Post-transcriptionally modified RNAs, including ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA), stabilize RNA tertiary structures during ribonucleoprotein biogenesis, regulate mRNA metabolism, and influence other facets of gene expression. RNA modifications located in the three-nucleotide anticodon of tRNAs contribute to accurate protein synthesis and expand the tRNA coding capacity by facilitating Watson–Crick-like base-pairs with mRNA codons. The most commonly modified tRNA anticodon position is at nucleotide 34 and the modifications to this nucleotide are functionally important for gene expression by facilitating base-pairing interactions with the mRNA codon (Agris et al., 2017; Boccaletto et al., 2018; Agris et al., 2018). Other tRNA nucleotides also contain highly conserved nucleotide modifications, but their precise roles in protein synthesis are not fully understood. For example, nucleotide 37 is adjacent to the anticodon (Figure 1A) and is modified in >70% of all tRNAs (Machnicka et al., 2014). The two most common modifications at nucleotide 37 are 6-threonylcarbamoyladenosine (t⁶A) and 1-methylguanosine (m¹G), which together account for >60% of nucleotide 37 modifications (Boccaletto et al., 2018). Both the t⁶A37 modification in tRNA^Lys and the m¹G37 modification in tRNA^Pro are thought to stabilize the tertiary structure of the anticodon loop for high-affinity binding to their cognate codons (Murphy et al., 2004; Sundaram et al., 2000; Nguyen et al., 2019). The m¹G37 modification also prevents the ribosome from shifting out of the three-nucleotide mRNA codon frame (‘frameshifting’) to ensure the correct polypeptide is expressed (Björk et al., 1989; Li et al., 1997; Urbonavicius et al., 2001; Gamper et al., 2015a). However, the molecular basis for how the m¹G37 modification maintains the mRNA reading frame is unknown.

Figure 1 with 1 supplement see all

Download asset Open asset

The m¹G37 modification is present in >95% of all three tRNA^Pro isoacceptors across all three domains of life (Boccaletto et al., 2018; Björk et al., 1989). The absence of m¹G37 in tRNA^Pro causes high levels of +1 frameshifting on so-called ‘slippery’ or polynucleotide mRNA sequences where four nucleotides encode for a single proline codon (Björk et al., 1989). In the case of the tRNA^Pro-CGG isoacceptor (anticodon is shown 5'-3'), a +1 slippery codon consists of the CCC proline codon and either an additional C or U to form a four-nucleotide codon, CCC-(U/C) (Sroga et al., 1992; Qian et al., 1998) (codons depicted 5’-3’). tRNA^Pro-CGG lacking m¹G37 results in the ribosome being unable to distinguish a correct from an incorrect codon–anticodon interaction during decoding at the aminoacyl (A) site (Nguyen et al., 2019; Maehigashi et al., 2014). However, this miscoding event does not cause the shift in the mRNA frame in the A site despite the tRNA^Pro-CGG anticodon stem-loop nucleotides become more mobile (Maehigashi et al., 2014). A post-decoding frameshift event is further supported by detailed kinetic analyses (Gamper et al., 2015a). The absence of the m¹G37 modification in tRNA^Pro causes a ~ 5% frameshifting frequency during translocation of the mRNA-tRNA pair from the A to the peptidyl (P) site and a ~40% frameshifting frequency once the mRNA-tRNA pair has reached the P site. When tRNA^Pro-CGG lacks m¹G37 and decodes a +1 slippery codon, both the process of translocation and the unique environment of the P site appear to contribute to the inability of the ribosome to maintain the mRNA frame.

In circumstances where mRNA frameshifting is caused by changes in the tRNA such as the absence of modifications or changes in the size of the anticodon loop as found in frameshift suppressor tRNAs, primer extension assays demonstrated that the shift into the +1 frame is observed upon direct tRNA binding at the P site (Phelps et al., 2006; Walker and Fredrick, 2006). These studies show that the nature of the interactions between the frameshift-prone mRNA-tRNA pair and the ribosomal P site directly permit frameshifting. Furthermore, the presence of a nascent polypeptide chain, the acylation status of the tRNA (deacylated or aminoacylated), and even the presence of a tRNA in the A site do not appear to contribute to the ability of these tRNAs to cause frameshifting. Structural studies of frameshift-prone or suppressor tRNAs have provided molecular insights into how such tRNAs may dysregulate mRNA frame maintenance (Maehigashi et al., 2014; Fagan et al., 2014; Hong et al., 2018). Frameshift suppressor tRNA^SufA6 contains an extra nucleotide in its anticodon loop and undergoes high levels of +1 frameshifting on slippery proline codons (Björk et al., 1989; Qian et al., 1998). The structure of the anticodon stem-loop (ASL) of the tRNA^SufA6 bound directly at the P site revealed that its anticodon engages the slippery CCC-U proline codon in the +1 frame (Hong et al., 2018). Further, the full-length tRNA^SufA6 bound at the P site reveals that the small 30S subunit head domain swivels and tilts, a movement that is similar to the one that is caused by the GTPase elongation factor-G (EF-G) upon translocation of the tRNAs through the ribosome that several groups have attempted to characterize (Ermolenko and Noller, 2011; Wasserman et al., 2016; Belardinelli et al., 2016a, Nguyen and Whitford, 2016, Guo and Noller, 2012). The process of mRNA-tRNA translocation is coupled to this head domain swivel and tilting which is distinct from other conformational changes the ribosome undergoes including intersubunit rotation (Wasserman et al., 2016; Ratje et al., 2010; Zhou et al., 2013; Zhou et al., 2014; Holtkamp et al., 2014; Belardinelli et al., 2016b; Guo and Noller, 2012, Nguyen and Whitford, 2016). However, the 70S-tRNA^SufA6 structure is in the absence of EF-G, suggesting that a +1 frameshift event caused by frameshift-prone tRNAs dysregulates some aspect of translocation. Although these studies demonstrate that +1 frameshift-prone tRNAs are good model systems to uncover mechanisms by which both the ribosome and the mRNA-tRNA pair contribute to mRNA frame maintenance, it is unclear whether normal tRNAs lacking modifications result in +1 frameshifting in the same manner. To address this question, here we solved six 70S structures of tRNA^Pro-CGG in the presence or absence of the m¹G37 modification and bound to either cognate or slippery codons in the P site. Our results define how the m¹G37 modification stabilizes the interactions of tRNA^Pro-CGG with the ribosome. We further show that ribosomes bound to mRNA-tRNA pairs that result in +1 frameshifts promoted by tRNA^Pro-CGG result in large conformational changes of the 30S head domain, consequently biasing the tRNA-mRNA pair toward the E site.

The importance of tRNA modifications in protein synthesis has been recognized for decades, yet the precise roles of modifications located outside the anticodon have been elusive. Modifications in the anticodon loop at position 37 of tRNAs have been implicated in mRNA frame maintenance (Urbonavicius et al., 2001; Yarian et al., 2002), but how the absence of a single methylation can dysregulate the mRNA frame remained unclear. Here, we elucidated the role of m¹G37 in tRNA^Pro-CGG, which was known to be important in stabilizing stacking interactions with anticodon nucleotides during decoding at the A site (Maehigashi et al., 2014) and in the prevention of frameshifting (Björk et al., 1989; Hagervall et al., 1993). We find that this single methyl group influences the overall stability of tRNA^Pro-CGG on the ribosome in an unexpected manner and causes large conformational changes between the tRNA and the 30S head domain, a domain known to move extensively during translocation of the tRNAs (Wasserman et al., 2016; Ratje et al., 2010; Zhou et al., 2013; Zhou et al., 2014). However, the methylation alone does not stabilize tRNA^Pro-CGG on the ribosome and, instead, its position is heavily influenced by interactions with a slippery proline codon. Our structures of different mRNA-tRNA^Pro-CGG pairs on the ribosome reveals the first detailed mechanistic insight into how mRNA frame maintenance is regulated by both the m¹G37 modification and the stability of mRNA-tRNA interaction.

The location on the ribosome where frameshifting can occur is likely dependent on the type of frameshift (in the positive or negative directionon the mRNA) and whether tRNA or mRNA causes the frameshift (Dinman, 2012; Dunkle and Dunham, 2015; Atkins et al., 2016; Korniy et al., 2019). The ribosome itself can prevent frameshifts through inherent differences in how the ribosome interacts with the tRNA-mRNA complex at the different tRNA binding sites and such differences could potentially influence frameshifting by relaxing interactions, for example, between the codon and anticodon. While the codon–anticodon interaction located in the 30S A site is strictly monitored by conserved 16S rRNA nucleotides to select for cognate tRNA (Ogle et al., 2002), there are comparatively few interactions with the codon–anticodon when positioned within the P and E sites, underscoring their different functional roles during the translation cycle. The relative absence of interactions in the P and E sites provides an opportunity for the mRNA to shift out of frame. Some tRNA^Pro isoacceptors frameshift either during translocation from the A to the P site or after the translocation step and once positioned in the P site (Gamper et al., 2015a; Gamper et al., 2015b). Therefore we sought to capture the interactions of tRNA^Pro during a frameshift event. The structure of ASL^Pro bound to a slippery codon in the P site reveals the codon–anticodon interaction has shifted into the +1 frame indicating that the anticodon stem-loop interaction with the mRNA codon alone is important for frameshifting (Figure 1D). This frameshift is likely possible because interactions with the P-site tRNA are limited to only 16S rRNA P/E loop nucleotides G1338 and A1339 with the anticodon stem and 16S rRNA C1400 with the anticodon nucleotide 34 (Figure 5A, Figure 5—figure supplement 1). The P/E loop appears to indirectly enforce the mRNA frame by gripping the P-site tRNA and these interactions are maintained as the tRNA moves from the P site to a hybrid P/E state (Dunkle et al., 2011) and to a translocation intermediate pe/E state (Zhou et al., 2013). In the structures of ASL^Pro interacting with the mRNA in the 0 frame, there is well-resolved density for G1338 and A1339 (Figure 1B and C, Figure 1—figure supplement 1A,D, G), while there is some disordering of these nucleotides in the context of ASL^Pro bound to the near-cognate, slippery codon in the +1 frame (Figure 1—figure supplement 1C, F and I). The inability for G1338 and A1339 to grip the anticodon stem when the codon–anticodon is in the +1 frame likely results in the destabilization of full-length tRNA^Pro in these frameshift-competent contexts (Figure 2C and D). Simulations of the ribosome undergoing large movements of the head domain during translocation implicate nucleotides G1338 and A1339 in the coupling of 30S head dynamics and displacement of the P-site tRNA towards the E site (Nguyen and Whitford, 2016). Therefore, the lack of gripping by the P/E loop in the P site when a destabilized mRNA-tRNA interaction is present, appears to bias tRNA^Pro toward the E site which, in turn, causes the 30S head domain to swivel and tilt as if EF-G were bound.

In addition to P/E loop nucleotides G1338 and A1339, 16S rRNA nucleotide G966 is also part of the 30S head domain and stacks with nucleotide 34 of the P-site tRNA anticodon (Figure 5A). The 30S head is connected to the body domain via interactions of G966 with nucleotide C1400; the G966-C1400 pair remains stacked beneath the P-site tRNA and follows the tRNA as it moves to a hybrid P/E state and to a translocation pe/E intermediate state upon EF-G binding (Figure 5B; Zhou et al., 2013; Zhou et al., 2014; Dunkle et al., 2011). In the case of P-site ASL^Pro interacting with a +1 slippery codon in the 0 or +1 frame, cis-Watson–Crick interactions form between C₊₃•C34 and U₊₄•C34, respectively (Figure 1—figure supplement 1K and L). Both the C₊₃•C34 and U₊₄•C34 interactions appear to result in reduced stacking with C1400. In structures of full-length tRNA^Pro-mRNA pairs that cause +1 frameshifting and destabilization at the P site, the G966-C1400 interaction is broken (Figure 5C). Together, these results suggest a connection between the reduced interactions of G1338-A1339 and G966-C1400 with the tRNA that appear to influence the movement of the P-site tRNA towards the E site.

Although neither C1400 nor G966 have previously been implicated in mRNA frame maintenance, there is a functional link between the P/E loop nucleotides G1338 and A1339 and C1400-G966. 16S rRNA nucleotides in the P site are generally more tolerable of mutations than the A-site 16S rRNA, although substitution of G966 substantially reduces ribosome activity to ~10% despite the mutation not being lethal (Abdi and Fredrick, 2005). This G966 mutant is suppressed by a G1338A mutation, indicating that the G1338A mutation can stabilize interactions with the P-site tRNA even in the absence of the C1400-G966 interaction.

The interactions observed between the codon and anticodon of ASL^Pro bound to either cognate or near-cognate codons reveal the process of shifting into the +1 frame (Figure 1D). In the context of full-length mRNA-tRNA pairs that cause +1 frameshifting, both the tRNA and mRNA move from the P site to occupy a position between the E and the P site (Figure 2C and D). This mRNA-tRNA placement is similar to the translocation intermediate containing EF-G (Zhou et al., 2013). In the structure presented here containing an e*/E tRNA^Pro as compared to the EF-G containing structures containing an pe/tRNA, e*/E tRNA^Pro is positioned slightly further away from the mRNA. In the e*/E tRNA^Pro-CGG structure, the first position of the codon–anticodon forms a Watson–Crick interaction but the second and third nucleotides of the codon–anticodon are not within hydrogen bonding distances (Figure 2G). Interestingly, in the EF-G-bound translocation intermediate structures, the codon–anticodon is also not within bonding distance (Zhou et al., 2013). These results suggest that it is the disruptive nature of moving between tRNA binding sites that perturbs the interactions between the codon and anticodon and these interactions likely reform once the transition to the next tRNA binding site is complete.

30S head domain conformational changes were first captured in ribosome structures of tRNAs moving between the A to the P sites (ap/ap state) and between the P and the E sites (pe/E state) on the 30S in the presence of EF-G (Zhou et al., 2013; Zhou et al., 2014; Dunkle et al., 2011; Ratje et al., 2010). Upon EF-G binding, the head domain is predicted to first tilt and then swivel in a counterclockwise manner (as viewed with the E, P, A tRNA sites from left to right shown in Figure 2) to translocate the two tRNAs to final E/E and P/P positions (Wasserman et al., 2016; Nguyen and Whitford, 2016). The departure of EF-G and reverse tilting of the head back towards the intersubunit space is followed by clockwise swivel, the rate-limiting step for translocation (Wasserman et al., 2016). Frameshift-prone tRNAs, such as tRNA^Pro-CGG, cause spontaneous head swiveling and tilting once the tRNA occupies the P site after the mRNA frameshift has occurred. The inability of the ribosome to hold the P-site tRNA is influenced by weakening of the interactions of the P/E loop with the anticodon stem and by disruption of the C1400-G966 interaction, both events likely major contributors to the dysregulation of the 30S head domain. In other words, the head domain is unable to maintain extensive interactions with the P-site tRNA and this failure leads to spontaneous translocation. Single molecule FRET (smFRET) studies of translocation events also show that upon tRNA movement from the P to the E site, there is increased dissociation of the tRNA from the ribosome, bypassing the post-translocation E/E state (Wasserman et al., 2016). These data seem to suggest that even during canonical translocation from the P to the E site, this is a highly dynamic process which leads to destabilized tRNA. Consistent with these observations, we would expect that once EF-G fully translocates tRNA^Pro-CGG to the post E/E state, there may be a further decrease in interactions between the tRNA and ribosome. Additionally, our structures show 30S head swiveling and tilting in the absence of EF-G, concurrent with the +1 frameshift event. This 30S head swivel and tilt movement would likely prevent EF-G from binding until the the head resets to a non-rotated state. EF-G residues located in the tip of domain IV interact with the anticodon stem-loop of the A-site tRNA during translocation to the P site (Zhou et al., 2013) and mutations in this domain slow the rate of translocation (Rodnina et al., 1997; Savelsbergh et al., 2000). Recent studies using slippery codon sequences where spontaneous frameshifting occurs, EF-G can restrict frameshifting and helps to maintain the mRNA frame (Peng et al., 2019). It is therefore an enticing hypothesis that EF-G may have a role in mRNA frame maintenance in addition to its established function in translocation.

Although all three prokaryotic tRNA^Pro isoacceptors have the capacity to frameshift, whether they use similar mechanisms of action is unknown (Björk et al., 1989; Gamper et al., 2015a; Qian et al., 1998; O'Connor, 2002). The m¹G37 modification minimally influences frameshifting in proline isoacceptor tRNA^Pro-GGG (proL) in contrast to tRNA^Pro-cmo⁵UGG (proM) (Gamper et al., 2015a). An additional difference is the dependency on elongation factor-P (EF-P) to reconcile +1 frameshifts. EF-P binds at the E site to overcome ribosome stalling induced by poly-proline codons (Ude et al., 2013; Huter et al., 2017) and is critical in suppressing frameshifts on poly-proline stretches (Gamper et al., 2015a). While EF-P reduces +1 frameshifts with tRNA^Pro-GGG G37(Δm¹) to an equivalent frequency as native tRNA^Pro-GGG, the absence or presence of m¹G37 has little influence on the ability of isoacceptor tRNA^Pro-cmo⁵UGG to frameshift (Gamper et al., 2015a). These mechanistic differences may be due to a combination of the codon–anticodon pairings on slippery codons (tRNA^Pro-cmo⁵UGG and tRNA^Pro-GGG are cognate with slippery codons while tRNA^Pro-CGG is near-cognate [Nasvall et al., 2004]) and/or the influence of other modifications such as the cmo⁵U34 modification in tRNA^Pro-cmo⁵UGG (Masuda et al., 2018). Further considerations may be the location of the slippery codon on the mRNA, which would have varying nascent chain lengths, and whether the following codon after the slippery codon is rare (Gamper et al., 2015a). Our structures show that both the m¹G37 modification status of tRNA^Pro-CGG and the CCC-U codon causes the tRNA to become destabilized and its position is biased towards the E site, which we predict is indicative of high levels of frameshifting. The possible synergistic effects of cmo⁵U34 and m¹G37 in tRNA^Pro-cmo⁵UGG in preventing frameshifts is unclear, but tRNA^Pro-UGG lacking all modifications exhibits higher levels of frameshifting as compared to tRNA^Pro-UGG lacking only the m¹G37 modification (Gamper et al., 2015b). Since the presence of m¹G37 in tRNA^Pro-cmo⁵UGG may restrict its movement to the e*/E position, this tRNA isoaccepor may no longer be an optimal substrate for EF-P, which is consistent with kinetic analyses (Gamper et al., 2015a). Together, our structures of tRNA^Pro-CGG in frameshifting contexts provides new insights into how RNA modifications impact tRNA stability on the ribosome.

Crystallography, atomic coordinates, and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB codes 6NTA, 6NSH, 6NUO, 6NWY, 6O3M, 6OSI).

1. 1. Hoffer ED
   2. Hong S
   3. Sunita S
   4. Maehigashi T
   5. Dunham CM

   (2020) RCSB Protein Data Bank

   ID 6NTA. Modified ASL proline bound to Thermus thermophilus 70S (cognate).

   https://www.rcsb.org/structure/6NTA
2. 1. Hoffer ED
   2. Hong S
   3. Sunita S
   4. Maehigashi T
   5. Dunham CM

   (2020) RCSB Protein Data Bank

   ID 6NSH. Modified ASL proline bound to Thermus thermophilus 70S (near-cognate, +1 sliipery codon).

   https://www.rcsb.org/structure/6NSH
3. 1. Hoffer ED
   2. Hong S
   3. Sunita S
   4. Maehigashi T
   5. Dunham CM

   (2020) RCSB Protein Data Bank

   ID 6NUO. Modified tRNA(Pro) bound to Thermus thermophilus 70S (cognate).

   https://www.rcsb.org/structure/6NUO
4. 1. Hoffer ED
   2. Hong S
   3. Sunita S
   4. Maehigashi T
   5. Dunham CM

   (2020) RCSB Protein Data Bank

   ID 6NWY. Modified tRNA(Pro) bound to Thermus thermophilus 70S (near-cognate, +1 slippery codon).

   https://www.rcsb.org/structure/6NWY
5. 1. Hoffer ED
   2. Hong S
   3. Sunita S
   4. Maehigashi T
   5. Dunham CM

   (2020) RCSB Protein Data Bank

   ID 6O3M. Unmodified tRNA(Pro) bound to Thermus thermophilus 70S (cognate).

   https://www.rcsb.org/structure/6O3M
6. 1. Hoffer ED
   2. Hong S
   3. Sunita S
   4. Maehigashi T
   5. Dunham CM

   (2020) RCSB Protein Data Bank

   ID 6OSI. Unmodified tRNA(Pro) bound to Thermus thermophilus 70S (near cognate, +1 slippery codon).

   https://www.rcsb.org/structure/6OSI

1. 1. Abdi NM
   2. Fredrick K

   (2005) Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli

   RNA 11:1624–1632.

   https://doi.org/10.1261/rna.2118105

   • PubMed
   • Google Scholar
2. 1. Adams PD
   2. Afonine PV
   3. Bunkóczi G
   4. Chen VB
   5. Davis IW
   6. Echols N
   7. Headd JJ
   8. Hung L-W
   9. Kapral GJ
   10. Grosse-Kunstleve RW
   11. McCoy AJ
   12. Moriarty NW
   13. Oeffner R
   14. Read RJ
   15. Richardson DC
   16. Richardson JS
   17. Terwilliger TC
   18. Zwart PH

   (2010) PHENIX : a comprehensive Python-based system for macromolecular structure solution

   Acta Crystallographica Section D Biological Crystallography 66:213–221.

   https://doi.org/10.1107/S0907444909052925

   • Google Scholar
3. 1. Agris PF
   2. Narendran A
   3. Sarachan K
   4. Väre VYP
   5. Eruysal E

   (2017) The importance of being modified: the role of RNA modifications in translational fidelity

   The Enzymes 41:1–50.

   https://doi.org/10.1016/bs.enz.2017.03.005

   • PubMed
   • Google Scholar
4. 1. Agris PF
   2. Eruysal ER
   3. Narendran A
   4. Väre VYP
   5. Vangaveti S
   6. Ranganathan SV

   (2018) Celebrating wobble decoding: half a century and still much is new

   RNA Biology 15:537–553.

   https://doi.org/10.1080/15476286.2017.1356562

   • PubMed
   • Google Scholar
5. 1. Atkins JF
   2. Loughran G
   3. Bhatt PR
   4. Firth AE
   5. Baranov PV

   (2016) Ribosomal frameshifting and transcriptional slippage: from genetic steganography and cryptography to adventitious use

   Nucleic Acids Research 243:gkw530.

   https://doi.org/10.1093/nar/gkw530

   • Google Scholar
6. 1. Belardinelli R
   2. Sharma H
   3. Caliskan N
   4. Cunha CE
   5. Peske F
   6. Wintermeyer W
   7. Rodnina MV

   (2016a) Choreography of molecular movements during ribosome progression along mRNA

   Nature Structural & Molecular Biology 23:342–348.

   https://doi.org/10.1038/nsmb.3193

   • PubMed
   • Google Scholar
7. 1. Belardinelli R
   2. Sharma H
   3. Peske F
   4. Wintermeyer W
   5. Rodnina MV

   (2016b) Translocation as continuous movement through the ribosome

   RNA Biology 13:1197–1203.

   https://doi.org/10.1080/15476286.2016.1240140

   • PubMed
   • Google Scholar
8. 1. Björk GR
   2. Wikström PM
   3. Byström AS

   (1989) Prevention of translational frameshifting by the modified nucleoside 1-methylguanosine

   Science 244:986–989.

   https://doi.org/10.1126/science.2471265

   • PubMed
   • Google Scholar
9. 1. Boccaletto P
   2. Machnicka MA
   3. Purta E
   4. Piatkowski P
   5. Baginski B
   6. Wirecki TK
   7. de Crécy-Lagard V
   8. Ross R
   9. Limbach PA
   10. Kotter A
   11. Helm M
   12. Bujnicki JM

   (2018) MODOMICS: a database of RNA modification pathways. 2017 update

   Nucleic Acids Research 46:D303–D307.

   https://doi.org/10.1093/nar/gkx1030

   • PubMed
   • Google Scholar
10. 1. Dinman JD

    (2012) Control of gene expression by translational recoding

    Advances in Protein Chemistry and Structural Biology 86:129–149.

    https://doi.org/10.1016/B978-0-12-386497-0.00004-9

    • PubMed
    • Google Scholar
11. 1. Dunkle JA
    2. Wang L
    3. Feldman MB
    4. Pulk A
    5. Chen VB
    6. Kapral GJ
    7. Noeske J
    8. Richardson JS
    9. Blanchard SC
    10. Cate JH

    (2011) Structures of the bacterial ribosome in classical and hybrid states of tRNA binding

    Science 332:981–984.

    https://doi.org/10.1126/science.1202692

    • PubMed
    • Google Scholar
12. 1. Dunkle JA
    2. Dunham CM

    (2015) Mechanisms of mRNA frame maintenance and its subversion during translation of the genetic code

    Biochimie 114:90–96.

    https://doi.org/10.1016/j.biochi.2015.02.007

    • PubMed
    • Google Scholar
13. 1. Emsley P
    2. Lohkamp B
    3. Scott WG
    4. Cowtan K

    (2010) Features and development of coot

    Acta Crystallographica 66:486–501.

    https://doi.org/10.1107/S0907444910007493

    • Google Scholar
14. 1. Ermolenko DN
    2. Noller HF

    (2011) mRNA translocation occurs during the second step of ribosomal intersubunit rotation

    Nature Structural & Molecular Biology 18:457–462.

    https://doi.org/10.1038/nsmb.2011

    • PubMed
    • Google Scholar
15. 1. Fagan CE
    2. Maehigashi T
    3. Dunkle JA
    4. Miles SJ
    5. Dunham CM

    (2014) Structural insights into translational recoding by frameshift suppressor tRNASufJ

    RNA 20:1944–1954.

    https://doi.org/10.1261/rna.046953.114

    • PubMed
    • Google Scholar
16. 1. Gamper HB
    2. Masuda I
    3. Frenkel-Morgenstern M
    4. Hou YM

    (2015a) Maintenance of protein synthesis reading frame by EF-P and m(1)G37-tRNA

    Nature Communications 6:7226.

    https://doi.org/10.1038/ncomms8226

    • PubMed
    • Google Scholar
17. 1. Gamper HB
    2. Masuda I
    3. Frenkel-Morgenstern M
    4. Hou YM

    (2015b) The UGG isoacceptor of tRNAPro is naturally prone to frameshifts

    International Journal of Molecular Sciences 16:14866–14883.

    https://doi.org/10.3390/ijms160714866

    • PubMed
    • Google Scholar
18. 1. Gao Y-G
    2. Selmer M
    3. Dunham CM
    4. Weixlbaumer A
    5. Kelley AC
    6. Ramakrishnan V

    (2009) The structure of the ribosome with elongation factor G trapped in the posttranslocational state

    Science 326:694–699.

    https://doi.org/10.1126/science.1179709

    • Google Scholar
19. 1. Grosjean H
    2. Söll DG
    3. Crothers DM

    (1976) Studies of the complex between transfer RNAs with complementary anticodons. I. origins of enhanced affinity between complementary triplets

    Journal of Molecular Biology 103:499–519.

    https://doi.org/10.1016/0022-2836(76)90214-x

    • PubMed
    • Google Scholar
20. 1. Guo Z
    2. Noller HF

    (2012) Rotation of the head of the 30S ribosomal subunit during mRNA translocation

    PNAS 109:20391–20394.

    https://doi.org/10.1073/pnas.1218999109

    • Google Scholar
21. 1. Gustilo EM
    2. Vendeix FA
    3. Agris PF

    (2008) tRNA's modifications bring order to gene expression

    Current Opinion in Microbiology 11:134–140.

    https://doi.org/10.1016/j.mib.2008.02.003

    • PubMed
    • Google Scholar
22. 1. Hagervall TG
    2. Tuohy TM
    3. Atkins JF
    4. Björk GR

    (1993) Deficiency of 1-methylguanosine in tRNA from Salmonella typhimurium induces frameshifting by quadruplet translocation

    Journal of Molecular Biology 232:756–765.

    https://doi.org/10.1006/jmbi.1993.1429

    • PubMed
    • Google Scholar
23. 1. Holtkamp W
    2. Cunha CE
    3. Peske F
    4. Konevega AL
    5. Wintermeyer W
    6. Rodnina MV

    (2014) GTP hydrolysis by EF-G synchronizes tRNA movement on small and large ribosomal subunits

    The EMBO Journal 33:1073–1085.

    https://doi.org/10.1002/embj.201387465

    • PubMed
    • Google Scholar
24. 1. Hong S
    2. Sunita S
    3. Maehigashi T
    4. Hoffer ED
    5. Dunkle JA
    6. Dunham CM

    (2018) Mechanism of tRNA-mediated +1 ribosomal frameshifting

    PNAS 115:11226–11231.

    https://doi.org/10.1073/pnas.1809319115

    • PubMed
    • Google Scholar
25. 1. Huter P
    2. Arenz S
    3. Bock LV
    4. Graf M
    5. Frister JO
    6. Heuer A
    7. Peil L
    8. Starosta AL
    9. Wohlgemuth I
    10. Peske F
    11. Nováček J
    12. Berninghausen O
    13. Grubmüller H
    14. Tenson T
    15. Beckmann R
    16. Rodnina MV
    17. Vaiana AC
    18. Wilson DN

    (2017) Structural basis for Polyproline-Mediated ribosome stalling and rescue by the translation elongation factor EF-P

    Molecular Cell 68:515–527.

    https://doi.org/10.1016/j.molcel.2017.10.014

    • Google Scholar
26. 1. Jenner LB
    2. Demeshkina N
    3. Yusupova G
    4. Yusupov M

    (2010) Structural aspects of messenger RNA reading frame maintenance by the ribosome

    Nature Structural & Molecular Biology 17:555–560.

    https://doi.org/10.1038/nsmb.1790

    • PubMed
    • Google Scholar
27. 1. Kabsch W

    (2010) Xds

    Acta Crystallographica 66:125–132.

    https://doi.org/10.1107/S0907444909047337

    • Google Scholar
28. 1. Korniy N
    2. Samatova E
    3. Anokhina MM
    4. Peske F
    5. Rodnina MV

    (2019) Mechanisms and biomedical implications of -1 programmed ribosome frameshifting on viral and bacterial mRNAs

    FEBS Letters 593:1468–1482.

    https://doi.org/10.1002/1873-3468.13478

    • PubMed
    • Google Scholar
29. 1. Li J
    2. Esberg B
    3. Curran JF
    4. Björk GR

    (1997) Three modified nucleosides present in the anticodon stem and loop influence the in vivo aa-tRNA selection in a tRNA-dependent manner

    Journal of Molecular Biology 271:209–221.

    https://doi.org/10.1006/jmbi.1997.1176

    • PubMed
    • Google Scholar
30. 1. Machnicka MA
    2. Olchowik A
    3. Grosjean H
    4. Bujnicki JM

    (2014) Distribution and frequencies of post-transcriptional modifications in tRNAs

    RNA Biology 11:1619–1629.

    https://doi.org/10.4161/15476286.2014.992273

    • PubMed
    • Google Scholar
31. 1. Maehigashi T
    2. Dunkle JA
    3. Miles SJ
    4. Dunham CM

    (2014) Structural insights into +1 frameshifting promoted by expanded or modification-deficient anticodon stem loops

    PNAS 111:12740–12745.

    https://doi.org/10.1073/pnas.1409436111

    • PubMed
    • Google Scholar
32. 1. Masuda I
    2. Takase R
    3. Matsubara R
    4. Paulines MJ
    5. Gamper H
    6. Limbach PA
    7. Hou YM

    (2018) Selective terminal methylation of a tRNA wobble base

    Nucleic Acids Research 46:e37.

    https://doi.org/10.1093/nar/gky013

    • PubMed
    • Google Scholar
33. 1. Mohan S
    2. Donohue JP
    3. Noller HF

    (2014) Molecular mechanics of 30S subunit head rotation

    PNAS 111:13325–13330.

    https://doi.org/10.1073/pnas.1413731111

    • PubMed
    • Google Scholar
34. 1. Murphy FV
    2. Ramakrishnan V
    3. Malkiewicz A
    4. Agris PF

    (2004) The role of modifications in Codon discrimination by tRNALysUUU

    Nature Structural & Molecular Biology 11:1186–1191.

    https://doi.org/10.1038/nsmb861

    • PubMed
    • Google Scholar
35. 1. Nasvall SJ
    2. Chen P
    3. Bjork GR

    (2004) The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAPro(cmo5UGG) promotes reading of all four proline codons in vivo

    RNA 10:1662–1673.

    https://doi.org/10.1261/rna.7106404

    • PubMed
    • Google Scholar
36. 1. Nguyen HA
    2. Hoffer ED
    3. Dunham CM

    (2019) Importance of a tRNA anticodon loop modification and a conserved, noncanonical anticodon stem pairing in tRNA CGG^Pro for decoding

    Journal of Biological Chemistry 294:5281–5291.

    https://doi.org/10.1074/jbc.RA119.007410

    • Google Scholar
37. 1. Nguyen K
    2. Whitford PC

    (2016) Steric interactions lead to collective tilting motion in the ribosome during mRNA-tRNA translocation

    Nature Communications 7:10586.

    https://doi.org/10.1038/ncomms10586

    • PubMed
    • Google Scholar
38. 1. O'Connor M

    (2002) Imbalance of tRNA(Pro) isoacceptors induces +1 frameshifting at near-cognate codons

    Nucleic Acids Research 30:759–765.

    https://doi.org/10.1093/nar/30.3.759

    • PubMed
    • Google Scholar
39. 1. Ogle JM
    2. Murphy FV
    3. Tarry MJ
    4. Ramakrishnan V

    (2002) Selection of tRNA by the ribosome requires a transition from an open to a closed form

    Cell 111:721–732.

    https://doi.org/10.1016/S0092-8674(02)01086-3

    • PubMed
    • Google Scholar
40. 1. Peng BZ
    2. Bock LV
    3. Belardinelli R
    4. Peske F
    5. Grubmüller H
    6. Rodnina MV

    (2019) Active role of elongation factor G in maintaining the mRNA reading frame during translation

    Science Advances 5:eaax8030.

    https://doi.org/10.1126/sciadv.aax8030

    • PubMed
    • Google Scholar
41. 1. Phelps SS
    2. Gaudin C
    3. Yoshizawa S
    4. Benitez C
    5. Fourmy D
    6. Joseph S

    (2006) Translocation of a tRNA with an extended anticodon through the ribosome

    Journal of Molecular Biology 360:610–622.

    https://doi.org/10.1016/j.jmb.2006.05.016

    • Google Scholar
42. 1. Qian Q
    2. Li JN
    3. Zhao H
    4. Hagervall TG
    5. Farabaugh PJ
    6. Björk GR

    (1998) A new model for phenotypic suppression of frameshift mutations by mutant tRNAs

    Molecular Cell 1:471–482.

    https://doi.org/10.1016/S1097-2765(00)80048-9

    • PubMed
    • Google Scholar
43. 1. Ratje AH
    2. Loerke J
    3. Mikolajka A
    4. Brünner M
    5. Hildebrand PW
    6. Starosta AL
    7. Dönhöfer A
    8. Connell SR
    9. Fucini P
    10. Mielke T
    11. Whitford PC
    12. Onuchic JN
    13. Yu Y
    14. Sanbonmatsu KY
    15. Hartmann RK
    16. Penczek PA
    17. Wilson DN
    18. Spahn CM

    (2010) Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites

    Nature 468:713–716.

    https://doi.org/10.1038/nature09547

    • PubMed
    • Google Scholar
44. 1. Rodnina MV
    2. Savelsbergh A
    3. Katunin VI
    4. Wintermeyer W

    (1997) Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome

    Nature 385:37–41.

    https://doi.org/10.1038/385037a0

    • PubMed
    • Google Scholar
45. 1. Savelsbergh A
    2. Matassova NB
    3. Rodnina MV
    4. Wintermeyer W

    (2000) Role of domains 4 and 5 in elongation factor G functions on the ribosome

    Journal of Molecular Biology 300:951–961.

    https://doi.org/10.1006/jmbi.2000.3886

    • PubMed
    • Google Scholar
46. Software

    1. Schrodinger LLC

    (2010)

    Schrodinger

    LLC.
47. 1. Selmer M
    2. Dunham CM
    3. Murphy FV
    4. Weixlbaumer A
    5. Petry S
    6. Kelley AC
    7. Weir JR
    8. Ramakrishnan V

    (2006) Structure of the 70S ribosome complexed with mRNA and tRNA

    Science 313:1935–1942.

    https://doi.org/10.1126/science.1131127

    • PubMed
    • Google Scholar
48. 1. Sherlin LD
    2. Bullock TL
    3. Nissan TA
    4. Perona JJ
    5. Lariviere FJ
    6. Uhlenbeck OC
    7. Scaringe SA

    (2001)

    Chemical and enzymatic synthesis of tRNAs for high-throughput crystallization

    RNA 7:1671–1678.

    • PubMed
    • Google Scholar
49. 1. Sroga GE
    2. Nemoto F
    3. Kuchino Y
    4. Björk GR

    (1992) Insertion (sufB) in the anticodon loop or base substitution (sufC) in the anticodon stem of tRNA(Pro)2 from Salmonella typhimurium induces suppression of frameshift mutations

    Nucleic Acids Research 20:3463–3469.

    https://doi.org/10.1093/nar/20.13.3463

    • PubMed
    • Google Scholar
50. 1. Stark MR
    2. Rader SD

    (2014) Efficient splinted ligation of synthetic RNA using RNA ligase

    Methods in Molecular Biology 1126:137–149.

    https://doi.org/10.1007/978-1-62703-980-2_10

    • PubMed
    • Google Scholar
51. 1. Sundaram M
    2. Durant PC
    3. Davis DR

    (2000) Hypermodified nucleosides in the anticodon of tRNA(Lys) stabilize a canonical U-turn structure

    Biochemistry 39:15652.

    https://doi.org/10.1021/bi005120y

    • PubMed
    • Google Scholar
52. 1. Ude S
    2. Lassak J
    3. Starosta AL
    4. Kraxenberger T
    5. Wilson DN
    6. Jung K

    (2013) Translation elongation factor EF-P alleviates ribosome stalling at Polyproline stretches

    Science 339:82–85.

    https://doi.org/10.1126/science.1228985

    • PubMed
    • Google Scholar
53. 1. Urbonavicius J
    2. Qian Q
    3. Durand JM
    4. Hagervall TG
    5. Björk GR

    (2001) Improvement of reading frame maintenance is a common function for several tRNA modifications

    The EMBO Journal 20:4863–4873.

    https://doi.org/10.1093/emboj/20.17.4863

    • PubMed
    • Google Scholar
54. 1. Walker SE
    2. Fredrick K

    (2006) Recognition and positioning of mRNA in the ribosome by tRNAs with expanded anticodons

    Journal of Molecular Biology 360:599–609.

    https://doi.org/10.1016/j.jmb.2006.05.006

    • PubMed
    • Google Scholar
55. 1. Wasserman MR
    2. Alejo JL
    3. Altman RB
    4. Blanchard SC

    (2016) Multiperspective smFRET reveals rate-determining late intermediates of ribosomal translocation

    Nature Structural & Molecular Biology 23:333–341.

    https://doi.org/10.1038/nsmb.3177

    • PubMed
    • Google Scholar
56. 1. Whitford PC
    2. Blanchard SC
    3. Cate JH
    4. Sanbonmatsu KY

    (2013) Connecting the kinetics and energy landscape of tRNA translocation on the ribosome

    PLOS Computational Biology 9:e1003003.

    https://doi.org/10.1371/journal.pcbi.1003003

    • PubMed
    • Google Scholar
57. 1. Yarian C
    2. Townsend H
    3. Czestkowski W
    4. Sochacka E
    5. Malkiewicz AJ
    6. Guenther R
    7. Miskiewicz A
    8. Agris PF

    (2002) Accurate translation of the genetic code depends on tRNA modified nucleosides

    Journal of Biological Chemistry 277:16391–16395.

    https://doi.org/10.1074/jbc.M200253200

    • PubMed
    • Google Scholar
58. 1. Zaher HS
    2. Green R

    (2009) Quality control by the ribosome following peptide bond formation

    Nature 457:161–166.

    https://doi.org/10.1038/nature07582

    • PubMed
    • Google Scholar
59. 1. Zhang Y
    2. Hong S
    3. Ruangprasert A
    4. Skiniotis G
    5. Dunham CM

    (2018) Alternative mode of E-Site tRNA binding in the presence of a downstream mRNA stem loop at the entrance channel

    Structure 26:437–445.

    https://doi.org/10.1016/j.str.2018.01.013

    • PubMed
    • Google Scholar
60. 1. Zhou J
    2. Lancaster L
    3. Donohue JP
    4. Noller HF

    (2013) Crystal structures of EF-G-ribosome complexes trapped in intermediate states of translocation

    Science 340:1236086.

    https://doi.org/10.1126/science.1236086

    • PubMed
    • Google Scholar
61. 1. Zhou J
    2. Lancaster L
    3. Donohue JP
    4. Noller HF

    (2014) How the ribosome hands the A-site tRNA to the P site during EF-G-catalyzed translocation

    Science 345:1188–1191.

    https://doi.org/10.1126/science.1255030

    • Google Scholar
62. 1. Zhou J
    2. Lancaster L
    3. Donohue JP
    4. Noller HF

    (2019) Spontaneous ribosomal translocation of mRNA and tRNAs into a chimeric hybrid state

    PNAS 116:7813–7818.

    https://doi.org/10.1073/pnas.1901310116

    • PubMed
    • Google Scholar

Author details

1. Eric D Hoffer

   Department of Biochemistry, Emory University School of Medicine, Atlanta, United States

   Contribution

   Formal analysis, Validation, Investigation, Visualization

   Competing interests

   No competing interests declared

2. Samuel Hong

   Department of Biochemistry, Emory University School of Medicine, Atlanta, United States

   Contribution

   Conceptualization, Formal analysis, Validation, Visualization, Methodology

   Competing interests

   No competing interests declared

3. S Sunita

   Department of Biochemistry, Emory University School of Medicine, Atlanta, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Investigation

   Competing interests

   No competing interests declared

4. Tatsuya Maehigashi

   Department of Biochemistry, Emory University School of Medicine, Atlanta, United States

   Contribution

   Conceptualization, Data curation, Formal analysis

   Competing interests

   No competing interests declared

5. Ruben L Gonzalez Jnr

   Department of Chemistry, Columbia University, New York, United States

   Contribution

   Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1344-5581

6. Paul C Whitford

   Department of Physics, Northeastern University, Boston, United States

   Contribution

   Funding acquisition, Investigation, Methodology

   Competing interests

   No competing interests declared

7. Christine M Dunham

   Department of Biochemistry, Emory University School of Medicine, Atlanta, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology

   For correspondence

   cmdunha@emory.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8821-688X

• 2,650

  views

• 371

  downloads

• 34

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.