An RNA modification prevents extended codon-anticodon interactions from facilitating +1 frameshifting

Abstract

RNA post-transcriptional modifications act by stabilizing the functional conformations of RNA. While their role in messenger RNA (mRNA) decoding is well established, it is less clear how transfer RNA (tRNA) modifications outside the anticodon contribute to tRNA stability and accurate protein synthesis. Absence of such modifications causes translation errors, including mRNA frameshifting. By integrating single-molecule fluorescence resonance energy transfer and cryogenic electron microscopy, we demonstrate that the N¹-methylguanosine (m¹G) modification at position 37 of Escherichia coli tRNA^ProL is necessary and sufficient for modulating the conformational energy of this tRNA on the ribosome so as to suppress +1 frameshifting otherwise induced by this tRNA. Six structures of E. coli ribosomal complexes carrying tRNA^ProL lacking m¹G37 show this tRNA forms four and even five codon-anticodon base pairs as it moves into the +1 frame, allowing direct visualization of the long-standing hypothesis that a four base pair codon-anticodon can form during +1 frameshifting.

Fig. 1. Mechanisms of +1 frameshifting by tRNA^Pro isoacceptors.

a The anticodon stem-loop (ASL; 18 nucleotides) of tRNA^Pro isoacceptors and their corresponding anticodons (blue), including the four nucleotide codons that induce +1 frameshifting in the absence of m¹G37. cmo⁵ denotes a 5-oxyacetic acid uridine modification. N denotes any A/U/G/C nucleotide. b The absence of m¹G37 in tRNA^ProL induces a +1 frameshift on proline slippery codons at two defined substeps during the elongation cycle: during the EF-G-mediated translocation of peptidyl-tRNA^Pro isoacceptor from the A site to the P site (middle) and after translocation, when the peptidyl-tRNA^Pro isoacceptor is positioned in the P site but the next A-site aa-tRNA hasn’t been delivered yet (right).

Fig. 2. Influence of G37 modification status and codon slipperiness on P-site tRNA^ProL conformational energy.

a Cartoon of the GS1⇄GS2 conformational equilibrium of a POST– complex containing Cy3- and Cy5-labeled ribosomal proteins bL9 and uL1, respectively, and carrying a P site-bound tRNA^ProL. Among other structural differences, GS1 features a P/P-configured tRNA and an open uL1 stalk, resulting in an E_FRET value of 0.55. In contrast, GS2 features a P/E-configured tRNA and a closed uL1 stalk, resulting in an E_FRET value of 0.35. b, c Surface contour plots are generated by superimposing numerous individual E_FRET vs. time trajectories (Supplemental Fig. S1) recorded using smFRET experiments conducted on eight POST– complexes. Contours are colored from white (lowest population) to red (highest population), as indicated, and N at the rightmost top of each surface contour plot specifies the number of E_FRET trajectories that were used to construct that plot. The eight POST– complexes carried either P-site native, unmodified, unmodified +m¹G37, or native –m¹G37 variants of tRNA^ProL, as specified by the tRNA cartoons along the top of the four columns of surface contour plots. In these cartoons, m¹G37 is indicated in blue, and other tRNA^ProL modifications are depicted in yellow. In addition, these POST– complexes were formed using mRNAs that place either a proline CCC-G non-slippery codon or a proline CCC-C slippery codon at the P site, as specified along the left of the two rows of surface contour plots. A detailed description of how the smFRET data were analyzed, including how the % GS1, % GS2, K_eq, k_GS1→GS2, and k_GS2→GS1 were calculated, can be found in the “Materials and Methods”.

Fig. 3. Four Watson-Crick base pairs form during +1 frameshifting of tRNA^ProL.

a A 2.9 Å cryo-EM structure of a ribosomal POST– complex carrying a P/E-configured, unmodified tRNA^ProL at a CCC-C slippery codon in the +1 frame. b Close-up of the codon-anticodon reveals four Watson-Crick base pairs and an interaction between U38 in the anticodon stem-loop and G₊₃ in the codon. The additional base pairs are highlighted in gray (and in c). c A schematic of these interactions.

Fig. 4. P site-bound, unmodified aa-tRNA^ProL can occupy the normal or +1 frames.

a 3.5 Å (normal frame) and 3.6 Å (+1 frame) cryo-EM structures of POST complexes carrying an unmodified Lys-tRNA^ProL bound to a CCC-C slippery codon in the P site. b ~32% of particles show that the P-site tRNA^ProL interacts with the slippery codon in the normal frame, with formation of the three expected Watson-Crick base pairs (left). A schematic of these interactions is shown (right). c ~28% of particles show that the P-site Lys-tRNA^ProL interacts with the slippery codon in the +1 frame, with formation of three Watson-Crick base pairs and movement of the C₊₄ codon nucleotide moving towards G37 of the Lys-tRNA^ProL. The additional base pairs are highlighted in gray. A schematic of these interactions is shown (right). d One major difference in the codon-anticodon interaction between POST complexes in the normal or +1 frames is the way the mRNA interacts with the unmodified G37. In the normal frame, G₊₃ is positioned away from G37, whereas in the +1 frame C₊₄ flips towards G37.

Fig. 5. Accommodation of a tRNA into the A site stabilizes P site-bound, unmodified tRNA^ProL in the +1 frame.

a A 3.0 Å cryo-EM structure of a POST– complex carrying an unmodified P-site tRNA^ProL paired to a CCC-C slippery codon in the +1 frame and an A-site, tRNA^Val bound to the GUU codon in the +1 frame. b Close-up of the codon-anticodon reveals four Watson-Crick base pairs and an interaction between U38 of the anticodon stem-loop and G₊₃ of the codon. The additional base pairs are highlighted in gray (and in c). c A schematic of these interactions.

Fig. 6. Mechanism by which m¹G37 influences the conformational energy of tRNA^ProL and modulates +1 frameshifting.

a Unmodified peptidyl-tRNA^ProL can undergo +1 frameshifting either during the substep of the elongation cycle in which it is translocated from the A site into the P site (substeps in white background) or at the substep in which it has been translocated into the P site but has not yet undergone transfer of its peptidyl moiety onto the incoming aa-tRNA at the A site (complexes in light green and light orange background and substeps connecting them). Delivery of an aa-tRNA into the A site in the +1 frame stabilizes the P site-bound, +1 frameshifted, unmodified tRNA^ProL via stabilization of four Watson-Crick (WC) base pairs and an additional hydrogen bond at a fifth pairing (complex in light pink background). b Close-up view of WC base pairing and hydrogen bonding interactions formed within the various substeps and complexes in (a).