Abstract
Modification of anticodon nucleotides allows tRNAs to decode multiple codons, expanding the genetic code. Additionally, modifications located in the anticodon loop, outside the anticodon itself, stabilize tRNA–codon interactions, increasing decoding fidelity. Anticodon loop nucleotide 37 is 3′ to the anticodon and, in , is methylated at the N1 position in its nucleobase (m1G37). The m1G37 modification in stabilizes its interaction with the codon and maintains the mRNA frame. However, it is unclear how m1G37 affects binding at the decoding center to both cognate and +1 slippery codons. Here, we show that the m1G37 modification is important for the association step during binding to a cognate CCG codon. In contrast, m1G37 prevented association with a slippery CCC-U or +1 codon. Similar analyses of frameshift suppressor tRNASufA6, a derivative containing an extra nucleotide in its anticodon loop that undergoes +1 frameshifting, reveal that m1G37 destabilizes interactions with both the cognate CCG and slippery codons. One reason for this destabilization is the disruption of a conserved U32·A38 nucleotide pairing in the anticodon stem through insertion of G37.5. Restoring the tRNASufA6 U32·A37.5 pairing results in a high-affinity association on the slippery CCC-U codon. Further, an X-ray crystal structure of the 70S ribosome bound to tRNASufA6 U32·A37.5 at 3.6 Å resolution shows a reordering of the anticodon loop consistent with the findings from the high-affinity measurements. Our results reveal how the tRNA modification at nucleotide 37 stabilizes interactions with the mRNA codon to preserve the mRNA frame.
Highlights
Modification of anticodon nucleotides allows tRNAs to decode multiple codons, expanding the genetic code
Because tRNAs decode mRNAs, these RNA molecules probably play a role in mRNA frame maintenance. tRNAs are ϳ76 –90 nucleotides in length and adopt an L-shaped tertiary structure allowing them to fit into ribosome-binding sites that span both subunits (Fig. 1). tRNAs undergo extensive posttranscriptional modifications important for the correct tertiary fold of the tRNA, including the conformation of the anticodon stem-loop (ASL) [3]
Modifications in the ASLs of tRNAs are critical, given that only 7 of the 61 sense codons are decoded by tRNAs that lack modifications at nucleotide 34 or 37 in E. coli [49, 50]
Summary
To assess the importance of the m1G37 modification in tRNACPrGoG in decoding, we used established filter binding assays to determine binding kinetics to the A site [39]. The dissociation of ASLSufA6 from a cognate CCG or a slippery CCC-U codon are all very similar regardless of the G37 modification status (0.013– 0.019 minϪ1) Together, these data indicate that the inserted G37.5 nucleotide in ASLSufA6 removes the dependence on the m1G37 modification required for tight association to the ribosome for the parent tRNACPrGoG. We found that potentially restoring the U321⁄7A37.5 base pair does not result in high-affinity binding to a cognate CCG codon in the absence or presence of the m1G37 modification (calculated Kd of 7.4 and 15 M, respectively; Fig. 5 (D and E) and Table 1). The A37.5 insertion in ASLSufA6 seems to orient the ASL to a conformation more similar to that of ASLCPrGoG than that of WT ASLSufA6 (Fig. S3, B and C). 16S rRNA nucleotides A1492 and A1493 flip from their internal position in helix 44, and G530 is positioned close to A1492, demonstrating recognition by the ribosome (Fig. S4)
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