FUNCTIONAL EXPRESSION OF EUKARYOTIC POLYPEPTIDE-CHAIN RELEASE FACTOR-1 AND FACTOR-3 BY MEANS OF BACULOVIRUS-INSECT CELLS AND COMPLEX-FORMATION BETWEEN THE FACTORS

Translation termination in eukruyotes is governed by termination codon s in mRNA and two release factors, eRF1 and eRF3. In this work, human eRF1 and eRF3 have been produced in insect cells using a recombinant b aculovirus expression system for the corresponding human cDNAs. Purifi cation of eRF1 has led to a homogeneous 50-kDa protein active in promo ting ribosome-dependent and termination-codon-dependent hydrolysis of formylmethionyl-tRNA(f)(Met). Purification of eRF3 yielded a full-leng th protein and shorter polypeptides. Microsequencing of the N-terminus of the shortest form detected a site of proteolytic cleavage between Arg91 and Gly92, probably due to exposed region(s) hypersensitive to p roteolysis. The mixture of full-length and truncated forms of eRF3 as well as bacterially expressed eRF3 lacking 138 N-terminal amino acids (eRF3Cp) are active as an eRF1-dependent and ribosome-dependent GTPase and in stimulating the GTP-dependent release activity of eRF1. Comple x formation between eRF1 and eRF3Cp was demonstrated by affinity and g el-filtration chromatographies and by native-gel electrophoresis. An a bnormal electrophoretic mobility observed for eRF1 as compared with th e complex points to a significant conformational change of either eRF1 or both factors in the complex. Go-expression of both factors in bacu lovirus-infected insect cells and a yeast two-hybrid assay were applie d to monitor complex formation in vivo. In yeast cells, both eRF1 and eRF3 are either in a monomeric or in a heterodimeric but not in a homo dimeric state.