Effect of the 5′ sequence on the heterogeneity of SLI RNAs transcribed as SLI-ARiBo precursors from the consensus T7 class III promoter. (A) Sequence of Stem Ia and numbering of SLI RNAs with different 5′ (and 3′) sequences. (B) Small-scale affinity batch purifications of each of the 16 SLI RNAs transcribed as SLI-ARiBo precursors from the T7 class III promoter using the wild-type T7 RNAP and analyzed on a 20% denaturing polyacrylamide gel stained with SYBR Gold. Only the E1 elution fractions are shown, and from the 400-μL elution volumes, 1.2-μL aliquots were loaded on the gel. (C,D) Small-scale affinity batch purifications of each of the 16 SLI RNAs transcribed as SLI-ARiBo RNAs from the T7 class III promoter using the wild-type (C) or P266L mutant (D) T7 RNAP. These E1 elution fractions were treated with calf alkaline phosphatase to remove phosphate heterogeneity at the 5′ end (in C and D only). From the 50-μL phosphatase reaction mixture, 6.3-μL aliquots were analyzed on 20% denaturing polyacrylamide gels stained with SYBR Gold. In B–D, gel lanes match the SLI numbering given in A.
