Disruption of evolutionarily correlated tRNA elements impairs accurate decoding
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Edited by Joseph D. Puglisi, Stanford University School of Medicine, Stanford, CA, and approved May 31, 2020 (received for review March 5, 2020)

Significance
Accurate gene expression relies on enzymes distinguishing correct from incorrect substrates that are chemically and structurally similar. During protein synthesis, correct base pairing between the tRNA anticodon and the mRNA codon is essential for accurate translation. Other tRNA elements outside of the anticodon also contribute to correct selection by the ribosome, but the structural basis for their contribution to selectivity is unknown. Here, we determine structures of the bacterial ribosome containing tRNAs with altered nucleotide pairings in the anticodon loop that are known to prevent the ribosome from distinguishing correct from incorrect tRNAs. Collectively, these structures reveal a previously unappreciated role for a ribosomal decoding site nucleotide in sensing the integrity of the tRNA that contributes to decoding fidelity.
Abstract
Bacterial transfer RNAs (tRNAs) contain evolutionarily conserved sequences and modifications that ensure uniform binding to the ribosome and optimal translational accuracy despite differences in their aminoacyl attachments and anticodon nucleotide sequences. In the tRNA anticodon stem−loop, the anticodon sequence is correlated with a base pair in the anticodon loop (nucleotides 32 and 38) to tune the binding of each tRNA to the decoding center in the ribosome. Disruption of this correlation renders the ribosome unable to distinguish correct from incorrect tRNAs. The molecular basis for how these two tRNA features combine to ensure accurate decoding is unclear. Here, we solved structures of the bacterial ribosome containing either wild-type
The ribosome orchestrates the binding of messenger RNA (mRNA), protein translation factors, and transfer RNAs (tRNAs) in a sequential manner for the synthesis of all cellular proteins. This process is remarkably complicated and involves numerous steps that have been evolutionarily optimized to select correct tRNAs from a pool of structurally and chemically similar tRNAs. The tRNAs are the so-called “adaptor” molecules that decode the genetic code on mRNA and carry an aminoacyl group (1). As noted in a recent review (2), the term “adaptor” implies plasticity between the aminoacyl group and anticodon; however, other nucleotide sequences and modifications in each tRNA are also evolutionarily tuned to permit comparable binding affinities to both EF-Tu and the ribosome for optimal accuracy (3⇓–5). This tuning occurs in all bacterial tRNAs (5), yet the structural basis for how optimized sequences contribute to efficient recognition on the ribosome is unclear.
All tRNAs adopt L-shaped structures that traverse both the small 30S and the large 50S subunits when bound to the ribosome. The aminoacyl group is attached to the 3′ end of the tRNA which is located ∼90 Å away from the anticodon (Fig. 1A). EF-Tu interacts extensively with the acceptor arm of tRNAs and surrounds the 3ʹ aminoacyl group. These aminoacyl groups represent a broad range of chemical diversity, yet all tRNAs bind to EF-Tu with the same relative affinities despite these intrinsic physical differences, due to compensatory binding contributions from the tRNA (2, 3, 6). Likewise, the anticodon regions of all tRNAs bind to their cognate mRNA codons on the ribosome with similar affinities, despite diverse codon−anticodon pairings that should exhibit differences in base pairing strengths (4). In both cases, the sequences of each tRNA have evolved to compensate for the chemical diversity of the aminoacyl group or the codon−anticodon strength to achieve similar rates of binding and optimal accuracy.
The
The evolution of tRNA sequences and modification patterns implicate previously unappreciated roles of specific tRNA regions in the process of translation, including decoding. During decoding, Watson–Crick base pairing between the three nucleotides of the mRNA codon and tRNA anticodon is monitored by the ribosome. Correct pairing causes conformational changes of the 30S subunit and GTPase activation of EF-Tu, leading to the release of the tRNA for elongation to proceed (7). Phylogenetic and biochemical analyses reveal that a universal feature of all bacterial tRNAs is a correlation between the nucleotide identities of the anticodon (nucleotides 34, 35, and 36) and nucleotides 32 and 38 located in the anticodon loop (5, 8⇓–10) (Fig. 1A). Specifically, strong GC-rich codon−anticodon interactions are always balanced by a weaker 32–38 pairing and, conversely, a weak AU-rich codon−anticodon interaction is coupled with a stronger 32–38 pairing. This coordination of nucleotide identities ensures uniform binding affinities of all tRNAs to their cognate codons (4, 11). Further, when the nucleotide identity of the 32–38 pair is disrupted, the ribosome is unable to distinguish correct from incorrect tRNAs, establishing that this correlation is important for translation fidelity (9, 10).
Mutations of the A32−U38 pair in
Motivated by these compelling biochemical and in vivo assays, we solved four X-ray crystal structures of Thermus thermophilus ribosomes containing either wild-type
Results
Near-Cognate Interactions between tRNA GGC Ala and the GCA Codon Influence the Position of 23S rRNA A1913.
To determine the structural basis for how
Cognate interactions between
Data collection and refinement statistics
We next solved a 3.2-Å structure of the T. thermophilus 70S wild-type
A near-cognate codon−anticodon interaction in
The near-cognate interaction between the codon−anticodon also causes the nucleobases of the A32−U38 base pair to become disordered, as indicated by the lack of electron density (Fig. 3C). This lack of electron density is notable because the cognate and the near-cognate, codon−anticodon-containing structures are at comparable resolutions (both 3.2 Å). The destabilization of the 32–38 pairing has also been seen when the tRNA−mRNA pairs are near cognate and cause mRNA frameshifts (20, 21). These data further emphasize the critical role of the 32–38 pair in the accurate decoding of cognate codons.
The ribosome closely monitors the codon−anticodon interaction (SI Appendix, Figs. S1A and S2), but the rest of the anticodon stem−loop (ASL) is minimally inspected in the A site, providing a conundrum in understanding how the correlation between the anticodon and the 32–38 pairing could tune tRNA binding and acceptance by the ribosome (8⇓–10). The closest ribosomal nucleotide or protein to anticodon stem nucleotides 32–38 is 23S rRNA nucleotide A1913 (Fig. 4A and SI Appendix, Fig. S2). A1913 is located in the loop of Helix 69 (H69) which is a universally conserved helix that forms an intersubunit bridge, contacts 16S rRNA nucleotide A1493 during decoding (22), and is also important for release factor recognition of stop codons (22, 23). A1913 typically packs against nucleotide 38 of the A-site tRNA and forms a hydrogen bond with the 2ʹ-OH of nucleotide 37 (SI Appendix, Fig. S1B). In the case of wild-type
Interaction of 23S rRNA A1913 with tRNA and its ablation when the 32–38 pair is destabilized. (A) The 23S rRNA A1913 (light pink) in helix 69 packs against the backbone of U38 of the A-site tRNA (purple) in the context of a cognate tRNA−mRNA pair. When a near-cognate tRNA−mRNA pair is present at the A site, A1913 (dark pink) moves away from the tRNA. (B) Interactions between A1913 showing its nucleobase is proximal to the U38−A32 pair in tRNA when
Disrupting the 32–38 Pair of tRNA GGC Ala Renders the Ribosome Unable to Distinguish Cognate from Near-Cognate Codon−Anticodon Pairs.
To understand the structural basis for how reversing the 32–38 pairing in tRNAs leads to miscoding, we solved a structure of the ribosome bound to an A-site
Reversing the 32–38 pair in
Discussion
To maintain efficient and accurate protein synthesis, tRNAs acquired diverse sequences and chemical modifications that enable their specific recognition by specific aminoacyl-tRNA synthetases and the decoding of cognate mRNA codons (24, 25). In addition, these tRNA elements are evolutionarily optimized to ensure that all tRNAs have similar binding affinities to both EF-Tu and the ribosome, thus preventing potential thermodynamic differences from contributing to the decoding process. As part of the tuning of tRNAs to bind uniformly to the ribosome, the nucleotide identities and strength of the 32–38 base pair and the anticodon nucleotides are correlated (9, 10). Since the ribosome closely monitors the codon−anticodon interaction but the rest of the ASL is minimally inspected in the A site, it was unclear how disrupting this correlation would influence the overall tRNA structure and whether this dysregulation affects how the ribosome interacts with the A-site tRNA. Here, we solved X-ray crystal structures of ribosome complexes containing
In this study, we report two observations that may explain the misreading propensity when the identity of the 32–38 pair and the anticodon nucleotides are dysregulated: In the context of the wild-type
A1913 adopts two different conformations that appear to be dependent on whether the codon−anticodon interaction is cognate or near cognate (Fig. 4 and SI Appendix, Fig. S1). In the case of a cognate codon−anticodon interaction, A1913 packs against the backbone of tRNA nucleotide 38 in a position that is observed in most ribosome structures solved to date (Fig. 4B and SI Appendix, Fig. S3). In contrast, in the case of a near-cognate, codon−anticodon interaction, A1913 moves ∼7 Å away from the tRNA (Fig. 4C). We propose that A1913 is part of the response of the ribosome to monitor the structural integrity of the A-site tRNA [previously termed “ON” (17)]. When the 32–38 nucleotides are reversed in
To our knowledge, the position of A1913 has never been seen to move away from the tRNA backbone in the context of a mismatched codon−anticodon interaction in the A site (Fig. 4). In structures containing single C•A, A•C, A•A, U•G, and G•U mismatches at the first (16, 26) or second (16, 17, 27) position of the codon−anticodon interaction, A1913 packs against the tRNA; in one structure with a U•G mismatch at the second A-site position, A1913 is conformationally dynamic and unresolvable in the electron potential map (17). In these cases, the mismatched interaction was formed by systematically changing the sequence of the mRNA codon using four standard tRNAs in the absence of prior biochemical knowledge of how these pairs impact the decoding process (SI Appendix, Fig. S7). It is now well appreciated that certain codon−anticodon pairs undergo high levels of misreading in vivo, while other pairings do not (28). Therefore, perhaps the movement of A1913 wasn’t previously identified because the mismatched complexes affect a different stage of initial tRNA selection than what was captured in the structure, or the mismatched codon−anticodon pair does not cause high levels of miscoding. Future studies are required to understand this previously unappreciated role of A1913 in maintaining the fidelity of the decoding process.
Materials and Methods
In Vitro Transcription of tRNA GGC Ala .
Two DNA oligos spanning the A32−U38
Crystallization, X-ray Data Collection, and Structural Determination.
The 70S ribosomes were purified from T. thermophilus using previously established protocols (14). The ribosome complex was formed by incubating 4.4 µM 70S with 8.8 µM mRNA (IDT) in buffer (5 mM Hepes-KOH, pH 7.5, 50 mM KCl, 10 mM NH4Cl, 10 mM Mg(CH3COO)2, 6 mM β-mercaptoethanol (β-Me)) at 55 °C for 5 min. Then 11 µM tRNAfMet (Chemical Block) and 22 µM tRNAAla were sequentially incubated at 55 °C for 15 min. The reaction was cooled to 37 °C, and 0.1 mM paromomycin was added and incubated at 37 °C. After equilibrating at 20 °C, a final concentration of 2.8 µM deoxy BigCHAP (Hampton Research) was added to the complex. Crystals grew from either a polyethylene glycol (PEG) condition (0.1 M Tris-HOAc pH 7.0, 0.2 M KSCN, 4–4.5% [weight/vol] PEG 20K, 4.5 to 5.5% [vol/vol] PEG 550MME, 10 mM Mg(CH3COO)2) or a 2-methyl-2,4-pentanediol (MPD) condition (0.1 M l-arginine HCl, 0.1 M Tris⋅HCl pH 7.5, 3% PEG 20K, 10 to 16.5% MPD, 1 mM β-Me). Data collection was performed at the SER-CAT 22-ID and NE-CAT 24ID-C beamlines at the Advanced Photon Source. Data were integrated and scaled using XDS (30), molecular replacement performed in PHENIX (31) using coordinates from Protein Data Bank (PDB) structure 4Y4O (32). Initial refinement was done using rigid-body restraints in PHENIX, followed by jelly-body refinement in REFMAC5 (33) in the CCP4i2 suite (34), and further iterative rounds of crystallographic refinements were performed in PHENIX. Model building was performed in Coot (35), and figures were generated using PyMol (36).
Data Availability.
Crystallography, atomic coordinates, and structure factors have been deposited in the PDB, https://www.wwpdb.org/ (PDB ID codes 6OF6, 6OJ2, 6OPE, 6ORD).
Acknowledgments
We thank Dunham laboratory member Dr. Eric Hoffer for technical assistance and helpful scientific insights, Dr. Graeme L. Conn and other members of the Dunham laboratory for critical reading of the manuscript, and staff members of the Northeast Regional Collaborative Access Team (NE-CAT) and Southeast Regional Collaborative Access Team (SER-CAT) beamlines for assistance during data collection. This work was supported by NIH Grant R01 GM093278. X-ray crystallography datasets were collected at the NE-CAT beamlines (funded by National Institute of General Medical Sciences (NIGMS) Grant P30 GM124165), using a Pilatus detector (RR029205) and an Eiger detector (OD021527), and at the SER-CAT beamlines (funded by its member institutions and NIH Equipment Grants S10_RR25528 and S10_RR028976). This research used resources of the Advanced Photon Source (APS), a US Department of Energy Office of Science User Facility operated by Argonne National Laboratory under Contracts DE-AC02-06CH11357 (NE-CAT) and W-31-109-Eng-38 (SER-CAT).
Footnotes
- ↵1To whom correspondence may be addressed. Email: christine.m.dunham{at}emory.edu.
Author contributions: S.S. and C.M.D. designed research; H.A.N. and S.S. performed research; H.A.N., S.S., and C.M.D. analyzed data; and H.A.N., S.S., and C.M.D. wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
Data deposition: Crystallography, atomic coordinates, and structure factors have been deposited in the Protein Data Bank (PDB ID codes 6OF6, 6OJ2, 6OPE, and 6ORD).
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2004170117/-/DCSupplemental.
Published under the PNAS license.
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