Proc Natl Acad Sci U S A 114(11): E2126-E2135

Rps3/uS3 promotes mRNA binding at the 40S ribosome entry channel and stabilizes preinitiation complexes at start codons

Supplementary Material

Supplementary File

^{Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892;}

^{Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892}

^{To whom correspondence may be addressed. Email:}vog.hin@hcsubenniha or vog.hin@hcsrol.noj.

Contributed by Alan G. Hinnebusch, January 24, 2017 (sent for review December 15, 2016; reviewed by Jamie H. D. Cate and Matthew S. Sachs)

Author contributions: J.D., C.E.A., J.R.L., and A.G.H. designed research; J.D. C.E.A., and A.T. performed research; B.-S.S. contributed new reagents/analytic tools; J.D., C.E.A., A.T., J.R.L., and A.G.H. analyzed data; and J.D., C.E.A., J.R.L., and A.G.H. wrote the paper.

Reviewers: J.H.D.C., University of California, Berkeley; and M.S.S., Texas A&M University.

Contributed by Alan G. Hinnebusch, January 24, 2017 (sent for review December 15, 2016; reviewed by Jamie H. D. Cate and Matthew S. Sachs)

Significance

In the initiation of protein synthesis, a preinitiation complex (PIC) of the 40S ribosomal subunit, initiation factors, and initiator tRNA_i scans the mRNA leader for an AUG codon in favorable context; and AUG recognition evokes a closed conformation of the PIC with more tightly bound tRNA_i. uS3 (Rps3 in yeast) is a protein in the 40S mRNA entry channel, whose function during initiation was unknown. Substituting uS3 arginine residues in contact with mRNA reduces initiation at suboptimal start codons (UUG and AUG in poor context), weakens ribosome–mRNA interaction specifically at the entry channel, and destabilizes tRNA_i binding selectively at UUG codons. Thus, uS3 promotes mRNA:40S interaction at the entry channel to enhance initiation accuracy.

Keywords: translation, initiation, uS3, ribosome, yeast

Significance

Abstract

The eukaryotic 43S preinitiation complex (PIC) bearing Met-tRNA_i^{in a ternary complex (TC) with eukaryotic initiation factor (eIF)2-GTP scans the mRNA leader for an AUG codon in favorable “Kozak” context. AUG recognition provokes rearrangement from an open PIC conformation with TC bound in a state not fully engaged with the P site (“P}_OUT”) to a closed, arrested conformation with TC tightly bound in the “P_IN” state. Yeast ribosomal protein Rps3/uS3 resides in the mRNA entry channel of the 40S subunit and contacts mRNA via conserved residues whose functional importance was unknown. We show that substitutions of these residues reduce bulk translation initiation and diminish initiation at near-cognate UUG start codons in yeast mutants in which UUG selection is abnormally high. Two such substitutions—R116D and R117D—also increase discrimination against an AUG codon in suboptimal Kozak context. Consistently, the Arg116 and Arg117 substitutions destabilize TC binding to 48S PICs reconstituted in vitro with mRNA harboring a UUG start codon, indicating destabilization of the closed P_IN state with a UUG–anticodon mismatch. Using model mRNAs lacking contacts with either the mRNA entry or exit channels of the 40S subunit, we demonstrate that Arg116/Arg117 are crucial for stabilizing PIC–mRNA contacts at the entry channel, augmenting the function of eIF3 at both entry and exit channels. The corresponding residues in bacterial uS3 promote the helicase activity of the elongating ribosome, suggesting that uS3 contacts with mRNA enhance multiple phases of translation across different domains of life.

Abstract

Accurate identification of the translation initiation codon in mRNA by ribosomes is crucial for expression of the correct cellular proteins. This process generally occurs in eukaryotic cells by a scanning mechanism wherein the small (40S) ribosomal subunit first recruits charged initiator tRNA (Met-tRNA_i^{) in a ternary complex (TC) with eukaryotic initiation factor (eIF)2-GTP in a reaction stimulated by eIFs 1, 1A, 3, and 5. The resulting 43S preinitiation complex (PIC) attaches to the 5′ end of the mRNA and scans the 5′ UTR with TC bound in a metastable state, “P}_OUT,” suitable for inspecting successive triplets for complementarity with the anticodon of Met-tRNA_i^{in the P site, to identify the AUG start codon. Nucleotides surrounding the AUG, particularly at the −3 and +4 positions (the Kozak context), further influence the efficiency of start-codon selection. In the scanning PIC, eIF2 can hydrolyze GTP, dependent on GTPase-activating protein eIF5, but P}_i release is blocked by eIF1, whose presence also impedes stable binding of Met-tRNA_i^{in the “P}_IN” state. Start-codon recognition triggers dissociation of eIF1 from the 40S subunit, allowing P_i release from eIF2-GDP•P_i and TC binding in the P_IN state of the 48S PIC (Fig. 1A). 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-tRNA_i^{base-paired to AUG in the P site (reviewed in ref.}1).

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Fig. 1.

Rps3/uS3 plays a critical role in promoting mRNA binding at the 40S entry site and stabilizing the preinitiation complex at the start codon. (A) Model describing known conformational rearrangements of the PIC during scanning and start-codon recognition. (A, i) eIF1 and the scanning enhancers (SEs) in the C-terminal tail (CTT) of eIF1A stabilize an open conformation of the 40S subunit to which TC rapidly binds. Rps3 (uS3) is located on the solvent-exposed surface of the 40S subunit near the entry channel; the bulk of eIF3 binds on the solvent-exposed surface, with a prominent domain at the mRNA exit channel. (A, ii) The 43S PIC in the open conformation scans the mRNA for the start codon with Met-tRNA_i^{bound in the P}_OUT state. eIF2 can hydrolyze GTP to GDP•P_i, but release of P_i is blocked. (A, iii) On AUG recognition, Met-tRNA_i^{moves from the P}_OUT to the P_IN state, clashing with eIF1 and the CTT of eIF1A, provoking displacement of the eIF1A CTT from the P site, dissociation of eIF1 from the 40S subunit, and P_i release from eIF2. The N-terminal tail (NTT) of eIF1A, harboring scanning inhibitor (SI) elements, adopts a defined conformation and interacts with the codon–anticodon helix. (Top) Arrows summarize that eIF1 and the eIF1A SE elements promote P_OUT and impede transition to the P_IN state, whereas the SI element in the NTT of eIF1A stabilizes the P_IN state. Results presented here indicate that uS3/Rps3 residues R116/R117, in contact with mRNA at the entry channel, stabilize the P_IN state and also promote PIC interaction with mRNA at the entry channel, augmenting the role of eIF3 in PIC–mRNA interactions at the exit channel (adapted from ref. 1). (B) Alignment of a portion of uS3 sequences from diverse eukaryotes and Escherichia coli using Clustal Omega (www.ebi.ac.uk/Tools/msa/clustalo/). Boundaries of secondary structure on the top line refer to the Saccharomyces protein; symbolic summary of sequence conservation applies only to the upper seven eukaryotic sequences. Six conserved residues of Rps3 at the mRNA entry channel analyzed in this study are highlighted in black or pink. (C) Position of uS3/Rps3 in the yeast 48S PIC, and locations of conserved residues at the entry channel. The solvent-exposed surface of the partial yeast 48S PIC [Protein Data Bank (PDB) ID code 3J81] is depicted (Left) in cartoon format highlighting uS3 (green), mRNA (black), Met-tRNA_i^{(pink), and rRNA residues of h18 or h34 that comprise the entry channel latch (blue). The boxed region is amplified (}Right) where the six uS3/Rps3 residues analyzed here, which interact with mRNA or the rRNA latch, are highlighted in black or magenta (only for ease of visualization), shown in stick format, and labeled. Other ribosomal proteins and eIFs 1, 1A, and 2 are hidden for clarity.

eIF1 plays a dual role in the scanning mechanism, promoting rapid TC loading in the P_OUT conformation while blocking rearrangement to P_IN at both near-cognate start codons (e.g., UUG) and cognate (AUG) codons in poor Kozak context; hence eIF1 must dissociate from the 40S subunit for start-codon recognition (Fig. 1A). Consistent with this, structural analyses of partial PICs reveal that eIF1 and eIF1A promote rotation of the 40S head relative to the body (2, 3), thought to be instrumental in TC binding in the P_OUT conformation, but that eIF1 physically clashes with Met-tRNA_i^{in the P}_IN state (2, 4), and is both deformed and displaced from its 40S location during the P_OUT-to-P_IN transition (3). Mutations that weaken eIF1 binding to the 40S subunit reduce the rate of TC loading and elevate initiation at near-cognate codons or AUGs in poor context as a result of destabilizing the open/P_OUT conformation and favoring rearrangement to the closed/P_IN state during scanning (5, 6). Moreover, decreasing wild-type (WT) eIF1 abundance reduces initiation accuracy, whereas overexpressing eIF1 suppresses initiation at near cognates or AUGs in poor context (5, 7 –10). In fact, cells exploit the mechanistic link between eIF1 abundance and initiation accuracy to autoregulate eIF1 expression: The AUG codon of the eIF1 gene (SUI1 in yeast) occurs in poor context, and the frequency of its recognition is inversely related to eIF1 abundance (5, 10).

The stability of the codon–anticodon duplex is an important determinant of initiation accuracy, as the rate of the P_OUT-to-P_IN transition is accelerated and the P_IN state is stabilized in the presence of AUG versus non-AUG start codons (11). Favorable Kozak context might also contribute to P_IN stability (5, 12), but the stimulatory effect of optimum context on initiation rate is not well-understood. It seems to require the α-subunit of eIF2 (12), and structural analyses of partial mammalian 43S (13) and yeast 48S PICs (3) place eIF2α domain 1 near the key −3 context nucleotide in the exit channel of the 40S subunit. The conserved β-hairpin of 40S protein uS7 (Rps5 in yeast) also occurs in this vicinity in a yeast partial 48S (py48S) PIC (3). We have shown that the β-hairpin of yeast Rps5 is important for both efficient and accurate translation initiation in vivo and the stability of P_IN complexes reconstituted in vitro (14). Approximately 7 additional mRNA nucleotides upstream of the −3 position occupy the 40S exit channel, and there is evidence that these 40S–mRNA interactions plus additional contacts between segments of eIF3 and mRNA nucleotides protruding from the exit channel also enhance PIC assembly at the start codon (15 –18).

eIF3 is a multisubunit complex that binds directly to the 40S subunit and promotes both recruitment and stable association of TC and mRNA with the PIC, and enhances scanning and accurate start-codon recognition in vivo (19). Recent structural analyses (20 –22), combined with earlier biochemical and genetic studies (19), reveal that different subunits/domains of yeast eIF3 interact with the PIC at multiple sites, effectively encircling the PIC and interacting with both the mRNA entry and exit pores on the solvent-exposed surface, as well as with the decoding center on the interface surface of the 40S subunit. The major point of contact involves binding of a heterodimer of the proteasome-COP9-initiation factor (PCI) domains of the eIF3a and c subunits to the 40S solvent side, below the platform and exit channel pore. A second contact occurs near the entry channel and involves segments of the eIF3a, b, g, and i subunits; at least some of these interactions appear to be dynamic, as alternative contacts with eIFs anchored in the decoding center have also been observed (20 –22). We recently presented evidence that the PCI domain of eIF3a mediates a key stabilizing interaction between the PIC and mRNA at the exit channel (18). Using a panel of m^{G-capped, unstructured model mRNAs to reconstitute 48S PICs, we determined that the eIF3a PCI domain interaction at the exit channel is functionally redundant with mRNA–PIC interactions at the entry channel, and is essential for stable 48S PIC assembly when the mRNA is truncated in a way that leaves the entry channel largely empty. Other eIF3 domains/subunits contribute to the functionally redundant contacts at the entry channel, but are not essential even when the opposite exit channel is unoccupied by mRNA. This suggests that other components of the PIC, including elements of the ribosome itself, participate in stabilizing mRNA binding at the entry channel.}

In fact, the cryo-EM structure of a partial yeast 48S PIC revealed predicted contacts between mRNA residues located 6 to 12 nt downstream (3′) of the AUG codon with particular amino acids of ribosomal protein Rps3/uS3 at the mRNA entry channel of the 40S subunit. Several Rps3 residues also appear to interact with 18S rRNA residues that form the “latch” on the entry channel, a noncovalent interaction between rRNA nucleotides in helices 18 and 34 (3). Interestingly, whereas the latch is closed in the yeast 48S complexes with AUG in the P site and in crystal structures of other partial PICs (2, 4, 23), the latch is open in a recent cryo-EM structure of a PIC formed with mRNA containing an AUC codon, owing to upward movement of the head away from the body of the 40S subunit. The P site is also widened in this open PIC conformation such that the tRNA_i^{is not fully engaged with rRNA residues in the body that contribute to the highly stable P}_IN conformation observed in the corresponding AUG complex (22). Hydroxyl radical probing of yeast PICs reconstituted with AUG or AUC mRNAs also revealed a more open conformation of the P site and less constricted mRNA entry channel in the AUC complex (24), consistent with a scanning-conducive conformation of the 40S subunit when a near-cognate triplet occupies the P site. Although Rps3 residues appear to interact with the mRNA and with rRNA residues of the entry channel latch, there is no functional evidence that these predicted contacts are important for the efficiency or fidelity of start-codon recognition. Here we provide strong genetic and biochemical evidence that these Rps3 residues enhance the stability of the P_IN state and promote recognition of poor initiation sites in vivo, and also mediate stabilizing mRNA interactions with the PIC at the entry channel that are functionally redundant with eIF3-dependent PIC–mRNA interactions at the exit channel.

Acknowledgments

We thank Jagpreet Nanda, Jyothsna Visweswaraiah, and Fan Zhang for advice on k_off determinations. We are grateful to members of our laboratories and Tom Dever’s group for helpful suggestions. This work was supported in part by the Intramural Research Program of the NIH (A.G.H. and J.R.L.) and NIH Grant GM62128 (previously to J.R.L.). C.E.A. was further supported by an NIH minority supplement to NIH Grant GM62128 and by a Leukemia & Lymphoma Society Career Development Program Fellowship.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1620569114/-/DCSupplemental.

Footnotes

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