Activation of the pyrrolysine suppressor tRNA requires formation of a ternary complex with class I and class II lysyl-tRNA synthetases.
Monomethylamine methyltransferase of the archaeon Methanosarcina barkeri contains a rare amino acid, pyrrolysine, encoded by the termination codon UAG. Translation of this UAG requires the aminoacylation of the corresponding amber suppressor tRNAPyl. Previous studies reported that tRNAPyl could be aminoacylated by the synthetase-like protein PylS. We now show that tRNAPyl is efficiently aminoacylated in the presence of both the class I LysRS and class II LysRS of M. barkeri, but not by either enzyme acting alone or by PylS. In vitro studies show that both the class I and II LysRS enzymes must bind tRNAPyl in order for the aminoacylation reaction to proceed. Structural modeling and selective inhibition experiments indicate that the class I and II LysRSs form a ternary complex with tRNAPyl, with the aminoacylation activity residing in the class II enzyme.
Polycarpo
Carla
C
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
Ambrogelly
Alexandre
A
Ruan
Benfang
B
Tumbula-Hansen
Debra
D
Ataide
Sandro F
SF
Ishitani
Ryuichiro
R
Yokoyama
Shigeyuki
S
Nureki
Osamu
O
Ibba
Michael
M
Söll
Dieter
D
D019843:Archaeal Proteins; D001483:Base Sequence; D018388:Codon, Terminator; D004247:DNA / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D008250:Lysine-tRNA Ligase / Q000737:chemistry*; D017021:Methanosarcina barkeri / Q000201:enzymology; D008780:Methyltransferases / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic
D019843:Archaeal Proteins; D018388:Codon, Terminator; D004247:DNA; D012343:RNA, Transfer; D008780:Methyltransferases; C113200:monomethylamine methyltransferase; D008250:Lysine-tRNA Ligase; C456839:pyrrolysine; D008239:Lysine