Importance of the m 1 G37 modification and 32–38 pairing in tRNA Pro (CCG) on decoding and tRNA stability

The ribosome is a complex and highly conserved molecular machine that is responsible for cellular protein synthesis (translation) in all three domains of life. During translation, the ribosome decodes the mRNA sequence three nucleotides at a time ( i.e . in a universal triplet reading code) with extreme precision to ensure accurate protein expression that is critical for cellular growth and function. However, it remains unclear how the ribosome maintains, or deliberately deviates from, this three‐nucleotide mRNA reading frame. Since the mRNA frame directly corresponds to the resulting protein sequence, the molecular mechanism of both frame maintenance and programmed frameshifting or frame dysregulation is an important question of the Central Dogma. While frameshifting events have been attributed to multiple factors, tRNAs alone can cause the ribosome to shift into noncanonical mRNA reading frames. We previously demonstrated that certain tRNA modifications and insertions alter how the ribosome decodes the mRNA sequence using structural biology approaches. We found that the N1 methylation at position G37 of the anticodon loop in tRNA Pro (CCG) has the same disruption of the anticodon loop as an inserted anticodon nucleotide as frameshift suppressor tRNA SufA6 (tRNA SufA6 is a variant of tRNA Pro (CCG) with a guanosine insertion between positions 37 and 38). Both situations cause a +1 shift in the mRNA frame whereby the ribosome decodes four instead of three nucleotides. Furthermore, widening of the anticodon loop ablates interactions between the 32–38 pair, a universally conserved interaction in all tRNAs. The identity of this pair is directly correlated to the anticodon sequence and disrupting this correlation prevents the ribosome from discerning correct from incorrect tRNAs. Here, I investigated how the tRNA Pro (CCG) modification and insertion influences decoding. I determined that the m 1 G37 methylation of tRNA Pro (CCG) is essential for binding to the cognate CCG proline codon and in contrast, destabilizes binding to the slippery CCCU codon. Modulation of the 32–38 pair in tRNA SufA6 shows a restoration of tight A‐site binding and thus selection by the ribosome, confirmed by X‐ray crystallography. These studies provide insights into tRNA recognition by the ribosome and further suggest rational approaches to engineer tRNAs for recoding of the genetic code with nonnatural amino acids. Support or Funding Information This work was supported by NIH GM093278 and NSF CHE 1808711. The X‐ray crystallography datasets were collected at the at the NE‐CAT beamlines (funded by the NIGMS from the NIH (P30 GM124165)), using a Pilatus detector (RR029205) and an Eiger detector (OD021527), and at the SER‐CAT beamlines (funded by its member institutions, and NIH equipment grants RR25528 and RR028976). This research used resources of the APS, a US Department of Energy Office of Science User Facility operated by Argonne National Laboratory under Contract DE‐AC02‐06CH11357 (NE‐CAT) and W‐31‐109‐Eng‐38 (SER‐CAT). Dr. Dunham is a Burroughs Wellcome Investigator in the Pathogenesis of Infectious Diseases.Frameshift suppressor tRNA SufA6 is a derivative of tRNA Pro (CCG) A , Tertiary structure of tRNA Pro (CCG) bound to its cognate mRNA with the first three nucleotides in the P‐site denoted as +1, +2, and +3 (PDB: 4LSK). B‐C , The anticodon stem loops of tRNA Pro (CCG) with its cognate codon and tRNA SufA6 showing the anticodons in green, they are both modified at position 37 (blue), but tRNA SufA6 has a G insertion between positions 37 and 38 (G37.5, red) and decodes a four‐nucleotide codon.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .