Fig. 4.
De novo and re-initiation of di-cistronic reporters using UAG as the initiation codon. A. Relative enzyme activity from E. coli CA274 transformed with CL or mutant di-cistronic reporters containing either a mutant CAT reporter (Cam1L) or a mutant fLuc reporter (CLam1) and expressing the U35A36 mutant initiator tRNA. Cell extracts were analysed for CAT (Cam1L, white bar) or fLuc activity (CLam1, grey bar) and compared with activity from CL. CAT activity was determined as described in Experimental procedures and normalized to total protein and β-lactamase activity (to normalize for plasmid copy number). fLuc activity was determined as described in Experimental procedures and normalized to cell number and CAT activity. B. Immunoblots of cell extracts from (A), using anti-CAT (top) or anti-fLuc (bottom) Ab. Different relative amounts of cell extract were analysed to facilitate comparison of protein levels between wild-type and mutant samples.