FIGURE 5.

Toeprinting of the ompA and ompF mRNAs. A, toeprinting of the ompA mRNA in the presence of YafO-His₆. The mRNA was synthesized in vitro from a 248-bp DNA fragment containing a T7 promoter using T7 RNA polymerase as described under “Experimental Procedures.” The sequence ladder shown to the right was obtained using the same primer used for toeprinting with pCR®2.1-TOPO®-ompA as template. B, toeprinting of the ompF mRNA in the presence of YafO-His₆. The mRNA was synthesized in vitro from a 224-bp DNA fragment containing a T7 promoter using T7 RNA polymerase as described under “Experimental Procedures.” The sequence ladder shown to the right was obtained using the same primer used for toeprinting with pCR®2.1-TOPO®-ompF as template. Lane 1, control; lane 2, with 0.34 μm YoeB-His₆; lane 3, with 12.3 μm YafO-His₆; lane 4, 1 μm tRNA_f^Met; lane 5, 0.34 μm YoeB-His₆ and 1 μm tRNA_f^Met; lane 6, 12.3 μm YafO-His₆ and 1 μm tRNA_f^Met; lane 7, 0.05 μm 70 S ribosomes; lane 8, 0.05 μm 70 S ribosomes and 0.34 μm YoeB-His₆; lane 9, 0.05 μm 70 S ribosomes and 12.3 μm YafO-His₆; lane 10, 0.05 μm 70 S ribosomes and 1 μm tRNA_f^Met; lane 11, 0.05 μm 70 S ribosomes and 1 μm tRNA_f^Met with 0.34 μm YoeB-His₆; lane 12, 0.05 μm 70 S ribosomes and 1 μm tRNA_f^Met with 12.3 μm YafO-His₆. Both mRNA sequences shown are complementary to the sequencing ladders. The initiation codon, AUG, is indicated with an arrow. SD, Shine-Dalgarno sequence. TP(O) is the band where toeprinting was stopped in the presence of YafO-His₆ (shown by an asterisks). TP(Y) is the band where toeprinting was stopped in the presence of YoeB-His₆. FL, the full-length of the mRNA; TP(r), the toeprinting site caused by normal ribosome binding to mRNA in the absence of YafO (indicated by black dots).