Fig 3.
In vitro transcription of 5′ ureB as a function of increasing amounts of purified 5′ureB-sRNA. (A) An 813-bp DNA fragment corresponding to 5′ ureB (nucleotides 46 to 859 of the ureB coding sequence) fused downstream of the cagA promoter (PcagA) was used as the template with E. coli RNA polymerase, [α-32P]UTP, and NTPs, without addition (lane 3) or with addition of increasing amounts of 5′ureB-sRNA from 100 to 400 fmol (lanes 4 to 10). The reaction mixtures were incubated with and without in vitro-synthesized 5′ureB-sRNA for 30 min at 37°C. (B) A similar in vitro transcription assay with a 515-bp DNA fragment corresponding to 3′ ureB (nucleotides 1123 to 1608 of the ureB coding sequence) fused downstream of PcagA was used as a control. Lanes 1, marker (ϕX174 DNA/HinfIII). Lanes 2, control with 1 μg rat liver mRNA but without 5′ureB-sRNA.