Biochemical analysis of an RNase T stall sequence in the 23S rRNA 3′ trailer. (A) Digestion of single-stranded regions by 3′ to 5′ exonucleases. A labeled RNA substrate containing both duplex and single-stranded regions, with the longer strand labeled at its 5′ end, is shown at the left. This substrate was treated with 100 ng of purified RNase T, RNase PH, RNase II, or PNPase. The RNase-treated reactions (lanes 3–6) and a control untreated reaction (lane 2) were electrophoresed on a denaturing gel, along with a labeled RNA oligonucleotide that corresponds to the product derived from the digestion of all single-stranded residues in this complex (lane 1). An RNA ladder, generated by alkaline hydrolysis of the labeled single-stranded RNA, was also included (lane 7). The positions of the undigested RNA and of the product derived from the digestion of all single-stranded residues are indicated by dotted lines. (B) Digestion of a 23S substrate by 3′ to 5′ exonucleases. An RNA substrate, RNA23S (shown on the right), which corresponds to the unprocessed 3′ end of 23S rRNA, was radiolabeled at its 5′ end and treated with 100 ng of PNPase, RNase T, RNase PH, or RNase II, followed by the fractionation of the treated or untreated reactions on a denaturing gel. The positions of the undigested substrate or a product generated by removal of one 3′ nucleotide are indicated by thick or thin arrows, respectively. Products generated by stalling of RNase T at an internal CC dinucleotide are indicated by brackets, and limit digestion products are indicated by an asterisk. (C) Effect of mutation of the CC dinucleotide in the 23S rRNA leader sequence. Oligonucleotides RNA23S or RNA23Smut (5′-GCUUAACCUUACAACGUUG-3′), a RNA23S variant that has the 3′ CC dinucleotide of RNA23S replaced with U residues (underlined), were treated with the indicated amounts of RNase T and fractionated on a denaturing gel. The migration of the undigested substrates is indicated by an arrow. (D) Digestion of ribosomal particles with RNase T. 50S large ribosomal subunit particles were isolated from a Δrnt strain and treated with 1 μg of RNase T. RNA was extracted from RNase T–treated or –untreated particles and analyzed by Northern blot, as described in Figure 1. The positions of the mature end and the +8 precursor are indicated.
