Domain IIb mutants interact with the 40S subunit properly. (A) RNase T1 footprinting gel with G82 cleavage indicated by the arrowhead. Lanes with and without bound 40S subunit are shown. A RNase T1 ladder made under denaturing conditions and a hydrolysis ladder are marked as T1 and OH, respectively. BKG lane is a no-RNase T1 control. (B) Quantified and normalized G82 bands from the gel in A show that the G82 of the ΔapexC mutant RNA is protected to the same degree as is WT. (C) Quantitated, normalized, and background-corrected modification data from two independent SHAPE probing experiments of the WT and dIIb mutant IRESs in the 40S subunit-bound form with an additional repeat (data not shown), validating the quantitated data shown here. Error bars represent one standard deviation from the mean of both independent experiments. The degree of modification is on the y-axis; each nucleotide is on the x-axis. Red bars indicate WT IRES; bars in different shades of blue indicate the dIIb mutant IRESs. The part of the gel corresponding to the tip of domain IIb (dIIb) is indicated.
