Protein synthesis in yeast Saccharomyces cerevisiae . Purification of Co-eIF-2A and "mRNA-binding factor(s)" and studies of their roles in Met-tRNA sub(f) multiplied by 40S multiplied by mRNA complex formation

Antibodies prepared against a homogeneous preparation of Co-eIF-2A super(20) reacted with several polypeptides including an 80-kDa polypeptide present in a crude yeast ribosomal salt wash. This 80-kDa polypeptide, containing Co-eIF-2A (Co-eIF-2A super(80)) activity, has been extensively purified using a two-step purification procedure involving an immunoaffinity column chromatograph prepared using antibodies against Co-eIF-2A super(20) (fraction II) and hydroxyapatite chromatography (fraction III). The factors, eIF-2 + homogeneous Co-eIF-2A super(80) (fraction III) promoted Met-tRNA sub(f) multiplied by 40S complex formation with an AUG codon but not with a physiological mRNA or a polyribonucleotide messenger poly(U,G) whereas eIF-2 + a partially purified Co-eIF-2A super(80) preparation (fraction II) promoted Met-tRNA sub(f) multiplied by 40S complex formation with an AUG codon as well as with globin mRNA and poly(U,G) messenger. This factor-promoted Met-tRNA sub(f) binding to 40S ribosomes depends absolutely on the presence of a polyribonucleotide messenger containing an initiation codon (such as AUG or GUG).