Fig. 3: HigB^TAC cleaves cspA mRNA at CCA codon during translation in vitro.

a HigB^TAC inhibits synthesis of CspA wild-type (WT with codon CCA at Pro2) but not of CspA with +1 (OOF1) and +2 (OOF2) out of frame CCA motifs. CspA WT, OOF1, and OOF2 were independently expressed in a cell-free translation system with or without HigB^TAC (1.5 µM) as described in Fig. 1d. CspA translation products were labeled with [³⁵S]methionine and reactions were performed for 1 h 30 min at 37 °C. After translation, samples were separated on SDS–PAGE and visualized by phosphorimager. b Cleavage of cspA wild-type is ribosome-dependent. cspA wild-type (CCA), OOF1, and OOF2 were independently expressed in a cell-free translation system for 2 h with or without HigB^TAC (1.5 µM). RNA was extracted and subjected to a primer extension with [³²P]-labeled cspA primer. In parallel, cspA mRNA was incubated for the same time with or without HigB^TAC (1.5 µM) in the absence of ribosomes (CCA-∆ribo), and also used for primer extension. The obtained labeled cDNAs were separated on denaturing urea-polyacrylamide gel and revealed by autoradiography. Arrows show the uncleaved (A, 126 nt) and cleaved (A*, 95 nt) cspA, and (m) stands for molecular ladder. Mutations in the CCA codon prevent both inhibition of CspA synthesis (c) and cleavage (d) of cspA by HigB^TAC in vitro. cspA wild type (CCA) and its mutant derivatives (mutations depicted in red) were independently expressed in a cell-free translation system with or without HigB^TAC (1.5 µM) and analyzed as described in panel (a) for CspA protein synthesis and in panel (b) for cspA cleavage. Representative results of triplicate experiments are shown.