subunit reduce the rate of TC loading, while elevating initiation at near-cognate codons or AUGs in poor context, by destabilizing the open/POUT conformation and favoring rearrangement to the closed/PIN state during scanning (Martin-Marcos et al., 2011, 2013). Moreover, decreasing wild-type (WT) eIF1 abundance reduces initiation accuracy, whereas overexpressing eIF1 suppresses initiation at near cognates or AUGs in poor context (Valasek et al., 2004; Alone et al., 2008; Ivanov et al., 2010; Saini et al., 2010; Martin-Marcos et al., 2011). This tight link between eIF1 abundance and initiation accuracy is exploited to autoregulate eIF1 expression, as the AUG start codon of the eIF1 gene (SUI1 in yeast) occurs in poor context—a feature conserved throughout eukaryotic evolution—and the frequency of recognizing its own start codon is inversely related to eIF1 abundance (Ivanov et al., 2010; Martin-Marcos et al., 2011). The stability of the codon-anticodon duplex is an important determinant of initiation accuracy, as the rate of the POUT to PIN transition and stability of the PIN state are both favored by AUG vs non-AUG start codons (Kolitz et al., 2009). It is possible that favorable context also contributes to the stability of PIN (Pisarev et al., 2006; Martin-Marcos et al., 2011), but the stimulatory effect of optimum context on initiation rate is not understood at the molecular level. There is evidence that the context nucleotides are recognized by the α-subunit of eIF2, as replacement of heterotrimeric eIF2 with the eIF2βγ heterodimer reduced the efficiency of AUG recognition and diminished the stimulatory effect of optimum context on 48S PIC assembly in a reconstituted mammalian system (Pisarev et al., 2006). Moreover, crosslinking experiments (Pisarev et al., 2006; Sharifulin et al., 2013) and structural analyses of a mammalian 43S PIC (Hashem et al., 2013) and a partial yeast (py48S) PIC (Hussain et al., 2014) indicate that the N-terminal domain (D1) of eIF2α is in proximity to the −3 nucleotide of the mRNA in the exit channel of the 40S subunit. These and other studies (Lomakin and Steitz, 2013) revealed that the conserved β-hairpin of the 40S protein uS7 (Rps5 in yeast) lies in the vicinity of eIF2α-D1 and the −3 nucleotide of mRNA in reconstituted 43S/48S PICs (Figure 2A,B); however, functional evidence that eIF2α-D1 and the Rps5 β-hairpin have important roles in start codon recognition in vivo is lacking. Figure 2 Download asset Open asset Location in the py48S PIC, and sequence conservation, of the Rps5 β-hairpin loop. (A, B) Depiction of the partial yeast 48S PIC (PDB 3J81) showing Rps5 (gold), mRNA (orange), Met-tRNAi (green), eIF2α (purple), eIF2γ (yellow), eIF1 (cyan) and eIF1A (blue). For clarity other ribosomal proteins, eIF2β and putative eIF5 densities are not shown. Residues implicated here in AUG recognition are shown with stick side-chains and highlighted (as in panel C) in blue or pink. (C) Alignment of Rps5 sequences from diverse eukaryotes using the Clustal Omega algorithm (http://www.ebi.ac.uk/Tools/msa/clustalo/). The β- hairpin loop is annotated below the alignment and residues implicated in this study in AUG recognition are highlighted in blue or pink and underlined. https://doi.org/10.7554/eLife.07939.004 In this report, we establish that the β-hairpin of Rps5 is critically required for both efficient and accurate translation initiation in vivo. Substituting Glu-144 (E144) in β-strand 1 of the hairpin, or the proximal C-terminal residue R225 (Figure 2B), confers a marked reduction in the efficiency of AUG recognition. This defect exacerbates the effect of poor context at the SUI1 mRNA start codon to reduce eIF1 cellular abundance and thereby increase recognition of near-cognate UUG codons as a secondary effect. Analysis in the yeast reconstituted system reveals striking destabilization of the PIN state formed at AUG codons by E144R mutant 40S subunits. Substitutions in the loop portion of the Rps5 β-hairpin also destabilize the PIN state, but produce this effect selectively for near-cognate UUG start codons, and thereby dampen UUG initiation in vivo. These findings indicate that the Rps5 β-hairpin functions on a par with soluble initiation factors, such as eIF1, eIF1A and eIF2, to insure efficient and accurate start codon recognition in eukaryotic cells. Results Substitutions E144R and R225K impair translation initiation and start codon selection in vivo To examine the role of the Rps5 β-hairpin in start codon recognition, we introduced single substitutions into 3 residues of β-strand 1 (144EDT146) and 8 of the 10 residues in the hairpin loop (147TR148 and 151GGGARRQ158) (Supplementary file 1). Residues in the β-strands, and the loop residues proximal to the β-strands, are among the most highly conserved in evolution (Figure 2C). We also substituted the last four residues of Rps5 (222KSNR225) in view of their strong conservation and proximity to the β-hairpin, and because invariant Glu144 in β-strand 1 (E144) appears to form a salt-bridge with C-terminal residue R225 in the yeast 80S ribosome (Ben-Shem et al., 2011) (Figure 2B). Residues were generally substituted with Ala to shorten the side-chain, or with basic or acidic residues to introduce or alter side-chain charge (Supplementary file 1). The mutations were generated in an RPS5 allele under its own promoter on a low-copy plasmid and examined in a yeast strain with WT chromosomal RPS5 under a galactose-inducible promoter (PGAL1). Mutant phenotypes were scored following a switch from galactose to glucose, where PGAL1-RPS5 expression is repressed. Despite strong sequence conservation of many β-hairpin residues (Figure 2C), only the G151S substitution was lethal and prevented growth on glucose medium; however, several substitutions conferred a slow-growth (Slg−) phenotype, including E144R and R225K (Figure 3A, glucose; Supplementary file 1). Figure 3 Download asset Open asset RPS5 mutations E144R and R225K impair translation initiation and elevate UUG initiation without reducing 40S subunit abundance in vivo. (A) 10-fold serial dilutions of transformants of PGAL1-RPS5 his4-301 strain (JVY07) with the indicated plasmid-borne RPS5 alleles were spotted on synthetic medium supplemented with histidine and containing galactose (SGal + His + Ura + Trp) or glucose (SD + His + Ura + Trp) as carbon source and incubated at 30°C for 3 days (Glucose) or 4 days (Galactose). (B) Strains from (A) also harboring HIS4-lacZ reporters with AUG or UUG start codons (plasmids p367 and p391, respectively) were cultured in SD + His + Trp at 30°C to A600 of ∼1 and β-galactosidase specific activities were measured in WCEs in units of nanomoles of o-nitrophenyl- β-D-galactopyranoside (ONPG) cleaved per min per mg of total protein. Ratios of mean expression of the UUG and AUG reporters calculated from four transformants are plotted with error bars (indicating S.E.M.s). (C) Strains from (A) were cultured in SD + His + Ura + Trp at 30°C to A600 of ∼1, and cycloheximide was added prior to harvesting. WCEs were separated by sucrose density gradient centrifugation and scanned at 254 nm to yield the tracings shown. Mean Polysome/Monosome and 40S/60S ratios (and S.E.M.s) from four replicates are indicated. Student's t-test indicates that the mean values for polysome/monosome ratio in the RPS5 mutants are reduced significantly from the WT (p < 0.0005). (D) Similar to (C) but the cultures were not treated with cycloheximide and lysed in buffers without MgCl2 to allow separation of the dissociated ribosomal subunits. https://doi.org/10.7554/eLife.07939.005 To identify effects on fidelity of start codon selection, the mutant strains were assayed for expression of otherwise identical HIS4-lacZ reporters containing an AUG or UUG start codon. Substantial (>twofold) increases in expression of the UUG relative to AUG reporter (UUG:AUG ratio) were observed only for E144R and three different mutations substituting Arg225. Mutations E144R and R225K elevated the UUG:AUG ratio by 5.9- and 3.6-fold, respectively (Figure 3B and Supplementary file 1). To test the importance of the Glu144/Arg225 salt-bridge, we constructed the E144R/R225E double mutant in which the salt-bridge should be reinstated. This strain displayed a Slg− phenotype and increased UUG:AUG ratio similar in magnitude to that of the R225K mutant but less severe than seen for the E144R strain (Figure 3A,B). The fact that combining these mutations did not produce more severe phenotypes than those conferred by E144R alone is consistent with the possibility that reinstating the salt-bridge mitigates the effects of the E144R single mutation, with the stipulation that the WT identity of E144 or R225 is needed for robust Rps5 function. Thus, although the salt-bridge is reinstated, the substitution of one or both residues still impairs growth and initiation fidelity in the double mutant. Additional experiments are needed to establish the importance of the salt bridge for Rps5 function. Mutations in various initiation factors are known that elevate the UUG:AUG ratio and restore translation of mutant his4-301 mRNA, which lacks the AUG start codon, by enabling initiation at the third, UUG codon, thereby suppressing histidine auxotrophy and conferring a Sui− (Suppressor of initiation codon mutation) phenotype. However, neither E144R nor R225K suppress the His− phenotype of his4-301 to confer the Sui− phenotype. Based on previous observations of Sui− mutants, it is possible that the Rps5 substitutions do not elevate the UUG:AUG ratio sufficiently to produce enough his4-301 product for adequate histidine biosynthesis (Dorris et al., 1995; Martin-Marcos et al., 2013). For example, the eIF1 mutations sui1-K37A and sui1-R33A increase the UUG:AUG ratio by 4.8- and 7.7-fold, but only the latter suppresses the His− phenotype of his4-301 (Martin-Marcos et al., 2013). Alternatively, the Rps5 mutations could interfere with an unknown aspect of histidine biosynthesis or utilization (Nanda et al., 2009). Consistent with their Slg− phenotypes, E144R and R225K conferred significant reductions in the polysome:monosome (P/M) ratio (p-value <0.0005) (Figure 3C), indicating a reduced rate of bulk translation initiation relative to elongation, with the greater reduction conferred by the mutation (E144R) with the stronger Slg− phenotype (Figure mutant significantly the ratio of 40S to (Figure that the initiation from 40S than in expression of 40S or stability of 40S subunits. Thus, it appears that E144R and R225K reduce the of Rps5 in the rate of translation initiation and accurate start codon selection. E144R and R225K elevate UUG initiation by exacerbating the effect of poor context of the SUI1 start codon and thereby reducing eIF1 abundance In to initiation from near-cognate UUG codons, Sui− mutations in eIF1, eIF1A, and eIF2β are known to initiation from AUG codons in poor context. suppress the effects of the suboptimal context of the AUG codon of SUI1 mRNA and increase expression of the eIF1 protein (Martin-Marcos et al., 2011). This phenotype is for the Sui− mutation in Figure 8 vs However, previously mutations that UUG recognition, and eIF1 abundance (Figure vs to a that with their elevated UUG:AUG HIS4-lacZ initiation ratios (Figure Consistently, E144R and R225K reduce expression of a reporter bearing the suboptimal context at the three nucleotides the AUG codon but not that of a reporter with an AUG context (Figure Thus, the mutations the effect of suboptimal context and AUG recognition on WT SUI1 mRNA. The reduction in eIF1 abundance that the mutations the of eIF1 expression, wherein eIF1 suppress the effect of poor context at the SUI1 AUG codon to eIF1 abundance (Ivanov et al., 2010; Martin-Marcos et al., 2011). Accordingly, it appears that the mutations a defect in recognition of the SUI1 AUG codon that at cellular of eIF1 that SUI1 translation. Figure 4 with 2 all Download asset Open asset RPS5 mutations E144R and R225K poor context at the SUI1 AUG to reduce eIF1 expression and confer phenotypes, but phenotypes when eIF1 abundance is (A) WCEs of strains from Figure 3A, and from strain were to using eIF1 or (as of by a of were in successive (B) Strains from Figure also harboring or reporters were cultured and assayed for β-galactosidase activities as in Figure Mean expression and from four transformants are and relative mean expression to that of the WT strain are below the Student's t-test indicates that the mean values for expression in the RPS5 mutants are reduced significantly from the WT < 0.0005). (C) WCEs of strains from Figure also harboring SUI1 plasmid or were to as in were and mean ratios are below the with (D) HIS4-lacZ reporters with AUG or UUG start codons were assayed in strains from (C) as in Figure his4-301 strains with the indicated WT or mutant RPS5 alleles Figure harboring SUI1 plasmid plasmid or were spotted on SD containing (SD + or histidine and incubated for 3 days and the poor context at SUI1 with reduced eIF1 expression a phenotype displayed by known (Suppressor of mutations in eIF1, eIF1A, and which suppress the elevated UUG:AUG ratio conferred by various Sui− mutations (Martin-Marcos et al., 2011). Hence, the fact that and poor context at SUI1 but elevate the UUG:AUG ratio However, their elevated UUG:AUG ratio phenotype could be by the reduced of eIF1 present in these mutants 2011). we that the of WT eIF1 by an plasmid-borne of WT SUI1 (Figure vs the Sui− phenotypes of both mutants, reducing their UUG:AUG ratios to WT (Figure also their Slg− phenotypes, for and for (Figure 1). The resulting strains reduced eIF1 to WT cells containing an of SUI1 (Figure and vs indicating that the mutations still the effect of poor AUG context at of eIF1 We conclude that the increased recognition of UUG start codons conferred by the mutations is an of their reduced expression of eIF1. reducing eIF1 expression by the poor context of the SUI1 AUG codon is a phenotype of known mutants (Martin-Marcos et al., 2011). mutations also reduced eIF1 expression in a we examined phenotypes by for the to suppress the Sui− phenotype of the of eIF5 et al., This test was using the RPS5 mutant strains harboring to for the reduced eIF1 expression for their Sui− on a plasmid conferred the phenotype in the his4-301 strain harboring WT RPS5 and eIF1 (Figure 1 with this phenotype was in the and mutants when eIF1 were by of as the reduction in growth on medium was greater than that seen on medium in the mutants vs the strain (Figure vs 1). of the phenotype of could from of mRNA with of however, at not reduce the expression of a reporter in cells containing an of SUI1 (Figure Hence, similar to known mutations in eIF1 or eIF1A (Martin-Marcos et al., 2011), the mutation appears to suppress recognition of the UUG start codon of his4-301 mRNA in to poor context at the SUI1 AUG codon. E144R and R225K confer scanning of an AUG start codon the mutations recognition of the SUI1 start codon and suppress UUG initiation when eIF1 are We also recognition of an AUG codon and allow scanning to the reporter was with a of that is to the This is for this because all scanning the AUG and at the AUG of the following of translation is that translation of the is et al., confers a increase in scanning elevating expression by for the containing optimum context at (Figure 1, WT vs This effect is to the increase in scanning seen in WT cells for the context of (Figure vs 1). also a increase in scanning for the with the context of but only a increase with the context of (Figure 3 and WT vs of the is not significantly by (Figure WT vs consistent with scanning the source of elevated expression for the various The R225K mutation also increases scanning of but to a for for and for (Figure 1, WT vs The increases in scanning conferred by the mutations were by the of WT eIF1 by (Figure vs 1, This was because the reduced of eIF1 in the mutants not be to reduce recognition and confer scanning, as eIF1 abundance is with increased start codon recognition for near-cognate start codons or AUG codons in poor (Pestova and Kolupaeva, Ivanov et al., 2010; Martin-Marcos et al., 2011). We conclude that the substitutions impair recognition of located in or poor surrounding sequence context, to allow increased translation of the Figure Download asset Open asset RPS5 mutations E144R and R225K confer strong scanning of in vivo. β-galactosidase activities were measured in WCEs of strains from Figure harboring the plasmid (as and reporters or the or poor context of or reporter with a Mean expression values with