Separation of formyl-methionyl transfer RNA, methionyl transfer RNA, and transfer RNAfmet using mixed-mode high-performance liquid chromatography on C6-modified aminopropylsilyl-hypersil.
Preparative amounts of formyl-methionyl-tRNAfmet, methionyl-tRNAfmet, and tRNAfmet were separated from each other with baseline resolution in 30 min using mixed-mode HPLC on hexanoic anhydride-modified aminopropylsilyl-Hypersil 2. Pure tRNAfmet was aminoacylated with [35S]methionine in the presence or absence of a formyl donor and was immediately fractionated on the column. Two isoacceptors, tRNA1fmet and tRNA2fmet, as well as aminoacyl-tRNA synthetases were clearly separated from each other. The purified f[35S]-methionyl-tRNA was biologically active in that as much as 98% could be bound to ribosomes in response to AUGUAA in vitro. Formyl-methionine was released from this complex by the action of termination factor and greater than 92% of bound formyl-methionine was released by puromycin.
Ricker
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RD
University of Pennsylvania, School of Medicine, Department of Microbiology, Philadelphia 19104-6076.
Kaji
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A
D002208:Caproates; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004926:Escherichia coli / Q000032:analysis; D012329:RNA, Bacterial / Q000302:isolation & purification; D012345:RNA, Transfer, Amino Acid-Specific / Q000302:isolation & purification*; D012346:RNA, Transfer, Amino Acyl / Q000302:isolation & purification*; D012358:RNA, Transfer, Met / Q000302:isolation & purification*; D012828:Silicones
D002208:Caproates; D012329:RNA, Bacterial; D012345:RNA, Transfer, Amino Acid-Specific; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; D012828:Silicones; C026464:tRNA(m)(Met), methionine-; C024988:tRNA, formylmethionine-