title: Transfer RNA misidentification scrambles sense codon recoding.
abstract: Sense codon recoding is the basis for genetic code expansion with more than two different noncanonical amino acids. It requires an unused (or rarely used) codon, and an orthogonal tRNA synthetase:tRNA pair with the complementary anticodon. The Mycoplasma capricolum genome contains just six CGG arginine codons, without a dedicated tRNA(Arg). We wanted to reassign this codon to pyrrolysine by providing M. capricolum with pyrrolysyl-tRNA synthetase, a synthetic tRNA with a CCG anticodon (tRNA(Pyl)(CCG)), and the genes for pyrrolysine biosynthesis. Here we show that tRNA(Pyl)(CCG) is efficiently recognized by the endogenous arginyl-tRNA synthetase, presumably at the anticodon. Mass spectrometry revealed that in the presence of tRNA(Pyl)(CCG), CGG codons are translated as arginine. This result is not unexpected as most tRNA synthetases use the anticodon as a recognition element. The data suggest that tRNA misidentification by endogenous aminoacyl-tRNA synthetases needs to be overcome for sense codon recoding.
authors.affiliation: Synthetic Biology and Bioenergy, J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850 (USA).
authors.forename: Radha
authors.initials: R
authors.lastname: Krishnakumar
authors.forename: Laure
authors.initials: L
authors.lastname: Prat
authors.forename: Hans-Rudolf
authors.initials: HR
authors.lastname: Aerni
authors.forename: Jiqiang
authors.initials: J
authors.lastname: Ling
authors.forename: Chuck
authors.initials: C
authors.lastname: Merryman
authors.forename: John I
authors.initials: JI
authors.lastname: Glass
authors.forename: Jesse
authors.initials: J
authors.lastname: Rinehart
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
mesh_terms: D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D003062:Codon / Q000235:genetics*; D016680:Genome, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D045623:Mycoplasma capricolum / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics
chemical_list: D003062:Codon; D012329:RNA, Bacterial; D012343:RNA, Transfer; D001616:beta-Galactosidase; D000604:Amino Acyl-tRNA Synthetases
keywords: aminoacyl-tRNA synthetase recognition; anticodons; gene technology; sense codon recoding; tRNA