Cyclone plus storage phosphor scanner

DNA fragments used in the binding assays were obtained by PCR, with the corresponding oligonucleotides pairs (Supplementary Table S4). DNA probes, including NtcA-binding consensus sequences, were cut with NotI restriction enzyme, generating fragments of ∼200 bp. These fragments were end-labeled with [α-³²P]dCTP, using Sequenase version 2.0 enzyme. The GST-NtcA fusion protein was expressed and purified, as previously described (39 (link)). DNA radiolabeled fragments (0.5 nM) were incubated with purified NtcA (0.1–0.4 μM), and with 2-OG (0.6 mM), when indicated. The binding reaction with the corresponding DNA fragment was carried out in a final volume of 15 μl within binding buffer (12 mM HEPES-NaOH pH 8.0, 8 mM Tris–HCl pH 8.0, 10% (w/v) glycerol, 0.5 mM ethylenediaminetetraacetic acid pH 8.0, 100 mM KCl, 2 mM MgCl, 0.05 μg/μl poly (dI–dC), 0.01 μg/μl bovine serum albumin and 1 mM dithiothreitol (DTT)). These mixtures were incubated at 25°C for 20 min, and the DNA–protein complexes were separated on non-denaturing 6% (w/v) polyacrylamide gel. Gels were dried and imaged using a Cyclone Plus storage phosphor scanner (PerkinElmer).

Total RNA was isolated from 30 ml samples of Synechocystis cultures in the mid-exponential growth phase (3 to 4 μg chlorophyll ml⁻¹). Extractions were performed by vortexing cells in presence of phenol–chloroform and acid-washed baked glass beads (0.25–0.3 mm diameter), as described by Garcia-Dominguez and Florencio (34 (link)). An aliquot of 5 μg of total RNA was loaded per lane, and electrophoresed in 1.2% agarose denaturing formaldehyde gels (35 ). For the ncRNA analysis, RNA samples (5–10 μg) were separated on 6% urea-polyacrylamide gels for 3 h at 25 mA. In both cases, migrated gels were later transferred to nylon membranes (Hybond N-Plus; GE Healthcare, Little Chalfont, UK). Prehybridization, hybridization and washes were carried out in accordance with GE Healthcare instruction manuals. Probes for northern blot hybridization were prepared by PCR, using primers shown in Supplementary Table S2. DNA probes were ³²P-labeled, with a random-primer kit (Amersham Biosciences, GE Healthcare), using [α-³²P] dCTP (3000 Ci/mmol). Hybridization signals were quantified with a Cyclone Plus storage phosphor scanner (PerkinElmer, Inc., Waltham, MA, USA). Each experiment was replicated at least twice.