Functional expression of eukaryotic polypeptide chain release factors 1 and 3 by means of baculovirus/insect cells and complex formation between the factors.
Translation termination in eukaryotes is governed by termination codons 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 baculovirus expression system for the corresponding human cDNAs. Purification of eRF1 has led to a homogeneous 50-kDa protein active in promoting ribosome-dependent and termination-codon-dependent hydrolysis of formylmethionyl-tRNAf(Met). Purification of eRF3 yielded a full-length 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 proteolysis. 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. Complex formation between eRF1 and eRF3Cp was demonstrated by affinity and gel-filtration chromatographies and by native-gel electrophoresis. An abnormal electrophoretic mobility observed for eRF1 as compared with the complex points to a significant conformational change of either eRF1 or both factors in the complex. Co-expression of both factors in baculovirus-infected insect cells and a yeast two-hybrid assay were applied 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 homodimeric state.
Frolova
L Y
LY
Department of Molecular and Structural Biology, University of Aarhus, Denmark.
Simonsen
J L
JL
Merkulova
T I
TI
Litvinov
D Y
DY
Martensen
P M
PM
Rechinsky
V O
VO
Camonis
J H
JH
Kisselev
L L
LL
Justesen
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010454:Peptide Termination Factors / Q000096:biosynthesis* / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D010454:Peptide Termination Factors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein; C489002:ETF1 protein, human; C087971:peptide-chain-release factor 3