CCA-containing precursor tRNA (pre-tRNAs) are processed exonucleolytically (Schurer et al., 2001). In cyanobacteria, the processing of CCA-containing pre-tRNAs has not been characterized. All tRNA precursors are processed at the 5′ side by RNase P. We have studied the expression and processing of the tRNAs encoded in the delta plasmid of Anabaena 7120, and we have determined that they are correctly processed and aminoacylated. During the study of the tRNA cluster, we have identified a variant tRNASerGCU that was not LY2157299 purchase annotated in the database. A structural analysis of this tRNA shows that it presents a tRNA-like structure, with a serine GCU codon, and other determinants of a

tRNASer. We demonstrate

that this newly identified tRNA is aminoacylated in vitro and in vivo. Anabaena 7120 (Rippka et al., 1979) was grown photoautotrophically p38 MAPK assay at 30 °C under white light (65–100 μE m−2 s−1). Medium used for growth on plates was BG11 (NaNO3 as the nitrogen source) or BG110 (N2 as the nitrogen source; Rippka et al., 1979). Liquid cultures were grown in the same media supplemented with 10 mM NaHCO3 and bubbled with 1% CO2-enriched air. Cells from cultures grown to 5 μg chlorophyll mL−1 were collected by filtration (filter type: 0.45 μm HA; Millipore HAWP05000) and washed in RNase-free TE buffer [10 mM Tris–HCl (pH 7.5), 1 mM EDTA]. Pelleted cells were reduced to dust after freezing in liquid nitrogen and resuspended in a buffer containing 50 mM HEPES-KOH (pH 7.5), 10 mM MgCl2, 5 mM CaCl2 and 20% glycerol, and the samples were centrifuged Nabilone at 2500 g for 10 min at 4 °C. Protein was quantified by Lowry’s method (Lowry et al., 1951). Cells pellets prepared as described above were resuspended in 100 μL of a lysozyme solution (3 mg mL−1) and subjected to three freeze–thaw cycles to facilitate cell lysis. RNA was isolated with 1 mL of Trizol reagent (Invitrogen), using manufacturer instructions. RNA was

extracted with phenol and with chloroform/isoamyl alcohol (24 : 1), precipitated with absolute ethanol and washed with 70% ethanol. Finally, RNA was resuspended in 30 μL of RNase-free water. To isolate RNA under acidic conditions, we used the method described by Varshney et al. (1991). Briefly, cells from a 25-mL culture were collected by filtration and resuspended in 300 μL of 0.3 M sodium acetate (pH 4.5) and 10 mM EDTA and subjected to two extractions with phenol equilibrated with the same buffer. The aqueous phase was then precipitated with absolute ethanol and resuspended in 60 μL of 0.3 M sodium acetate (pH 4.5) and 1 mM EDTA. The RNA was again precipitated with absolute ethanol and resuspended in the same buffer. A total of 10 μg of total RNA was treated with 2 units of RQ1 DNase (Promega), in 20 μL, for 1 h at 37 °C. Reaction was stopped with 2 μL of the stop buffer provided and heated for 10 min at 70 °C.