Coupled transcription-translation assay.

In vitro coupled transcription-translation assays using PURExpress (New England BioLabs) followed a published procedure (31). Plasmid pYH258 contains a T7 promoter driving transcription of the rpoE translational fusion from near the P2 transcription start site (nt −75 to +26 relative to the rpoE start codon). Plasmids pYH259 and pYH260 are identical to pYH258, except that they contain a GGA-to-CCA mutation in BS1 or in both BS1 and BS3, respectively (Fig. 1). pYH310 is identical to pYH258, except that it contains a TAA stop codon in ORF51 (Fig. 1). Plasmid pYH309 contains a T7 promoter that drives transcription from near the P1 transcription start site (nt −215 to +26 relative to the rpoE start codon). Plasmids pYH314 and pYH315 are identical to pYH309, except that they contain a TGA stop codon in ORF51 and a GGA-to-GCA mutation in BS1, respectively (Fig. 1). These plasmids were used as the templates for coupled transcription-translation reactions using the PURExpress in vitro protein synthesis kit, according to the manufacturer's instructions. Each 6.7-μl reaction mixture contained 250 ng of plasmid DNA template, various concentrations of purified CsrA-His6, 1 U of RNase inhibitor (Promega), 2.5 mM DTT, 2.7 μl of solution A, and 2 μl of solution B. The mixtures were incubated for 2.5 h at 37°C, and β-galactosidase activity was determined according to the manufacturer's instructions.