Effect of the 5′ sequence on the heterogeneity of SLI RNAs transcribed as SLI-ARiBo precursors from the T7 class II φ2.5 promoter. (A) Sequence of Stem Ia and numbering of SLI RNAs with different 5′ (and 3′) sequences. (B,C) Small-scale affinity batch purifications of each of the 16 SLI RNAs transcribed as SLI-ARiBo RNAs from the T7 class II φ2.5 promoter using the wild-type (B) or P266L mutant (C) T7 RNAP. The E1 elution fractions were treated with calf alkaline phosphatase to remove phosphate heterogeneity at the 5′ end. From the 50-μL phosphatase reaction mixture, 6.3-μL aliquots were analyzed on 20% denaturing polyacrylamide gels stained with SYBR Gold. In B and C, gel lanes match the SLI numbering given in A.
