Figure 3.

The mccA transcript is stable and ribosome-bound. (A) An McC-producing culture of E. coli harboring the pp70 plasmid was treated with rifampicin. At times indicated, aliquots of the culture were withdrawn, total RNA was purified and amounts of mccA and mccB transcripts were measured by Northern blotting and primer extension, respectively, as described in Figure 2B legend. The results were quantified and the mean values and standard deviations in mRNA abundance obtained in three independent experiments are presented. (B) Lysates of McC-producing cells were subjected to sucrose gradient centrifugation, fractions were collected and the amounts of mccA RNA were determined by Northern blotting. Gradient profile of sucrose centrifugation is shown below the gel. (C) Total RNA was purified from McC-producing E. coli culture harboring the pp70 plasmid or from equally grown cultures harboring pp70 derivatives with a mutated mccA gene initiating codon (ATG¹->TAA) or multiple substitutions in the SD sequence of mccA and amounts of the mccA transcript (top), mccB transcript (middle) or bla transcript (bottom) were determined by Northern blot (mccA) or primer extension (mccB, bla) using appropriate primers.