Open AccessGenetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons
Author(s) -
Jeffery M. Tharp,
Oscar Vargas-Rodriguez,
Alanna Schepartz,
Dieter Söll
Publication year - 2021
Publication title -
acs chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.899
H-Index - 111
eISSN - 1554-8937
pISSN - 1554-8929
DOI - 10.1021/acschembio.1c00120
Subject(s) - transfer rna , bioorthogonal chemistry , stop codon , aminoacyl trna synthetase , amino acid , genetic code , tyrosine , biology , mutant , biochemistry , genetics , chemistry , gene , rna , combinatorial chemistry , click chemistry
We recently described an orthogonal initiator tRNA ( i tRNA Ty2 ) that can initiate protein synthesis with noncanonical amino acids (ncAAs) in response to the UAG nonsense codon. Here, we report that a mutant of i tRNA Ty2 ( i tRNA Ty2 AUA ) can efficiently initiate translation in response to the UAU tyrosine codon, giving rise to proteins with an ncAA at their N-terminus. We show that, in cells expressing i tRNA Ty2 AUA , UAU can function as a dual-use codon that selectively encodes ncAAs at the initiating position and predominantly tyrosine at elongating positions. Using i tRNA Ty2 AUA , in conjunction with its cognate tyrosyl-tRNA synthetase and two mutually orthogonal pyrrolysyl-tRNA synthetases, we demonstrate that UAU can be reassigned along with UAG or UAA to encode two distinct ncAAs in the same protein. Furthermore, by engineering the substrate specificity of one of the pyrrolysyl-tRNA synthetases, we developed a triply orthogonal system that enables simultaneous reassignment of UAU, UAG, and UAA to produce proteins containing three distinct ncAAs at precisely defined sites. To showcase the utility of this system, we produced proteins containing two or three ncAAs, with unique bioorthogonal functional groups, and demonstrate that these proteins can be separately modified with multiple fluorescent probes.