In vivo deletion analysis of the architecture of a multiprotein complex of translation initiation factors.
Protein complexes play a critical role in virtually all cellular processes that have been studied to date. Comprehensive knowledge of the architecture of a protein complex of interest is, therefore, an important prerequisite for understanding its role in the context of a particular pathway in which it participates. One of the possible approaches that has proven very useful in characterizing a protein complex is outlined in this chapter using the example of the eukaryotic initiation factor 3 (eIF3) and some of its binding partners. eIF3 is one of the major players in the translation initiation pathway because it orchestrates several crucial steps that ultimately conclude with formation of the 80S ribosome where the anticodon of methionyl-tRNA(i)(Met) base-pairs with the AUG start codon of the mRNA in the ribosomal P-site. We previously demonstrated that, in the budding yeast Saccharomyces cerevisiae, eIF3 closely cooperates with several other eIFs to stimulate recruitment of methionyl-tRNA(i)(Met) and mRNA to the 40S ribosome and that it forms, together with eIFs 1, 2, and 5, an important intermediate in translation initiation called the multifactor complex (MFC). Here, we summarize the fundamental procedure that allowed in-depth characterization of the MFC composition and identification of protein-protein interactions among its constituents. Primarily, we describe in detail in vivo purification techniques that, in combination with systematic deletion analysis, produced a 3D subunit interaction model for the MFC. Site-directed clustered-10-alanine-mutagenesis (CAM) employed to investigate the physiological significance of individual interactions is also presented. The general character of the entire procedure makes it usable for first-order structural characterization of virtually any soluble protein complex in yeast.
Nielsen
Klaus H
KH
Department of Molecular Biology, University of Aarhus, Aarhus, Denmark.
Valásek
Leos
L
D000465:Algorithms; D000595:Amino Acid Sequence; D002457:Cell Extracts / Q000032:analysis / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods*; D039621:Eukaryotic Initiation Factor-3 / Q000737:chemistry / Q000235:genetics; D039642:Eukaryotic Initiation Factors / Q000737:chemistry* / Q000235:genetics*; D017353:Gene Deletion; D006639:Histidine / Q000737:chemistry; D021621:Imaging, Three-Dimensional; D008954:Models, Biological; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009532:Nickel / Q000378:metabolism; D021122:Protein Subunits / Q000032:analysis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics
D002457:Cell Extracts; D039621:Eukaryotic Initiation Factor-3; D039642:Eukaryotic Initiation Factors; D046912:Multiprotein Complexes; D021122:Protein Subunits; C033223:polyhistidine; D006639:Histidine; D009532:Nickel