Figure 1

Preparation of E. coli strains and TAP tag purification. (A) Schematic representation of λ Red recombination strategy. PCR cassettes containing TAP tag sequence followed by kanamycin resistance gene (Kan) and flanked by FRT (flip recombinase targets) sites were prepared using primers with overhangs homologous to the sequences surrounding STOP codon of the chosen gene (gene X). After recombination TAP tag forms C-terminal translational fusion with the protein product of chosen gene. (B) Accuracy of the fusion proteins was monitored by western blot. Total bacterial proteins were subjected to western blot using α-RNase R antibodies (αRNR) or α- Calmodulin Binding Protein antibody (αCBP). Due to protein A in the TAP tag sequence the signal from RpoC-TAP fusion can be observed using α-RNase R antibodies. (C) Level of RNase R-TAP increases in a similar fashion as RNase R upon cold shock. Total bacterial proteins were subjected to western blot using α-RNase R (αRNR) antibody. Ponceau stain is provided as the loading control. ex- cells grown at 37°C until OD 0,5; cs- cells grown at 37°C until OD 0,5 and subsequently moved to 15°C for 4 h. (D) TAP tag purification of fusion proteins. Proteins from strains expressing RNase R-TAP, RpoC-TAP, or RNase R-GFP were purified[15], final elutions from calmodulin resin were separated on SDS-PAGE gel. Chosen bands (indicated by frames) were extracted and proteins were identified using mass spectrometry. Position of fusion proteins in the gel is indicated with stars.