FIG. 2.
PAGE analysis of 5′-32P-labeled WT RNAs subjected to limited nuclease digestion in the presence and absence of tRNATrp primer. Bands (arrows) in the marker lanes are WT RNA fragments digested with 5 U of T1 under denaturing conditions of 7 M urea and 60°C. Numbers to the left of each arrow describe the length of each digestion product from the 5′-32P label. To the left of each figure are brackets designating in which RNA secondary structure cleavage occurs. At the top of the figure are brackets designating in which lanes various components were added to the reaction mixture. (A) Synthetic WT RNA (0.7 pmol) with or without 5 pmol of synthetic tRNATrp was digested with increasing amounts of RNase T1. Lanes: 1 and 6, T1 concentration of 0.003 U/μl; 2 and 5, T1 concentration of 0.0009 U/μl; 3 and 4, negative controls where the RNAs were incubated in the absence of T1; 7, alkaline hydrolysis ladder of WT RSV RNA. (B) Synthetic WT RNA (0.7 pmol) with or without 5 pmol of synthetic tRNATrp was digested with increasing amounts of RNase A. Lanes: 1 and 8, RNase A concentration of 3 pg/μl; 2 and 7, RNase A concentration of 1 pg/μl; 3 and 6, RNase A concentration of 0.3 pg/μl; 4 and 5, negative controls where the RNAs were incubated in the absence of RNase A. (C) Synthetic WT RNA (0.7 pmol) with or without 5 pmol of synthetic tRNATrp was digested with increasing amounts of RNase V1. Lanes: 1 and 8, V1 concentration of 5 × 10−6 U/μl; 2 and 7, V1 concentration of 5 × 10−5 U/μl; 3 and 6, V1 concentration of 1 × 10−4 U/μl; 4 and 5, negative controls where no exogenous V1 was added.