Expression of the tRNAs in the delta plasmid was analysed by RT-PCR with a set of primers
designed to generate overlapping fragments encompassing the whole tRNA cluster (Fig. 1a and b). RT-PCR products were detected for all primer pairs used, indicating that the cluster is transcribed as a single RNA. However, no full-length RNA could be detected with primers F7 and R1, suggesting quick processing of the primary transcript. The presence of a single active promoter upstream of the tRNA cluster has been confirmed recently by RNASeq (Mitschke et al., 2011). Individual tRNAs were also detected by Northern blot PARP inhibitor (Fig. 1c). The sizes of the bands were the expected for correctly processed tRNAs. Correct 5′ ends were confirmed by primer extension for tRNASerGCU(2) and tRNAGlnCUG (not shown). In addition to tRNAs, an RNA corresponding to an intergenic region (Int2) was also detected by Northern blot, indicating stable accumulation of this RNA, generated after processing of the flanking tRNAs. To study tRNA processing within the cluster, we prepared three different pre-tRNAs by in vitro transcription
(Fig. 2a). These precursors were incubated with purified RNase Z from Synechocystis (Ceballos-Chávez & Vioque, 2005), which would cleave at the 3′ side of CCA-lacking pre-tRNAs. In the three cases, the expected processing products were detected. No products corresponding to cleavage at the 3′ ends of the CCA-encoding tRNAAsnGUU(3) and tRNAGlnCUG by RNase Z were observed (Fig. 2b), confirming the previously described inhibition of cyanobacterial RNase Z activity by the presence of CCA at the 3′-end of tRNAs (Ceballos-Chávez & Vioque, 2005). Selleck BAY 80-6946 The pre-tRNAs were also incubated with Glycogen branching enzyme the RNA subunit of the Anabaena 7120 RNase P in
a high-salt buffer, reaction conditions appropriate for catalytic activity of the RNase P RNA in the absence of the protein cofactor (Pascual & Vioque, 1999), as well as with the complete RNase P holoenzyme in low-salt buffer. In both cases, the expected products were detected for all three pre-tRNAs (Fig. 2c). These results indicate that there is no specific cleavage order for RNase P and RNase Z, because both RNases can generate the expected final products. The results described previously indicate that the tRNAs encoded in the cluster are expressed and processed to mature tRNAs. We next analysed whether they were aminoacylated in vivo. For this purpose, we used the OXOPAP method (Gaston et al., 2008). We could detect aminoacylation for most tRNAs encoded in the cluster (Fig. 3), including several classified as pseudogenes by tRNAscan-SE: tRNASerGCU(2), and tRNAArgUCU(2). Also, tRNAs whose genes contain the CCA sequence were aminoacylated [tRNAGlnCUG, tRNALeuCAA(2), tRNALysUUU(2), , tRNAValUAC(2)]. This confirms that CCA-containing pre-tRNAs are processed correctly at the 3′ side in vivo to generate mature functional tRNAs, despite the inability of RNase Z to carry out the reaction in our in vitro assay.