title: Natural mRNA is required for directing Met-tRNA(f) binding to 40S ribosomal subunits in animal cells: involvement of Co-eIF-2A in natural mRNA-directed initiation complex formation.
abstract: Two protein factors, eIF-2 as well as a high molecular weight protein complex from reticulocyte ribosomal high-salt wash which we term Co-eIF-2, promote Met-tRNA(f) binding to 40S ribosomes. This binding is dependent on the presence of an AUG codon or natural mRNAs [Roy et al. (1984) Biochem. Biophys. Res. Commun. 122, 1418-1425]. Co-eIF-2 contains two component activities, Co-eIF-2A and Co-eIF-2C. Previously, we have purified an 80-kDa polypeptide containing Co-eIF-2A activity and showed that this polypeptide is a component of Co-eIF-2 and is responsible for Co-eIF-2A activity in Co-eIF-2 [Chakravarty et al. (1985) J. Biol. Chem. 260, 6945-6949]. We now report purification of a protein complex (subunits of Mr 180K, 110K, 65K, 63K, 53K, 50K, 43K, and 40K) containing Co-eIF-2C activity and devoid of Co-eIF-2A activity. In SDS-PAGE, the purified Co-eIF-2C preparation and an eIF-3 preparation (purified in Dr. A. Wahba's laboratory) separated into seven similar major polypeptides (Mr 110K, 65K, 63K, 53K, 50K, 43K, and 40K). The 50-kDa polypeptide in Co-eIF-2C was immunoreactive with a monoclonal antibody against eIF-4A (50 kDa). We have studied the roles of purified Co-eIF-2A and Co-eIF-2C activities in ternary and Met-tRNA(f).40S ribosome complex formation. The results are as follows: (1) At low and presumably physiological factor concentration (30 nM), eIF-2 did not form detectable levels of ternary complex. Moreover, such complex formation was totally dependent on the presence of Co-eIF-2A and/or Co-eIF-2C.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry, University of Nebraska, Lincoln 68588-0304.
authors.forename: A L
authors.initials: AL
authors.lastname: Roy
authors.forename: D
authors.initials: D
authors.lastname: Chakrabarti
authors.forename: B
authors.initials: B
authors.lastname: Datta
authors.forename: R E
authors.initials: RE
authors.lastname: Hileman
authors.forename: N K
authors.initials: NK
authors.lastname: Gupta
mesh_terms: D000818:Animals; D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D007700:Kinetics; D010448:Peptide Initiation Factors / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D010448:Peptide Initiation Factors; D011506:Proteins; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-