Maintaining the translational reading frame poses difficulty for the ribosome. Slippery mRNA sequences such as CC[C/U]-[C/U], read by isoacceptors of tRNA^Pro, are highly prone to +1 frameshift (+1FS) errors. Here we show that +1FS errors occur by two mechanisms, a slow mechanism when tRNA^Pro is stalled in the P-site next to an empty A-site and a fast mechanism during translocation of tRNA^Pro into the P-site. Suppression of +1FS errors requires the m¹G37 methylation of tRNA^Pro on the 3' side of the anticodon and the translation factor EF-P. Importantly, both m¹G37 and EF-P show the strongest suppression effect when CC[C/U]-[C/U] are placed at the second codon of a reading frame. This work demonstrates that maintaining the reading frame immediately after the initiation of translation by the ribosome is an essential aspect of protein synthesis.

Significance: In bacterial tRNAs with the ³⁶ GG ³⁷ sequence, where positions 36 and 37 are, respectively, the third letter of the anticodon and 3′ adjacent to the anticodon, the modification of N ¹ -methylguanosine (m ¹ G) at position 37 prevents +1 frameshifts on the ribosome. The m ¹ G37 modification is introduced by the enzyme TrmD, which harbors a deep trefoil knot within the S -adenosyl-L-methionine (AdoMet)-binding site. We determined the crystal structure of the TrmD homodimer in complex with a substrate tRNA and an AdoMet analog. The structure revealed how TrmD, upon AdoMet binding in the trefoil knot, obtains the ability to bind the substrate tRNA, and interacts with G37 and G36 sequentially to transfer the methyl moiety of AdoMet to the N ¹ position of G37.

Significance: Mg ²⁺ is an important signal for the regulation of virulence and thermotolerance in Salmonella enterica . Transcription of the mgtA gene, which encodes a transporter for Mg ²⁺ , is highly induced by Mg ²⁺ limitation. The 5′ leader of the mgtA mRNA encodes mgtL , a 17-codon, proline-rich open reading frame, whose translation controls the transcription of mgtA : efficient translation of mgtL results in termination of transcription upstream of the mgtA protein-coding region, whereas slow or incomplete translation of mgtL antagonizes this termination. We show that the proline codons in mgtL present an impediment to translation at low but not high Mg ²⁺ concentrations, and thereby, we provide a model for how Mg ²⁺ is coupled to the regulation of mgtA transcription.

N¹-methylation of G37 is required for a subset of tRNAs to maintain the translational reading-frame. While loss of m¹G37 increases ribosomal +1 frameshifting, whether it incurs additional translational defects is unknown. Here, we address this question by applying ribosome profiling to gain a genome-wide view of the effects of m¹G37 deficiency on protein synthesis. Using E coli as a model, we show that m¹G37 deficiency induces ribosome stalling at codons that are normally translated by m¹G37-containing tRNAs. Stalling occurs during decoding of affected codons at the ribosomal A site, indicating a distinct mechanism than that of +1 frameshifting, which occurs after the affected codons leave the A site. Enzyme- and cell-based assays show that m¹G37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in m¹G37 deficiency similar to those in the stringent response that is typically induced by deficiency of amino acids. This work demonstrates a previously unrecognized function of m¹G37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.