Direct charging of tRNA(CUA) with pyrrolysine in vitro and in vivo.
Pyrrolysine is the 22nd amino acid. An unresolved question has been how this atypical genetically encoded residue is inserted into proteins, because all previously described naturally occurring aminoacyl-tRNA synthetases are specific for one of the 20 universally distributed amino acids. Here we establish that synthetic L-pyrrolysine is attached as a free molecule to tRNA(CUA) by PylS, an archaeal class II aminoacyl-tRNA synthetase. PylS activates pyrrolysine with ATP and ligates pyrrolysine to tRNA(CUA) in vitro in reactions specific for pyrrolysine. The addition of pyrrolysine to Escherichia coli cells expressing pylT (encoding tRNA(CUA)) and pylS results in the translation of UAG in vivo as a sense codon. This is the first example from nature of direct aminoacylation of a tRNA with a non-canonical amino acid and shows that the genetic code of E. coli can be expanded to include UAG-directed pyrrolysine incorporation into proteins.
Department of Microbiology, 484 West 12th Avenue, The Ohio State University, Columbus, Ohio 43210, USA.
Sherry K
SK
Blight
Ross C
RC
Larue
Anirban
A
Mahapatra
David G
DG
Longstaff
Edward
E
Chang
Gang
G
Zhao
Patrick T
PT
Kang
Kari B
KB
Green-Church
Michael K
MK
Chan
Joseph A
JA
Krzycki
D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D000926:Anticodon / Q000235:genetics; D001105:Archaea / Q000201:enzymology; D019843:Archaeal Proteins; D002474:Cell-Free System; D003062:Codon / Q000235:genetics; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005815:Genetic Code; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism*; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013489:Suppression, Genetic / Q000235:genetics
D000926:Anticodon; D019843:Archaeal Proteins; D003062:Codon; D011756:Diphosphates; D012345:RNA, Transfer, Amino Acid-Specific; D000255:Adenosine Triphosphate; D008780:Methyltransferases; C113200:monomethylamine methyltransferase; D000604:Amino Acyl-tRNA Synthetases; C456839:pyrrolysine; D008239:Lysine