Expression and functional analysis of Euglena gracilis chloroplast initiation factor 3

A portion of a cDNA predicted to encode the mature form of Euglena gracilis chloroplast translational initiation factor 3 (IF-3 (chl)M, molecular mass, 46 402) and the portion of this factor homologous to bacterial IF-3 (IF-3 (chl)H, molecular mass 22-829) have been cloned and expressed in Escherichia coli as histidine-tagged proteins. The homology domain can be expressed in reasonable levels in E. coli. However, IF-3(chl)M is quite toxic and can only be produced in small amounts. Both forms of the chloroplast factor are associated with E. coli ribosomes. Purification procedures have been developed for both IF-3(chl)M and IF-3(chl)H using Ni-NTA affinity chromatography followed by ion exchange chromatography. IF-3(chl)M and IF-3(chl)H are active in promoting ribosome dissociation and in promoting the binding of fMet-tRNA to E. coli ribosomes. However, IF-3(chl)H has at least 5-fold more activity than either native IF-3(chl) or IF-3(chl)M in promoting initiation complex formation on chloroplast 30S ribosomal subunits in the presence of a mRNA carrying a natural translational initiation signal. This observation suggests that regions of IF-3(chl) lying outside of the homology domain may down-regulate the activity of this factor.