PROTEIN FACTORS IN POLYPEPTIDE CHAIN INITIATION IN EUKARYOTIC SYSTEMS

Abstract

A model in vitro system for polypeptide chain initiation in mammalian calf liver cytoplasms has been developed. Two protein factors, designated eIF-2 and eIF-5, have been purified from calf liver ribosomal salt-wash proteins that are required for GTP-dependent, AUG-directed binding of Met-tRNA(,f) to ribosomes to form a functional 80S polypeptide chain initiation complex. Both eIF-2 and eIF-5 have been extensively purified, and the properties of the partial reactions catalyzed by each of the factors have been studied.;eIF-2, purified to apparent electrophoretic homogeneity, has been shown to be composed of one 48,000 and one 38,000 dalton polypeptide chains corresponding to an M(,r) of 86,000 for the native factor. The purified factor forms stable ternary complexes with Met-tRNA(,f) and GTP, and binary complexes with GDP. Both complexes can be observed by either Millipore or gel filtration. Stable complex formation between GTP and eIF-2 cannot be detected by either technique. In contrast, formation of stable binary complexes between Met-tRNA(,f) and eIF-2 can be easily demonstrated.;eIF-2 binds a wide variety of nucleic acids--reoviral mRNAs, bacteriophage T3 mRNA, E. coli rRNAs, E. coli tRNA and bacteriophage 0X174 DNA. However, the factor only demonstrates high affinity for binding to eukaryotic mRNA and this affinity is similar to the affinity the factor exhibits for GTP-dependent binding of Met-tRNA(,f).;The purified eIF-2 can be phosphorylated by heme controlled translational repressor (eIF-2 kinase) and the site of phosphorylation has been identified to be the 38,000 dalton polypeptide chain.;The ternary complex (eIF(.)2-GTP(.)Met-tRNA(,f)) efficiently transfers both Met-tRNA(,f) and intact GTP to 40S subunits in the presence of AUG codon. This transfer reaction does not require any additional protein factor. The 40S initiation complex thus formed can join a 60S subunit in the presence of purified eIF-5 to form the 80S initiation complex that is active in peptidyl transfer. GTP present in the 40S initiation complex is hydrolyzed during the 60S subunit joining reaction and the products of hydrolysis, GDP and P(,i) are released from the ribosomes.;The function of GTP and its hydrolysis in polypeptide chain initiation have been investigated by comparing initiation reactions carried out with GTP to those which either did not contain GTP or contained the non-hydrolyzable analogue of GTP, 5'-guanylyl methylene diphosphonate (GDPCP). It has been observed that 40S initiation complexes formed either with GTP or in the absence of GTP can efficiently form 80S initiation complexes upon addition of 60S subunits and eIF-5. In contrast, 40S initiation complexes formed with GDPCP are unable to join 60S subunits to form 80S initiation complexes.;It is postulated that GTP hydrolysis provides a means by which the bound nucleotide is removed from the ribosomal complex resulting in a conformational change of the 40S ribosomal initiation complex to allow interaction with 60S subunits to form the 80S initiation complex. The energy of hydrolysis of GTP is presumably not required for the subunit joining reaction.