Fig. 6. RNase P RNA cleavage of pre-24^otRNA and the 5′-leader ribozyme-catalyzed tRNA aminoacylation in trans. (A) Cleavage of pre-24^otRNA by RNase P RNA. The ³²P-body-labeled pre-24^otRNA was treated with RNase P RNA for 2 h, resulting in the cleavage of 23% of pre-24^otRNA (lane 1). The absence of RNase P RNA yielded no cleaved product (lane 2). The marker RNAs (5′-leader segment in lane 3 and otRNA in lane 4) were prepared by in vitro transcription using the corresponding DNA segments. (B) Trans-aminoacylation of otRNA. The autoradiogram displays the time course of 5′-leader ribozyme-catalyzed aminoacylation of 5′-³²P-labeled otRNA. a, Biotin-Phe-otRNA complexed with SAv; b, otRNA. The RNase P-digested RNA fragments of pre-24^otRNA (2 µM) were used for aminoacylation of 5′-³²P-labeled otRNA (0.5 µM), giving k_obs = 1.0 × 10^–3 min^–1. (C) Trans-aminoacylation of tRNA variants. rtRNA, v1, v3 and otRNA are the fragment of the tRNA domain described in Figures 1B and 5A. a, Biotin-Phe-otRNA or tRNA variants complexed with SAv; b, otRNA or tRNA variants. tRNA variants were prepared by in vitro transcription using the corresponding DNA segments. Controls: lane 5, the absence of SAv; lane 6, otRNA treated with NaIO₄ was used for aminoacylation; lane 7, otRNA lacking its 3′-A was used for aminoacylation. (D) Trans-aminoacylation of a minihelix RNA. The autoradiogram depicts the time course of 5′-leader ribozyme-catalyzed aminoacylation of a minihelix RNA. a, Biotin-Phe-minihelix RNA complexed with SAv; b, minihelix RNA.