Effects of lengthening helix 45 by two base pairs on methylation by KsgA. (A) A mutant composed of the addition of two base pairs in helix 45 was constructed and tested for substrate activity. (B) Sucrose gradient analysis for the add2bp mutant. Material corresponding to the 30S peak, indicated by arrows, was used for the activity assay. (C) KsgA time course activity assay of 30STag,UM (red) and mutant 30STag,UM[add2bp] (green) subunits. (D) Nucleoside analysis of the 2-hr end point reaction product. The top trace is measured absorbance at 254 nm of a collection of nucleoside standards including m6A and m62A, while the bottom trace is measured 3H levels.