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. 2014 Nov;20(11):1715–1724. doi: 10.1261/rna.047241.114

FIGURE 3.

FIGURE 3.

Mycobacterium smegmatis YrlA contains a tRNA-like module that can undergo RNase P cleavage and CCA addition. (A) Map of the M. smegmatis rsr locus. (B,C) Lysates from M. smegmatis expressing Flag3-Rsr on a plasmid, or the empty vector, were subjected to immunoprecipitation using anti-Flag. YrlA and YrlB were detected by Northern blotting. In B, the oligonucleotide probes were complementary to sequences in the 3′ (lanes 1–5) or 5′ half of the RNA (lanes 6–9). (D,E) Predicted secondary structures of YrlA and YrlB. The structure of the YrlA “acceptor stem” after cleavage and CCA addition is indicated by the arrow in D. Conserved nucleotides within the GNCGAAN0-1G motif and the tRNA-like domain are colored red as in Figure 1. (F) 5′-labeled M. smegmatis YrlA was incubated with RNase P RNA under conditions that support cleavage. At the indicated times, aliquots were removed and fractionated in denaturing gels (lanes 3–7). (Arrow) Cleavage product. Lanes 1 and 2, alkaline hydrolysis and T1 ribonuclease ladders.