Molecular cloning and expression of mammalian translation initiation factors involved in the binding and hydrolysis of GTP

Abstract

Eukaryotic translation initiation factor, eIF-5, a monomeric phosphoprotein of about 50 kDa, plays an essential role in initiation of protein synthesis. The factor specifically interacts with the 40S ribosomal initiation complex (40S{dollar}\cdot{dollar}mRNA{dollar}\cdot{dollar}Met-tRNA{dollar}\sb{lcub}\rm i{rcub}\cdot{dollar}eIF-2{dollar}\cdot{dollar}GTP) to mediate the quantitative hydrolysis of ribosome-bound GTP. GTP hydrolysis is essential for the release of eIF-2 and guanine nucleotide (as an eIF-2{dollar}\cdot{dollar}GDP complex) from the 40S subunit and the subsequent joining of the 60S ribosomal subunit to form a functional 80S initiation complex (80S{dollar}\cdot{dollar}mRNA{dollar}\cdot{dollar}Met-tRNA{dollar}\sb{lcub}\rm i{rcub}).{dollar}.;In this thesis, a rat cDNA encoding eIF-S has been isolated, sequenced and characterized. The entire coding region of the cDNA, with a calculated molecular mass of 48,926 dalton, was expressed in Escherichia coli in soluble form and the recombinant protein was purified to homogeneity in a catalytically active form. Analysis of the amino acid sequence of eIF-S revealed the presence of a somewhat imperfect sequence motif similar to proteins of the GTPase superfamily. Site directed mutagenesis studies demonstrated that these sequence motifs do not play an essential role in the hydrolysis of GTP mediated by eIF-S. These results along with earlier observations made in this laboratory that eIF-S neither binds nor hydrolyzes free GTP, suggest that the function of eIF-S is to activate the intrinsic GTPase activity of eIF-2 that is bound to the 40S initiation complex.;In this thesis, we have also carried out further characterization of eIF-2, a heterotrimeric protein consisting of {dollar}\alpha,\ \beta{dollar} and {dollar}\gamma{dollar} subunits, that binds guanine nucleotides, GTP and GDP during initiation of protein synthesis. A human cDNA clone containing the entire coding region of the {dollar}\gamma{dollar}-subunit of eIF-2 was isolated and characterized. Amino-acid sequence of eIF-2{dollar}\gamma{dollar} revealed the presence of consensus GTP binding domains, characteristic of proteins of the GTPase superfamily including guanine nucleotide-binding translation factors. To investigate the role of the {dollar}\gamma{dollar}-subunit of eIF-2 in the binding of GTP and GDP, we have expressed the coding region of the {dollar}\gamma{dollar}-subunit in E. coli. The recombinant protein which forms inclusion bodies was solubilized in 8M urea, renatured and then purified to near homogeniety. The purified recombinant {dollar}\gamma{dollar}-subunit did not form a stable millipore retainable-complex with either GDP or GTP even in the presence of initiator Met-tRNA{dollar}\sb{lcub}\rm i{rcub}{dollar}. A variety of factors that may account for our failure to detect guanine nucleotide binding to the bacterially-expressed eIF-2{dollar}\gamma{dollar} protein are discussed.