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-3chlM, molecular mass, 46 402) and the portion of this factor homologous to bacterial IF-3 (IF-3chlH, 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-3chlM 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-3chlM and IF-3chlH using Ni-NTA affinity chromatography followed by ion exchange chromatography. IF-3chlM and IF-3chlH are active in promoting ribosome dissociation and in promoting the binding of fMet-tRNA to E. coli ribosomes. However, IF-3chlH has at least 5-fold more activity than either native IF-3chl or IF-3chlM 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-3chl lying outside of the homology domain may down-regulate the activity of this factor.
Lin
Q
Q
Department of Chemistry CB 3290, University of North Carolina, Chapel Hill 27599-3290, USA.
Yu
N J
NJ
Spremulli
L L
LL
D000818:Animals; D002736:Chloroplasts / Q000235:genetics* / Q000502:physiology; D003001:Cloning, Molecular; D004926:Escherichia coli; D005056:Euglena gracilis / Q000235:genetics* / Q000502:physiology; D006639:Histidine; D010448:Peptide Initiation Factors / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D039665:Prokaryotic Initiation Factor-3; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology
D010448:Peptide Initiation Factors; D039665:Prokaryotic Initiation Factor-3; D011994:Recombinant Proteins; D006639:Histidine