FIG. 2.

Structure probing of the MHV PS. (A) Secondary-structure model of the MHV PS. Nucleotides are numbered from the 5′ end of the first cytosine in the PS. Bulges, the internal loop, and the pentaloop are indicated. Additional base pairs in the internal loop are indicated by a dotted line. (B to D) Enzymatic and chemical probing. Susceptible nucleotides were identified by a reference sequencing ladder. For probing reactions, 0.1-μg aliquots of RNA transcripts were treated with nuclease-free water (N), 0.0001 U of RNase T₁ (T₁), 0.0001 U of RNase V₁ (V₁), 4 U of RNase S₁ (S₁), 10 pg of RNase A (Ra), 0.01% (vol/vol) DEPC (P), and 0.05% (vol/vol) DMS (D) for 20 min at room temperature in a total volume of 50 μl. DMS and DEPC probing shown in Fig. 2D were done on ice. The incubation buffer contained 10 mM Tris, 100 mM KCl, 10 mM MgCl₂, and, optionally, 10 mM of zinc acetate (indicated by the asterisk above these lanes), pH 6.0. Primer extension reactions were carried out with 0.01 μg of treated transcripts, 0.5 μl of a 0.1 mM concentration of the MHV1 primer, 1 μl of 5 mM dGAT, 1 μl of 25 μM dCTP, 0.1 μl of α-³²P-labeled dCTP (10 mCi/ml), 1 μl of 5× reverse transcriptase buffer (Promega), and 20 U of Moloney murine leukemia virus reverse transcriptase (Promega).