Figure 3.

A truncated AapA1 mRNA species revealed by rifampicin treatment is efficiently translated in vitro (A) AapA1 and IsoA1 transcripts half-lives were determined in the 26695 H. pylori strain (left panel) or in an isogenic mutant deleted for aapA1 promoter (Δ-10 box, right panel). After rifampicin addition (at OD₆₀₀ = 1.7), aliquots of cultures were collected at several time points (as indicated on top of the gel), RNA extracted and subjected to Northern blot analysis. The same membrane was successively probed with FD47 and FD198 labeled oligonucleotides to detect either AapA1 or IsoA1 transcripts, respectively. Arrows indicate the different transcripts: AapA1_FL (one star), AapA1_Tr1 (two stars), AapA1_Tr2 (three stars) and IsoA1 full-length and processed transcripts. A labeled DNA marker (lane M) was used for size estimation. Proper loading was assessed by staining the membrane with methylene blue (level of 5S rRNA is shown). The Northern blot shown here is a representative of more than three independent experiments using several biological replicates. (B) RNA decay was determined by plotting normalized intensities (RNA signal relative to time 0) of band corresponding to AapA1_FL* and IsoA1 transcripts, either in the wild-type or Δ-10 box mutated strains, as function of time after rifampicin addition. Approximate half-lives (t_1/2, in min) are indicated for each transcript analyzed. (C) In vitro translations assays of AapA1_FL (left) and AapA1_Tr1 (right). Increasing amounts of in vitro synthesized mRNAs (μg) were added to E. coli S30 extracts in presence of ³⁵S methionine. Control lane (Ctrl) shows the translation background obtained without exogenous mRNA. Here is a representative experiment out of several independent assays. (D) The amount of AapA1 peptide produced from both transcripts (AapA1_FL and Tr1) was quantified and the intensity of the band at the highest AapA_Tr1 quantity (1 μg) was set to 100%. Data are the average and standard deviations of three independent experiments.