tRNA(2Gln) mutants that translate the CGA arginine codon as glutamine in Escherichia coli.
We present a novel missense suppression system for the selection of tRNA(2GIn) mutants that can efficiently translate the CGA (arginine) codon as glutamine. tRNA(2Gln) mutants were cloned from a partially randomized synthetic gene pool using a plasmid vector that simultaneously expresses the tRNA gene and, to ensure efficient aminoacylation, the glutamine aminoacyl-tRNA synthetase gene (glnS). tRNA mutants that insert glutamine at CGA were selected as missense suppressors of a lacZ mutant (lacZ625(CGA)) that contains CGA substituted for an essential glutamine codon. Preliminary characterizations of four suppressors is presented. All of them contain two anticodon mutations: C-->U at position 34 and U-->C at position 35, which allow for cognate translation of CGA. U35 was previously shown to be an important determinant for glutaminylation of tRNA(2Gln) in vitro; suppression in vivo requires overexpression of the glutaminyl-tRNA synthetase gene (glnS). One tRNA variant contains no further mutations and has the highest missense suppression activity (8%). Three other isolates each contain an additional point mutation that alters suppression efficiency. This system will be useful for further studies of tRNA structure and function. In addition, because relatively efficient translation of the rare CGA codon as glutamine is not toxic for Escherichia coli, it may be possible to translate this sense codon with other alternate meanings, a property which could greatly facilitate protein engineering.
Tsai
F
F
Department of Biology, Wake Forest University, Winston-Salem, North Carolina 27109, USA.
Curran
J F
JF
D000483:Alleles; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics; D001120:Arginine / Q000235:genetics*; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics*; D016153:Genes, Suppressor; D005973:Glutamine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012351:RNA, Transfer, Gln / Q000737:chemistry / Q000235:genetics*; D013329:Structure-Activity Relationship; D001616:beta-Galactosidase / Q000378:metabolism
D003062:Codon; D012351:RNA, Transfer, Gln; D005973:Glutamine; D001120:Arginine; D001616:beta-Galactosidase; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase