Identification and characterization of large, complex forms of chloroplast translational initiation factor 2 from Euglena gracilis.
Chromatography of partially purified preparations of Euglena gracilis chloroplast initiation factor 2 (IF-2chl) on gel filtration resins indicates that this factor is present in high molecular mass forms ranging from 200 to 700 kDa. The higher molecular weight complexes can be separated from the 200,000 Mr form of this factor by chromatography on DEAE-cellulose. Further purification indicates that the majority of the IF-2chl is present as dimeric, tetrameric, and probably hexameric complexes of polypeptides of 97,000-110,000 in molecular weight. In addition, one form consisting of subunits of about 200,000 Mr has been detected. All of these species are active in promoting fMet-tRNA binding to chloroplast 30 S subunits in a message-dependent reaction. Initiation complex formation promoted by IF-2chl requires the presence of GTP. Similar levels of binding are obtained when GTP is replaced by a nonhydrolyzable analog suggesting that IF-2chl is acting stoichiometrically rather than catalytically under the conditions used. The activity of this factor is stimulated by the presence of either Escherichia coli or chloroplast IF-3. None of the forms of IF-2chl detected is active on E. coli ribosomes.
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Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290.
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D000818:Animals; D002499:Centrifugation, Density Gradient; D002736:Chloroplasts / Q000378:metabolism*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005056:Euglena gracilis / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000302:isolation & purification* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met; D012270:Ribosomes / Q000378:metabolism; D013816:Thermodynamics
D015852:Eukaryotic Initiation Factor-2; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D006160:Guanosine Triphosphate