Researchers are exploring whether these ubiquitous fluorinated molecules might worsen infections or hamper vaccine effectiveness.
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Edited by Rachel Green, Johns Hopkins University, Baltimore, MD, and approved July 10, 2014 (received for review May 21, 2014)
Significance
Biological fitness is dependent on the accurate flow of genetic information from DNA to mRNA to protein. Breakdown in ribosome translational fidelity is detrimental because of its central role in the production of proteins. Altering the 3-base genetic code usually results in the expression of aberrant or nonsense proteins that are degraded. Here, we describe molecular snapshots of the ribosome in the process of decoding a 4-base codon by a frameshift suppressor tRNA that results in a +1-nt shift of the mRNA reading frame. Conformational dynamics of the anticodon stem loop seem to drive remodeling of the tRNA–mRNA interaction to promote the +1 movement, which we predict occurs after accommodation of the tRNA onto the ribosome.
Abstract
Maintenance of the correct reading frame on the ribosome is essential for accurate protein synthesis. Here, we report structures of the 70S ribosome bound to frameshift suppressor tRNASufA6 and N1-methylguanosine at position 37 (m1G37) modification-deficient anticodon stem loopPro, both of which cause the ribosome to decode 4 rather than 3 nucleotides, resulting in a +1 reading frame. Our results reveal that decoding at +1 suppressible codons causes suppressor tRNASufA6 to undergo a rearrangement of its 5′ stem that destabilizes U32, thereby disrupting the conserved U32–A38 base pair. Unexpectedly, the removal of the m1G37 modification of tRNAPro also disrupts U32–A38 pairing in a structurally analogous manner. The lack of U32–A38 pairing provides a structural correlation between the transition from canonical translation and a +1 reading of the mRNA. Our structures clarify the molecular mechanism behind suppressor tRNA-induced +1 frameshifting and advance our understanding of the role played by the ribosome in maintaining the correct translational reading frame.
The three polymerase reactions of DNA replication, RNA transcription, and protein translation are essential to life and all involve a delicate balance between speed and fidelity. The bacterial ribosome decodes three mRNA nucleotides into a single amino acid at a rate of ∼20 residues/s with high fidelity (103–104) (1, 2). High translational fidelity requires the strict maintenance of the triplet mRNA reading frame with shifts in either the 5′ or 3′ direction (positive or negative shifting, respectively) occurring infrequently (∼1 in 30,000) (3). Upon shifting of the mRNA, the proteins expressed are nonsense polypeptides and typically degraded (4). However, frameshifting rates may be underestimated because of rapid halting of protein synthesis by the postpeptidyl transfer quality control mechanism, which recognizes near- and noncognate interactions in the P site (5). In this case, the identification of such incorrect proteins would be difficult to detect.
Although it was hypothesized almost 50 years ago that the triplet genetic code was immutable, the identification of mutagen-induced genomic insertions that still yielded the expression of the correct protein implied otherwise (6⇓–8). This alternative reading of mRNA indicated that the ribosome had the ability to decode a nontriplet reading frame under certain circumstances. It was reasoned that suppressors to these genomic insertions should be located adjacent to the original mutation site. However, most identified frameshift suppressors were extragenic; genetic mapping showed that the majority of these suppressors were nucleotide insertions, deletions, and modifications in tRNA genes, typically at the anticodon stem loop (ASL) (9, 10). Over the next several decades, genetic characterization of these suppressors played a large role in the fundamental understanding of tRNA structure, decoding, and other general features of ribosome function (reviewed in ref. 11).
The first bacterial +1 suppressor sequenced was tRNASufD (suppressor of frameshift D), a derivative of tRNAGlyCCC that contained a cytosine insertion in its ASL and decoded an expanded glycine codon 5′-GGG-G-3′ (inserted guanosine follows the GGG glycine codon and is preceded by a hyphen; all codons are denoted 5′ to 3′) (12). Given the Watson–Crick complementarity between the inserted nucleotide at the anticodon (C) and codon (G), a 4-bp interaction between the codon and anticodon was proposed as the mechanistic basis for the +1 mRNA reading frame (13). Additional evidence to support this hypothesis included the isolation of suppressor tRNAs from both bacteria (7, 12, 14⇓⇓–17) and yeast (18, 19), all containing additional nucleotides in their anticodon loops that had the ability to Watson–Crick pair with the corresponding expanded mRNA codon. This 4-bp decoding or quadruplet model was further expanded upon to state that the size of the tRNA anticodon loop dictated the mRNA length decoded by the ribosome rather than the number of potential interactions that form between the tRNA–mRNA pair (20). Subsequently, this elegant model, termed the “yardstick” model, became dogma in textbooks.
However, emerging evidence began to call both models into question. The first indication that a 4-nt codon–anticodon interaction is not essential for +1 decoding was the identification of suppressor tRNASufJ (a tRNAThrGGU derivative), which lacks Watson–Crick complementarity between the inserted nucleotide in the anticodon stem and the additional residue in the mRNA (21, 22). The second indication was that mutational studies of tRNASufD and other +1 tRNA suppressors showed that Watson–Crick complementarity between the anticodon and codon was not essential for suppression of the +1 site (19, 23⇓⇓–26). Moreover, the isolation of +1 suppressors with normal 7-nt anticodon loops illustrated that factors other than an increased loop size may influence frameshifting (27, 28).
High-resolution X-ray crystal structures of the 70S ribosome have provided molecular insights at multiple steps during protein synthesis (reviewed in ref. 29). Despite these advances, very few structural studies have been able to provide a molecular understanding of mRNA frameshifting. Here, we investigated the structural basis for +1 decoding by a well-studied suppressor, tRNASufA6 (17, 30). Frameshift suppressor tRNASufA6 is a derivative of tRNAProCGG and contains an inserted guanosine (G37.5) between positions 37 and 38 in the anticodon loop (Fig. 1A). tRNASufA6 suppresses a mutagen-induced cytosine insertion in the Salmonella typhimurium histidine operon by decoding CCC-N codon as proline (where N represents any nucleotide) (17, 30) (Fig. 1B). This 4-nt decoding or +1 frameshifting event restores the correct reading frame and reverts a nonsense truncated protein back to the WT protein. The presence of an N1-methylation at position 37 (m1G37) in tRNASufA6 precludes base pairing with the cytosine-rich proline codon and thus, prevents a 4-base codon–anticodon interaction (30). We investigated both how tRNASufA6 mediates frameshifting and what role the modification plays in mRNA frame maintenance by determining nine X-ray crystal structures of either the ASLSufA6 or ASLPro bound to the Thermus thermophilus 70S A site at resolutions of 2.9–3.9 Å (Tables S1 and S2). Our results distinguish between the various models of +1 frameshifting and provide molecular insights into the frameshifting process.
Fig. 1.
Frameshift suppressor tRNASufA6 decodes a 4-nt codon to produce a +1 frameshift. (A) Secondary structure representations of the ASLs of tRNAPro and tRNASufA6 show base pairing to their respective mRNA codons (blue). The insertion in the anticodon loop of +1 frameshift suppressor tRNASufA6 is between nucleotides 37 and 38; G37.5 is red, whereas the m1G37 modification is shown in both. (B) Frameshift suppressor tRNASufA6 decodes the proline codon as CCC-U (blue) rather than the cognate 3-nt proline codon CCC (0 frame), suppressing the insertion N nucleotide (any nucleotide but depicted as a U for simplicity) and bypassing a premature stop codon UGA in the zero frame. The +1 reading of hisD3018 by tRNASufA6 restores the correct amino acid sequence with Glu (green) read as the next codon.
Results
Structures of ASLSufA6 Bound to Three +1 Suppressible Codons in the Ribosomal A Site.
We solved three structures of 70S ribosomes programmed with ASLSufA6 and mRNAs containing A-site codons that support +1 frameshifting: CCC-A, CCC-G, and CCC-U (where the fourth nucleotide is decoded as part of the CCC proline codon) (17) (Figs. 1A and 2A and Table S1). The antibiotic paromomycin was used in all 70S structures determined to promote better diffraction. Two structures of 70S programmed with ASLSufA6 decoding the +1 suppressible codon CCC-U were solved with and without paromomycin, revealing identical structures (Table S1). In all three 70S-ASLSufA6 structures, a canonical 2-nt codon–anticodon interaction forms with no interactions between ASLSufA6 and the fourth nucleotide of the codon (codon position 7 with numbering beginning with the P-site codon) (Fig. 2A and Fig. S1). Despite the potential for a Watson–Crick base pair between the ASL wobble nucleotide C34 and the fourth nucleotide in the expanded CCC-G codon, only a 3-nt codon–anticodon interaction is observed (Fig. 2A). Similar to structures of tRNA decoding cognate mRNAs on the 30S and 70S ribosomes, Watson–Crick base pair interactions form at the first and second positions of the codon–anticodon helix (31, 32). Unexpectedly, at the wobble position, a C•C mismatch base pair between ASL C34 and mRNA position C6 forms containing a single hydrogen bond between the N4 amino group of C34 and the N3 atom of C6 of the mRNA (Fig. 2 A and B). The C34•C6 mismatch pairing is a similar width as a Watson–Crick C-G base pair (measured from Cl′ to C1′) (Fig. 2B) and preserves the base-stacking interactions with adjacent codon–anticodon nucleotides (Fig. 2A). Additionally, the C34•C6 mismatch contains a similar geometry as an ASL-modified cmo5U•U mRNA wobble interaction, providing a possible rationale for why the ribosome views both as cognate (33). However, to accommodate the C34•C6 mismatch, the tip of ASL nucleotide C34 (N4 atom) moves toward the mRNA by 1.6 Å (Fig. 2C). The wobble mRNA C6 position is rigidly held in place, possibly because of the coordination of a Mg2+ to its 2′-OH and the base of 16S rRNA residue G530 as seen in other cognate tRNA–mRNA structures (32) (Fig. 2D). The coordination of the Mg2+ by both mRNA and 16S rRNA may restrict the movement of the A-site mRNA, necessitating the ASL conformational changes that we observe. The length of the codon–anticodon helix is maintained by 16S rRNA C1054, which stacks beneath C34 of the ASL, indicating that base stacking is not affected by the C•C mismatch pairing.
Fig. 2.
Details of the interaction of ASLSufA6 with mRNA in the ribosomal A site. (A) Watson–Crick base pairs at the first two positions of the A-site codon (C4 and C5) form when ASLSufA6 decodes a CCC-N codon, whereas a mismatch interaction occurs between ASL C34•C6 codon at the wobble position (shown with codon G7; codon numbering starts from the P-site codon). The inserted nucleotide G37.5 is 3′ of the anticodon (red), whereas the nucleotide that lacks any electron density (U32) is shown in blue. (B) A detailed view of the base pairing between C34 of ASLSufA6 and the C6 or wobble position of the mRNA. (Upper) ASLSufA6 forms the C•C mismatch pair in the context of a +1 frameshift CCC-G codon but (Lower) can also form a Watson–Crick C-G base pair with a cognate CCG codon. 2Fo-Fc electron density map contoured at 1.0 σ is shown in blue. The overall width of the C34•C6 mismatch and C34–G6 base pair is similar at 10.7 and 11.0 Å, respectively, as measured between C1′ atoms as depicted by the bar. (C) A comparison of the position of ASLSufA6 C34 when decoding either a +1 suppressible codon (purple) or a cognate proline codon (green) shows an ∼1.6 Å movement of C34 (N4 of the base) to allow the C34•C6 pairing. (D) Direct coordination between the 2′-OH of C6 of the mRNA, a Mg2+ ion (green sphere), and 16S rRNA residue G530 (gray) fixes the position of C6 requiring C34 movement to accommodate the C•C pair. Fo-Fc difference electron density maps are contoured at 3 σ (green mesh), indicating the presence of the Mg2+ ion.
During tRNA selection, three highly conserved 16S rRNA nucleotides (A1492, A1493, and G530) monitor the codon–anticodon interaction in the A site. These three 16S rRNA nucleotides directly inspect the minor groove formed by the codon–anticodon helix to probe for Watson–Crick pairing at the first and second positions (31). In our structure, despite the near-cognate interaction with a single mismatch at the wobble position, rRNA interacts identically with the codon–anticodon helix as observed in other ribosome structures containing cognate tRNA–mRNA pairs (31, 32) (Fig. S2) and even other near-cognate pairs (34, 35). Additionally, 23S rRNA residue 1913 interacts directly with the 2′-OH of ASL nucleotide 37, despite the adjacent inserted nucleotide.
A 7-nt anticodon loop is highly conserved across all domains of life and known to diverge in only rare cases, such as in yeast mitochondrial tRNAThr (36). The G37.5 insertion leads to an increase in the minor groove width of the anticodon loop, which is most pronounced (∼5 Å) immediately adjacent to the insertion between nucleotides 31–33 (Fig. 3A). This increased minor groove width leads to an increase in RNA helical twist, which is distributed over the length of the entire anticodon stem (Fig. 3 A and B). Although there is a large conformational movement of the phosphate backbone, base pairing of the stem seems to be back in standard register by the nucleotides 29–41 pairing (Fig. 3A). On the other side of the anticodon loop, the structural integrity of the conserved U-turn motif is maintained. Because the phosphate backbone movement of the 5′ stem is distributed over the length of the ASL, structural superpositioning of ASLSufA6 onto ASLPro reveals little variation in its global position (Fig. 3A and Fig. S3).
Fig. 3.
Remodeling of ASLSufA6 structure induced by the G37.5 insertion. (A) Overview of the 8-nt anticodon loop of ASLSufA6 (green) compared with the canonical 7-nt anticodon loop of ASLPro (gray). The G37.5 insertion (red) causes a widening of the ASL minor groove by ∼5 Å between nucleotides 30 and 32 located on the opposite side of the insertion site. In ASLSufA6, G31 and U32 span the distance of 3 nt (G37.5, A38, and C39) on the 3′ side of the ASL; however, in canonical tRNAs, such as tRNAPro, G31 and U32 span only 2 nt (A38 and C39) on the 3′ side of the ASL. In both cases, the G31–C39 base pair forms the boundary of the base-paired stem region. The ribose and base of ASLSufA6 nucleotide U32 (blue) are not observed in electron density maps, although its phosphate and the following phosphate are visible, allowing its position to be approximated. (B) Secondary representations of both ASLPro and ASLSufA6 show that expansion of the ASLSufA6 anticodon loop to 8 nt abrogates the conserved hydrogen bond between U32 and A38 (boxed) and alters the incline of base pairs in ASLSufA6. (C) The U32–A38 interaction in ASLPro (gray) shown alongside U32 (blue) and G37.5 (red) of ASLSufA6, emphasizing the lack of interaction.
ASLSufA6 Undergoes a Context-Dependent Conformational Change at U32.
Important tertiary features govern ASL stability, including a hydrogen bond between the O2 of a pyrimidine at position 32 and the N6 of A38 (Fig. 3 B and C). The insertion of G37.5 in ASLSufA6 impedes the formation of the U32–A38 interaction, because G37.5 occupies the equivalent physical position of A38 in the ASL (Fig. 3A). Despite excellent electron density for most of the defining features of the ASL in all three structures of 70S-ASLSufA6 decoding +1 suppressible codons, there is a distinct absence of electron density for U32, which is located on the opposite side of the loop from the G37.5 insertion (Fig. 4A).
Fig. 4.
Dynamics of ASL nucleotide U32 are controlled by the ASL and mRNA context. The ASLSufA6 G37.5 insertion (red) interferes with the U32–A38 base pair, increasing the dynamics of U32 (blue) such that it undergoes an ordered to disordered conformational switch depending on whether it is bound to (A) a +1 frameshifting codon CCC-A/G/U or (B) a cognate CCG codon. Anticodon nucleotides 34, 35, and 36 that interact with the mRNA codon are indicated by an arc, whereas U32 is indicated with an arrow. (C) The absence or (D) presence of the m1G modification at G37 in ASLPro that is also known to promote +1 frameshifting controls the dynamics of the 5′ stem of the ASL when decoding a cognate CCG codon. 2Fo-Fc electron density contoured at 1.0 σ is shown in blue.
To determine if this disordering of U32 is an inherent property of ASLSufA6, we solved a structure of 70S ribosome-ASLSufA6 decoding a cognate proline codon (CCG) (Fig. 4B and Table S1). Surprisingly, in this mRNA–ASL context, the electron density for U32 is clearly visible (Fig. 4B). The presence of U32 electron density in this context indicates that disordering of U32 is not inherently caused by the G37.5 insertion in tRNASufA6. Rather, it is the combination of the suppressor tRNA and mRNA sequence context that seems to drive the observed flexibility of U32.
We next examined whether the disordering of U32 is structurally characteristic of the +1 suppressible or C•C near-cognate base pair. We solved two structures of 70S bound to ASLPro, which contains a 7-nt anticodon loop and an m1G37 modification, decoding both +1 suppressible codons CCC-G and CCC-U (Fig. S4 and Table S2). An identical C34•C6 mismatch pair forms at the wobble position while electron density for U32 is observed (Fig. S5). These results indicate that U32 disordering is the consequence of a specific context: the noncanonical 8-nt anticodon loop of ASLSufA6 binding to a suppressible codon with the pattern CCC-N within the ribosomal A site.
Modification State of G37 in ASLPro Also Controls U32 Dynamics.
RNA modifications of tRNAs are widespread, and ASL modifications are known to influence decoding (37). Both the anticodon nucleotides 34 and 37 are highly modified, and the lack of modifications increases frameshifting (38, 39). Modifications at the ASL wobble nucleotide 34 mainly facilitate unusual base pairing to expand the genetic code (33, 37, 40), whereas nucleotide 37 modifications participate in stacking interactions with other anticodon nucleotides and may preorder anticodon nucleotide 36 for mRNA recognition (41, 42). Specifically, the lack of m1G37 modification in tRNAPro promotes +1 frameshifting on near-cognate codons (43, 44). The potential mechanistic similarities of this phenomenon to ASLSufA6-mediated frameshifting led us to investigate how the m1G37 modification of tRNAPro affects its structure at the decoding center. We first solved the structure of ASLProm1G37 decoding its cognate CCG codon bound to the 70S ribosome (Fig. 4D). This complex adopts a conformation consistent with the ribosome recognizing a cognate codon–anticodon pair (31, 32). Next, we determined the structure of 70S bound to ASLPro lacking the m1G37 modification with the same CCG codon (Fig. 4C). In this structure, nucleotides 30–32 of the ASL have poor electron density compared with the 70S-ASLProm1G37 structure (Fig. 4 C and D). This lack of electron density suggests that the loss of the m1G modification at position 37 disrupts the U32–A38 interaction in a manner similar to when ASLSufA6 decodes +1 suppressible codons (Fig. 4A). The structural similarities in modification-deficient ASLPro and suppressor ASLSufA6 suggest that they share a common mechanism to induce +1 frameshifting.
Discussion
The work here provides a structural basis for the roles of tRNA structure, mRNA sequence, and RNA modifications in the noncanonical reading of the genetic code. Although ASLSufA6 contains an additional nucleotide insertion within the anticodon loop, this extra nucleotide does not cause a rearrangement of the anticodon that allows for a 4-nt interaction between the codon and anticodon. Instead, an ASL C34•C6 mismatch forms at the wobble position upon ASLSufA6 decoding one of the +1 suppressible proline codons: CCC-A, CCC-G, or CCC-U (30). The overall geometry of the C34•C6 pairing is nearly superimposable with modified wobble base interactions as seen in 30S structures containing an ASL cmo5U34 decoding either C or U (33). Both our results and the 30S-cmo5U34•U/C structures show movements of the anticodon nucleotides toward the mRNA to accommodate these unusual pairings, suggesting a mechanism by which the wobble position achieves plasticity. In the case of ASLSufA6, the phosphate backbone on the opposite strand to the G37.5 insertion undergoes a conformational rearrangement that considerably widens the minor groove of the ASL by a maximum of 5 Å (Fig. 3A). In contrast, with the 70S-cmo5U•U/C pairings, the anticodon only shifts closer to the codon to facilitate this pairing, and no additional movements are noted elsewhere in the ASL (33).
Modifications 3′ to the anticodon at tRNA position 37 are found on the same isoacceptor tRNAs throughout all three kingdoms, implying their evolutionary importance (45). Deletion of the methyltransferase that modifies nucleotide 37 in tRNAProGGG causes substantial increases in +1 frameshifting (43, 44). Our structures of ASLPro decoding its cognate codon showed striking differences depending on the presence of the m1G37 modification (Fig. 4 C and D). Without the modification, U32 is presumed to be conformationally dynamic because of the lack of electron density that we observe (Fig. 4D), similar to the 70S-ASLSufA6 structures where the disorder of U32 correlates with this suppressor decoding +1 suppressible codons CCC-A/G/U (disordered) or a cognate CCC codon (ordered) (Fig. 4 A and B). In support of the importance of U32 in maintaining the integrity of the ASL are previous 30S structural studies of engineered ASLs containing an extra nucleotide 5′ of the anticodon that also cause a +1 reading of specific mRNA codons (46). Structures of these ASLs revealed a similar destabilization of the 5′ anticodon stem, also disrupting the U32–A38 pair (46).
The structural changes that we observe in ASLSufA6 are distinct from another suppressor tRNA, the Hirsh suppressor. The Hirsh suppressor tRNA mediates UGA stop codon readthrough by decoding a C34•A6 mismatch pairing at the wobble position facilitated by a distortion in the tRNA body caused by a G24A mutation (34, 47). A comprehensive tRNA distortion seen with both A9C and G24A mutations extends to both extreme ends of the tRNA (34): to the decoding center to promote stop codon readthrough and to the GTPase center, where an increase in the rate of GTP hydrolysis by EF-Tu occurs during tRNA selection (48). In contrast, although the ASLSufA6 G37.5 insertion in the anticodon loop affects the structure of the base-paired stem, we predict that structural perturbations are not propagated along the entire tRNA body, because the ASL is almost entirely back into register by the 28–41 bp (Fig. 3A).
Instead, what seems to promote +1 decoding by ASLSufA6 is the lack of a 32–38 interaction, which has previously been shown to be important for correct tRNA selection (49, 50). The nucleotide identity of the 32–38 pair is finely tuned to both the strength of the codon–anticodon interaction and the corresponding aminoacyl group to ensure uniform binding of tRNAs to the ribosome. Any changes to this pair increase near-cognate tRNA incorporation (49, 50). Our structural results, along with structural studies of 30S-extended ASL complexes (46), imply that insertions that perturb the structural integrity of the anticodon loop of suppressor tRNAs may direct recoding by destabilization of the 32–38 pairing.
Many models, including both the quadruplet and yardstick models, argued for shifting of the mRNA frame during decoding in the A site of the ribosome (25). However, structural studies of the ribosome showed close monitoring of the codon–anticodon helix and the anticodon loop by rRNA, reflecting the importance of mRNA frame maintenance in the A site (32). Conversely, there are no ribosomal interactions with the ASL stem. Based on our results and other structural studies of the ribosome (32, 51), we propose that, by virtue of the space and size restrictions in the A site, the ribosome exclusively decodes three nucleotides in the A site (Fig. 5A). This three-nucleotide decoding occurs regardless of whether there is an insertion in the anticodon loop, the absence of tRNA modifications, or the propensity to near-cognate base pair. Most, if not all, frameshifting models that depict a larger than 3-nt interaction between the codon and the anticodon in the A site are incompatible with our structural understanding of the decoding center.
Fig. 5.
A model for +1 frameshifting for tRNASufA6 and m1G37-deficient tRNAPro. (A) In the A site, the ASL (gray) interacts with a 3-nt codon (yellow) with the additional nucleotide (7) that is read as the preceding codon shown in red. The conserved 32–38 are depicted as blue and green, respectively. The anticodon nucleotides 34–36 and mRNA codons 4–6 are closely monitored by 16S rRNA residues G530, C1054, A1492, and A1493 (light purple) and 23S rRNA A1913 (yellow). The architecture of the A site implies that a 3-nt codon–anticodon interaction is exclusively read by the ribosome. (B) Domain IV of EF-G (teal) interacts with the ASL 5′ stem and nucleotide 32, potentially inspecting the integrity of the anticodon loop. (C) On movement into the P site by EF-G, the 16S rRNA residues A1338, G1339 (light purple), S9 (purple), and S13 (pink) inspect the stem of the ASL, whereas very few interactions are made with the codon–anticodon helix, which is now in a +1 frame.
Upon tRNA–mRNA translocation to the P site, the interactions between the tRNA and the ribosome entirely readjust to allow strict inspection of the anticodon stem with minimal contact with the codon–anticodon helix (Fig. 5C) (32). The P-site tRNA is held rigidly in place by 16S rRNA residues A1338 and G1339 and ribosomal proteins S9 and S13 to properly orient the tRNA for peptide bond formation. Additionally, 16S rRNA G1338, A1339, and A790 are proposed to function as a gate between the P and E sites (32, 52), but they may also inspect the structural integrity of the P-site tRNA. Upon superpositioning of ASLSufA6 into the P site, the 16S rRNA residues and the C-terminal tail of S9 interact with the ASL stem directly adjacent to the U32–A38 pair; these interactions may alter the dynamics of U32 (Fig. 5C). Indeed, mutation of the terminal residue of S9 alone (Arg128 in Escherichia coli and Arg130 in T. thermophilus) has been shown to promote +1 frameshifting, implicating its important role in frame maintenance (53). Although it is clear that after the mRNA–tRNA pair is translocated to the P site, the mRNA is in the +1 frame based on toeprint primer extension assays of suppressor tRNAs (54, 55), it is unclear precisely how and where the +1 frameshift event occurred.
Based on decades of genetic, biochemical, and structural studies, two likely possibilities exist to explain the molecular basis of +1 frameshifting (11). The first possibility is an alteration in translocation of the tRNA–mRNA pair on the 30S from the A to P site by translation factor EF-G (Fig. 5B). The second possibility is that after canonical 3-nt translocation, the ribosome loses its grip on the P-site tRNA stem and slips in the +1 direction. Although our structures here are of +1 frameshifting-prone ASLs in the A site, there are hints as to how +1 frameshifting occurs in this particular context. The loss of the U32–A38 base pair in two +1 frameshifting contexts, modification-deficient ASLPro and ASLSufA6, implies that the integrity of this base pair plays a significant role in +1 frameshifting (Fig. 5A). In support of this concept, the U32–A38 base pair has been shown to dramatically affect tRNA discrimination of cognate vs. near-cognate codons both in vitro and in vivo, demonstrating an important role in stability and function (49, 50). Although the ribosome does not directly contact U32–A38, the destabilization of the stem caused by the removal of the U32–A38 interaction may be recognized by EF-G during translocation (Fig. 5B). Evidence for this comes from a recent cryo-EM pretranslocation structure of 70S bound to EF-G, where domain IV residues appear to interact with the 5′ stem of the ASL proximal to U32 (56). The process of translocation involves the dynamic remodeling of intersubunit bridge interactions and the ∼20-Å relocation of the mRNA–tRNA pair. Therefore, it is tempting to hypothesize that EF-G causes a rearrangement of the anticodon loop by virtue of its interactions with the ASL stem, and then, direct gripping with 16S rRNA and S9 residues in the P site facilitates conformational rearrangements of the stem. The remodeling of the P-site tRNA results in changes that are propagated to the codon–anticodon interaction, allowing a readjustment into the +1 frame. These data are consistent with and provide a new molecular basis for previously proposed repairing models, whereby mRNA–tRNA repairing is initiated by the ribosome gripping the ASL stem (30).
Although it is unlikely that a single mechanism exists to explain all occurrences of mRNA frameshifting (11, 57), this study represents a first step toward understanding the molecular details required for +1 frameshifting. We envisage that most +1 frameshift suppressor tRNAs adopt noncanonical conformations in the 5′ region of the ASL that mediate a rearrangement in the P site.
Materials and Methods
Ribosome Purification and Crystallization.
All mRNAs and unmodified ASLs were chemically synthesized and purchased from IDT (Table S3), m1G modified ASLs were purchased from Dharmacon, and E. coli tRNAfMet was purchased from Chemical Block Ltd. Purification of T. thermophilus HB8 70S ribosomes, the formation of 70S complexes, crystallization trials, and cryoprotection are as previously described (32).
Structural Studies of 70S Ribosome Complexes.
X-ray diffraction data were collected at either the Southeast Regional Collaborative Access Team 22-ID beamline or the Northeastern Collaborative Access Team ID24-C or ID24-E beamlines at the Advanced Photon Source. Data were integrated and scaled using the program XDS (58), and solved by molecular replacement using coordinates from a 70S structure containing mRNA and tRNAs (Tables S1 and S2) (Protein Data Bank ID codes 3I9D and 3I9E) (59). Crystallographic refinement was performed using the PHENIX software suite (60) followed by iterative rounds of manual building in Coot (61). Additional refinement details can be found in SI Materials and Methods. All figures were prepared in PyMOL (www.pymol.org).
Structural Comparisons.
Comparisons of RNA helical parameters for ASLSufA6 bound to the CCC-U codon vs. ASLPro bound to the CCC-U codon were made using the program 3DNA (62). The modeling of ASLSufA6 in the ribosomal P site was done by superpositioning the A-site ASLSufA6 model onto the P-site tRNAfMet using the least-squares fit in Coot (61).
Acknowledgments
We thank Frank M. Murphy IV and staff members of the NE-CAT beamlines for assistance during data collection and Graeme L. Conn, Crystal E. Fagan, and Marc A. Schureck for critical reading of the manuscript. Research reported in this publication was supported by National Institute of General Medical Sciences of the National Institutes of Health Grant R01GM093278 (to C.M.D.). This work is based on research conducted at the Advanced Photon Source on the Northeastern Collaborative Access Team beamlines, which is supported by National Center for Research Resources National Institutes of Health Grant RR-15301, and the Southeast Regional Collaborative Access Team beamline. Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy Office of Science by the Argonne National Laboratory, was supported by US Department of Energy Contract DE-AC02-06CH11357. C.M.D. is a Pew Scholar in the Biomedical Sciences.
Footnotes
↵1T.M. and J.A.D. contributed equally to this work.
- ↵2To whom correspondence should be addressed. Email: christine.m.dunham{at}emory.edu.
Author contributions: T.M., J.A.D., and C.M.D. designed research; T.M., J.A.D., and S.J.M. performed research; T.M., J.A.D., and C.M.D. analyzed data; and T.M., J.A.D., and C.M.D. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The crystallography, atomic coordinates, and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4L47, 1VVJ, 4L71, 4LEL, 4LFZ, 4LNT, 4LSK, 4LT8, and 4P70).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1409436111/-/DCSupplemental.
References
Structures of 70S-+1 frameshift-prone tRNAs
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Image credit: Ceri Shipton.
Jonathon Yuly, David Beratan, and Peng Zhang investigate how electron bifurcation reactions work.
A study finds that giant pandas roll in horse manure to increase their cold tolerance.
Image credit: Fuwen Wei.