Fig. 3.

The fLuc reporter gene is translated by re-initiating ribosomes. A. Relative fLuc activity from mutant di-cistronic reporters containing premature UAG stop codons placed at various distances upstream of the normal geneV UAA stop codon. SupF suppressor or U35A36 mutant initiator tRNA was also coexpressed. fLuc activity from CL was set at 100%. nt, number of nucleotides separating the two reporter genes. fLuc activity is defined as RLU/OD₆₀₀. B. Immunoblot of total-cell extract from (A) using anti-CAT or anti-fLuc Ab. The mutant di-cistronic reporter containing a 40-nucleotide intercistronic region (C_am219L, lane 4) encodes a CAT protein that is ∼1.4 kDa smaller than the full-length CAT. Lane 6 represents a sample from E. coli transformed with empty vector. C. Relative fLuc activity from mutant di-cistronic reporters without or with a SD sequence upstream of the fLuc start codon (C_rbsL). fLuc activity from CL was set at 1. fLuc activity is defined as RLU/OD₆₀₀.