Purification and characterization of yeast mitochondrial initiation factor 2.
Yeast mitochondrial initiation factor 2 (ymIF2) is encoded by the nuclear IFM1 gene. A His-tagged version of ymIF2, lacking its predicted mitochondrial presequence, was expressed in Escherichia coli and purified. Purified ymIF2 bound both E. coli fMet-tRNA(f)(Met) and Met-tRNA(f)(Met), but binding of formylated initiator tRNA was about four times higher than that of the unformylated species under the same conditions. In addition, the isolated ymIF2 was compared to E. coli IF2 in four other assays commonly used to characterize this initiation factor. Formylated and nonformylated Met-tRNA(f)(Met) were bound to E. coli 30S ribosomal subunits in the presence of ymIF2, GTP, and a short synthetic mRNA. The GTPase activity of ymIF2 was found to be dependent on the presence of E. coli ribosomes. The ymIF2 protected fMet-tRNA(f)(Met) to about the same extent as E. coli IF2 against nonenzymatic deaminoacylation. In contrast to E. coli IF2, the complex formed between ymIF2 and fMet-tRNA(f)(Met) was not stable enough to be analyzed in a gel shift assay. In similarity to other IF2 species isolated from bacteria or bovine mitochondria, the N-terminal domain could be eliminated without loss of initiator tRNA binding activity.
Garofalo
Cristiana
C
Department of Chemistry & Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.
Trinko
Richard
R
Kramer
Gisela
G
Appling
Dean R
DR
Hardesty
Boyd
B
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002855:Chromatography, Thin Layer; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D039642:Eukaryotic Initiation Factors / Q000737:chemistry* / Q000302:isolation & purification*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors
D000596:Amino Acids; D039642:Eukaryotic Initiation Factors; C451339:IFM1 protein, S cerevisiae; D024101:Mitochondrial Proteins; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D006160:Guanosine Triphosphate; D012343:RNA, Transfer