title: Identification of the sites in the eukaryotic elongation factor 1 alpha involved in the binding of elongation factor 1 beta and aminoacyl-tRNA.
abstract: In this article we report the identification of the sites which are involved in the binding of the GDP-exchange factor EF-1 beta and aminoacyl tRNA to the alpha-subunit of the eukaryotic elongation factor 1 (EF-1) from Artemia. For this purpose the polypeptide chain of EF-1 alpha, having 461 amino acid residues, was proteolytically cleaved into large fragments by distinct proteases. Under well defined conditions, a mixture of two large fragments, free from intact EF-1 alpha and with molecular masses of 37 kDa and 43 kDa, was obtained. The 37-kDa and 43-kDa fragments comprise the residues 129-461 and 69-461, respectively. However, in aqueous solution and under non-denaturing conditions, the mixture still contained a short amino-terminal peptide, encompassing the residues 1-36, that remained tightly bound. The ability of the mixture of the 37+43-kDa fragments, including this amino-terminal peptide 1-36, to bind GDP or to facilitate aminoacyl tRNA binding to salt-washed ribosomes was severely reduced, compared to intact EF-1 alpha. However, both of these complexes were able to bind to the GDP-exchange-stimulating subunit EF-1 beta. A 30-kDa fragment, comprising the residues 1-287, was generated after treatment of the protein with endoproteinase Glu-C. This fragment contained the complete guanine nucleotide binding pocket. Although it was able to bind GDP and to transport aminoacyl tRNA to the ribosome, no affinity towards EF-1 beta was observed. We propose that the guanine-nucleotide-exchange stimulation by EF-1 beta is induced through binding of this factor to the carboxy-terminal part of EF-1 alpha. As a result, a decreased susceptibility towards trypsin of the guanine-nucleotide-binding pocket of EF-1 alpha, especially in the region of its presumed effector loop is induced.
authors.affiliation: Department of Medical Biochemistry, Sylvius Laboratory, University of Leiden, The Netherlands.
authors.forename: H T
authors.initials: HT
authors.lastname: van Damme
authors.forename: R
authors.initials: R
authors.lastname: Amons
authors.forename: W
authors.initials: W
authors.lastname: Möller
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D015977:Eukaryotic Initiation Factor-1 / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D014357:Trypsin
chemical_list: D015977:Eukaryotic Initiation Factor-1; D006150:Guanine Nucleotides; D012346:RNA, Transfer, Amino Acyl; D014357:Trypsin