RNA synthesis and enzymatic structure probing RNA transcripts were generated by in vitro transcription using T7 RNA polymerase. The purified RNAs of desired length were then dephosphorylated by calf intestine alkaline phosphatase, 5 0 -end labeled with [g-32 P] ATP using T4 polynucleotide kinase, and then separated by a 20% sequencing gel for recovery. All the RNase protection experiments were performed with 50 000 to 70 000 cpm of 5 0 -end labeled RNA in the presence of RNase cleavage buffer (30 mM Tris-HCl, pH 7.5; 3 mM EDTA; and 100 mM LiCl) for each reaction, and 10 mM MgCl 2 were included in the same buffer for RNase V1 experiments. Before the addition of probing enzymes, the RNAs were denatured by heating at 65 C for 5 min and followed by slow cooling to 20 C for structural mapping. Finally, 0.02-0.5 U of RNase T2 (USB) or 0.02-0.5 U of RNase V1 (Amersham Pharmacia) was added to each reaction. The hydrolysis RNA ladders were obtained by incubation of RNA in the hydrolysis buffer at 100 C for 2 min, and parallel RNA sequencing products were obtained by the treatment of unfolded RNA with RNases T1 or A. They were used as markers for the assignment of guanines and pyrimidines, respectively. All reactions were incubated at 20 C for 10 min unless specified. The reactions were terminated by addition of gel loading dye, and the cleavage products were resolved by a 20% denaturing gel and visualized by phosphorimagery.