FIG. 6.
Primer extension inhibition assays (toeprints) of fepB leader transcripts. Each primer extension reaction was performed in the presence of tRNAfmet with (+) and without (−) 30S subunits using the fepB mRNA indicated. The following major primer extension inhibition products (described in text) are indicated by arrows: FL, full-length product; BoxC, product corresponding to the 3′ end of the BoxC repeat region; TP, 30S subunit-dependent toeprint products. (A and B) Toeprint assays of fepB leader constructs containing the native GUG start codon (A) or the mutant AUG start codon (B). The length and number of 30S subunit-dependent bands (TP) were verified with 8% polyacrylamide-urea gels (not shown), which provided better resolution of shorter fragments than the 6% polyacrylamide-urea gel shown provided. (C) Quantitation of toeprint assays was done with the Bio-Rad Quantity One software. The bars indicate the toeprint percentage for each lane obtained by dividing the toeprint product band value by the total lane value. The open and solid bars indicate reactions performed without (−) and with (+) 30S subunits, respectively. The bars indicate the averages of three experiments, and the data are expressed relative to the value obtained for the WT with 30S subunits (WT solid bar). The error bars indicate standard deviations.