Phosphoserine aminoacylation of tRNA bearing an unnatural base anticodon.
An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) could expand the genetic alphabet and allow the incorporation of non-standard amino acids into proteins at defined positions. For this purpose, we synthesized tRNAs bearing Pa at the anticodon and tested non-standard amino acid phosphoserine aminoacylation by the wild-type and various engineered phosphoseryl-tRNA synthetases (SepRSs). The D418N D420N T423V triple mutant of SepRS efficiently charged phosphoserine to tRNA containing the PaUA anticodon with a K(m)=47.1muM and a k(cat)=0.151s(-1), which are comparable to the values of the wild-type SepRS for its cognate substrate, tRNA(Cys) with the GCA anticodon (26.9muM and 0.111s(-1)). The triple mutant SepRS and the tRNA with the PaUA anticodon represent a specific pair for the site-specific incorporation of phosphoserine into proteins in response to the UADs codon within mRNA.
Fukunaga
Ryuya
R
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Harada
Yoko
Y
Hirao
Ichiro
I
Yokoyama
Shigeyuki
S
D049148:Aminoacylation; D000926:Anticodon / Q000737:chemistry*; D020029:Base Pairing; D008956:Models, Chemical; D009154:Mutation; D009690:Nucleic Acid Conformation; D010768:Phosphoserine / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011743:Pyrimidines / Q000737:chemistry; D011758:Pyrroles / Q000737:chemistry*; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012714:Serine-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013876:Thiophenes / Q000737:chemistry
C522077:7-(2-thienyl)imidazo(4,5-b)pyrimidine; D000926:Anticodon; D011743:Pyrimidines; D011758:Pyrroles; D012346:RNA, Transfer, Amino Acyl; D013876:Thiophenes; C043354:tRNA, O(beta)-phosphoserine-; C051266:pyrrole-2-carboxaldehyde; D010768:Phosphoserine; D012714:Serine-tRNA Ligase