Tandem translation of E.coli initiation factor IF2β: Purification and characterization in vitro of two active forms

Abstract

Two forms of E.coli initiation factor IF2, IF2α and IF2β, have been known for several years. Both forms are products of the gene infB with translational initiation at codon 1 (AUG) and codon 158 (GUG) in the same reading frame. In this work we demonstrate that IF2β exists in two forms, IF2β and IF2β′ with initiation codons 158 (GUG) and 165 (AUG) and molecular masses of 79.7 kDa and 78.8 kDa respectively. We have recently described a fast purification method for IF2α, using an FPLC procedure consisting of ion-exchange liquid chromatography on Q Sepharose HP, Mono Q and Mono S. After the Mono Q step, an apparently homogeneous IF2β was observed when analyzed by SDS-PAGE. However the chromatography on Mono S results in the elution of two peaks containing IF2β. The N-terminal amino acid sequence of the two proteins identified the first peak to be IF2β and the second as a protein which we term IF2β′ starting seven residues downstream at the AUG codon 165. The activity in vitro of the two purified forms of IF2β was tested by measuring the stimulation of binding of the initiator fMet-tRNA f Met to 70S ribosomes in the presence of GTP and poly(A,U,G) as messenger-RNA. In this assay no difference in activity is detected.