IMPROVED RECOMBINANT TANDEM EXPRESSION OF TRANSLATION INITIATION-FACTOR IF2 IN RNASE-E DEFICIENT ESCHERICHIA-COLI-CELLS

The prokaryotic translation initiation factor IF2 exists in a varying number of nested forms in different species. In E. coli three natural forms exist, IF2 alpha, IF2 beta and IF2 gamma differing only in the N -terminal: IF2 beta and IF2 gamma lack 158 and 165 amino acid residues , respectively, as compared to IF2 alpha. We have earlier shown that t he smaller forms of IF2 are not the result of a specific proteolysis o f IF2 alpha, but produced from individual translation initiation sites in the mRNA. However it has not been known whether the expression in E. coli of IF2 beta and IF2 gamma is dependent on or related to a post transcriptional processing of the polycistronic nusA operon, containin g infB, the gene for IF2. Here we have used S1 mapping to study the ex istence of such mRNA processing in the region between the initiation s ites for IF2 alpha and IF2 beta/IF2 gamma. The results show a Ribonucl ease E cleavage site at position +200 in the infB mRNA between the tra nslation initiation sites. However, studies of the overexpression of t he different forms of IF2 show that the relative expression of IF2 alp ha and IF2 beta/IF2 gamma is independent of RNase E activity. Thus E. coli exhibits a true tandem translation of intact infB mRNA with multi ple in-frame translation initiation sites resulting in gene products o f different sizes. An additional observation is a significant increase in the level of overexpression of IF2 in cells devoid of RNase E acti vity. We conclude that due to lack of RNase E activity, the amount of plasmid-transcribed infB mRNA available for translation is accumulated , resulting in an elevated amount of recombinant IF2. This observation may have a more general application within the field of recombinant p rotein production and expression efficiency. (C) 1995 Academic Press, Inc.