FIG. 2.
Enzyme structure probing of the mRNA 5 IS region. (A) Predicted secondary structure of the mRNA 5 IS region by the Zuker algorithm and a summary of the single-stranded (ss) and double-stranded (ds) regions as determined by enzyme probing. The canonical upstream IS (site 1) and noncanonical downstream sequence (site 2) are shown in bold type. (B) Pattern of end-labeled extended primer after separation on a DNA sequencing gel. The 207-nt-long T7 RNA polymerase-generated transcript containing 193 nt of the 199-nt-long mRNA 5 IS region was treated with RNases as indicated, and a 5′ end-labeled oligonucleotide binding at the 3′ end of the transcript was used for primer extension. Lanes: 1 through 5, CV1 digestion with 0.0001, 0.001, 0.01, 0.1, and 0.5 U/ml, respectively; 6, undigested RNA; 7 through 9, RNase T2 digestion with 0.5, 0.1, and 1.0 U/ml, respectively; 10 through 13, sequencing ladder generated from the same transcript using the same primer. Base positions noted are those for the pDrepIS12.7 DI RNA. Positions of the IS1 and IS2 motifs are noted, as are the deduced stem-loop structures.