T4 pnk kinase

The RNA-protein interactions were determined by the Electrophoretic Mobility Shift Assay (EMSA). We used the affinity purified GST-fused Com and [³²P] labeled synthetic RNAs. RNAs were labeled with γ ³³P ATP (Hartmann Analytic) using T4 PNK Kinase (Thermo Fisher Scientific), phenol/chloroform extracted, precipitated, desalted and separated from the unincorporated label on MicroSpin G-25 Columns (GE Healthcare Life Sciences). RNA samples were annealed in buffer containing 50 mM Tris, pH 7, 2.5 mM MgCl₂ by heating at 80°C for 15 min and cooling 1°C per min until it reached room temperature (performed in Thermal Cycler BioRad).
Each binding reaction consisted of about 4 pmole of annealed RNA and appropriate Com concentration: 0, 8, 16, 32, 64, 125, 250, 500, 1000, 2000 nM (the RNA IA and IB motif tests were performed in the presence of 2000 nM Com). The binding reaction was carried out for 1 hour at room temperature in the reaction buffer containing: 50 mM Tris, pH 7, 50 mM NaCl, 1 μg of BSA, 1 mM DTT, 10% glycerol, 0.1% Igepal, 2.5 mM MgCl₂, 20 μM ZnSO₄, 2 μg dI-dC. The samples were then loaded on a 15% non-denaturing polyacrylamide gel and resolved in 0.5x Tris-borate-EDTA (TBE) buffer at 1 W for 1.5 hours. The separated RNA samples were visualized using Typhoon Phosphorimager.

pUC18 expression vectors carrying the wild-type codon-optimized RbcL gene coding sequence of R. rubrum (GenBank: CAA25080) or S. microadriaticum (GenBank: OLP96161) were ordered from Genomed S.A., Warsaw, Poland. The latter was disposed of its chloroplastic signal peptide coding sequence. The R. rubrum Δloop and the S. microadriaticum Δloop mutants were generated by PCR-based site-directed mutagenesis of the expression vector (loop nucleotide sequence insertion and deletion, respectively), followed with the PCR product phosphorylation by the T4 PNK kinase (Thermofisher, Waltham, MA, USA), and a subsequent ligation by the T4 ligase (Thermofisher, Waltham, MA, USA). DNA sequences of all the resulting constructs used in this study were confirmed by sequencing (Genomed S.A., Warsaw, Poland). Primers for the PCR reaction were as follows in Table 5 (expression vector complement primer sequences are capitalized).