Northern analysis of in vivo mutant 6S RNA expression. (A) Total RNA was extracted from E. coli pEcoli-R9-33 cells under denaturing conditions. Five OD600 units were taken starting at 60 min of incubation in LB+Amp with IPTG and at intervals until 1320 min (22 h). An 8% denaturing PAGE gel imaged by SYBR Green is shown in the top panels. On the left, in vitro-synthesized 6S RNA was loaded alongside in vitro-transcribed T1 and R9-33 RNA (using plasmid cut [pc] with ClaI as DNA template) that served as a control for probe specificity and also as size reference for plasmid-derived RNA. The nucleic acid in this gel was transferred onto a membrane and probed with radiolabeled R9-33-specific probe (middle panel; Supplemental Table S2). (B) Total RNA was extracted from BL21 pEcoli-T1 cells using previously described conditions. The same positive controls were loaded in this gel along with a sample extracted from BL21 pEcoli-T1 cells grown in the absence of IPTG. In all cases, hybridization to 5S rRNA was used as an internal loading control (bottom panels).
