N6-methylation of adenosine (m6A) is the most abundant post-transcriptional modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m6A in mRNA decoding. Although m6A base pairs with uridine during decoding as shown by x-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements employing an Escherichia coli translation system revealed that m6A modification of mRNA can act as a barrier to tRNA accommodation and translation elongation. The interaction between an m6A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, as delay in tRNA accommodation depends on the position and context of m6A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics.

During protein synthesis, the ribosome selects aminoacyl-tRNAs with anticodons matching the mRNA codon present in the A-site of the small ribosomal subunit. The aminoglycoside antibiotic streptomycin disrupts decoding by binding close to the site of codon recognition. Here we use X-ray crystallography to define the impact of streptomycin on the decoding site of the Thermus thermophilus 30S ribosomal subunit in complexes with cognate or near-cognate anticodon stem-loop analogs (ASLs) and mRNA. Our crystal structures display a significant local distortion of 16S rRNA induced by streptomycin, including the crucial bases A1492 and A1493 that participate directly in codon recognition. Consistent with kinetic data, we observe that streptomycin stabilizes the near-cognate ASL complex, while destabilizing the cognate ASL complex. These data reveal how streptomycin disrupts the recognition of cognate ASLs and yet improves recognition of a near-cognate ASL.

In this work, a concentric-flow electrokinetic injector delivered microcrystals of Geobacillus stearothermophilus thermolysin (2.2 Å structure), Thermosynechococcus elongatus photosystem II (< 3 Å diffraction) and Thermus thermophilus small ribosomal subunit (3.4 Å structure). The first ambient-temperature X-ray crystal structure of the 30S subunit bound to the antibiotic paromomycin was obtained in its native mother liquor. Compared to previous cryo-cooled structures, this new structure showed that paromomycin binds to the decoding center in a different conformation.