Fig. 2.

Effects of different SymR RNA levels. A. Quantitative Northern analysis of symE and SymR RNA levels. Total RNA (10 μg) isolated for the Northern analysis in Fig. 1B and total RNA (0.5 μg) isolated from the samples used for the Western analysis in Fig. 2B was separated alongside in vitro synthesized RNA (0.1, 0.3, 1 and 3 fmol) on 1.2% agarose gels. The cellular levels of symE mRNA and SymR RNA were determined from the ratio of the signals of control RNAs to the cellular RNAs. B. SymR RNA expressed in trans can repress SymE synthesis. The symE-SPA −10 mutant carrying pACYC and pACYC-SymR was grown to OD₆₀₀∼0.3 in LB medium containing tetracycline at 37°C. Cell lysates prepared at 0, 30, 60, 90, 150 and 300 min after treatment with 1 μg ml⁻¹ mitomycin C were analysed by immunoblot assays using monoclonal anti-FLAG M2-AP antibodies. C. Expression of an anti-antisense RNA leads to increased SymE-SPA synthesis. MG1655 P_CP18-araE symE-SPA and MG1655 P_CP18-araE symE-SPA −10 mutant strains carrying pAZ3-anti-SymR were grown to OD₆₀₀∼0.2 in LB medium containing chloramphenicol at 37°C. The cultures were split and half of each culture was treated with 0.02% arabinose for 30 min (0 min). All cultures were then treated with 1 μg ml⁻¹ mitomycin C for 90 min (90 min). Cell extracts prepared at the 0 and 90 min time points were analysed by immunoblot assays using monoclonal anti-FLAG M2-AP antibodies.