Overproduction and purification of Escherichia coli tRNA(2Gln) and its use in crystallization of the glutaminyl-tRNA synthetase-tRNA(Gln) complex.
We describe the genetically engineered overproduction of Escherichia coli tRNA(2Gln), its purification by high pressure liquid chromatography (HPLC), and its subsequent use in the growth of crystals of the E. coli glutaminyl-tRNA synthetase-tRNA(Gln) complex. The overproduced tRNA represents 60 to 70% of the total tRNA extracted from the engineered strain. A single anion exchange HPLC column is then sufficient to increase the purity of this isoacceptor to 90 to 95%. Crystals of this material complexed with the monomeric E. coli glutaminyl-tRNA synthetase enzyme were obtained by vapor diffusion from solutions containing sodium citrate as the precipitating agent. The crystals diffract to beyond 2.8 A resolution (1 A = 0.1 nm) and are of the orthorhombic space group C222(1) with unit cell parameters a = 240.5 A, b = 93.9 A, c = 115.7 A. Gel electrophoresis of dissolved crystals demonstrates the presence of both protein and tRNA.
Perona
J J
JJ
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Swanson
R
R
Steitz
T A
TA
Söll
D
D
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D012329:RNA, Bacterial / Q000096:biosynthesis* / Q000302:isolation & purification; D012345:RNA, Transfer, Amino Acid-Specific / Q000096:biosynthesis*; D012351:RNA, Transfer, Gln / Q000096:biosynthesis* / Q000302:isolation & purification; D014961:X-Ray Diffraction
D012329:RNA, Bacterial; D012345:RNA, Transfer, Amino Acid-Specific; D012351:RNA, Transfer, Gln; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase