Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion

• Published in: FEBS letters Vol. 586; no. 21; pp. 3931 - 3937
• Main Authors: O’Donoghue, Patrick, Prat, Laure, Heinemann, Ilka U., Ling, Jiqiang, Odoi, Keturah, Liu, Wenshe R., Söll, Dieter
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 02-11-2012
• Subjects: 4-Base codon; Anticodon; Boc-lysine; Codon; Escherichia coli; Escherichia coli - genetics; Genetic Code; Lysine - analogs & derivatives; Lysine - genetics; Orthogonal translation systems; Protein Biosynthesis; Pyrrolysyl-tRNA synthetase; RNA, Transfer, Amino Acyl - genetics; Spectrometry, Mass, Electrospray Ionization; Suppression, Genetic; Synthetic biology; tRNA; Orthogonal translation systems; Pyrrolysyl-tRNA synthetase; Synthetic biology; Boc-lysine; tRNA; 4-Base codon
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2012.09.033

► Nonsense suppression by natural amino acids hinders genetic code expansion. ► Higher levels of near-cognate suppression found in opal versus amber codons. ► E. coli BL21 and MG1655 display higher background suppression than TOP10. ► PylRS/tRNAUCAPyl cannot outcompete Trp incorporation at opal codons. ► PylRS/tRNAUCCUPyl is outcompeted by Arg-tRNAArg in quadruplet decoding.
Over 300 amino acids are found in proteins in nature, yet typically only 20 are genetically encoded. Reassigning stop codons and use of quadruplet codons emerged as the main avenues for genetically encoding non-canonical amino acids (NCAAs). Canonical aminoacyl-tRNAs with near-cognate anticodons also read these codons to some extent. This background suppression leads to ‘statistical protein’ that contains some natural amino acid(s) at a site intended for NCAA. We characterize near-cognate suppression of amber, opal and a quadruplet codon in common Escherichia coli laboratory strains and find that the PylRS/tRNAPyl orthogonal pair cannot completely outcompete contamination by natural amino acids.