FIG. 5.

Quantification of RNA-protein interactions. (A) Representative gel mobility shift assay with increasing amounts of the hLa protein and a constant amount of 3′ UTR PAM mRNA. Lane 1, free probe; lanes 2 to 11, probe plus His-hLa in the amounts indicated; M, multimer of the probe; fp, free probe. (B) The percent ³²P-PAM 1 RNA bound was plotted against His-hLa concentration to generate a theoretical saturation binding curve (inset). The stoichiometry of the interaction of hLa with PAM 1 RNA, as determined by the slope of the line in the inset graph, was about 1:1. The dissociation constant was calculated using the following equation: log (percent bound/percent unbound) + 2 = n{log[His-hLa (nM)] + 1} − log K_D. The K_D was estimated to be 155 nM for hLa-PAM 1 RNA. (C) Representative gel mobility shift assay with increasing amount of His-hLa protein and a constant amount of oligo(U). Lane 1, free probe; lanes 2 to 9, probe plus hLa in the amounts indicated. (D) The percent ³²P-oligo(U) bound was plotted against the concentration of His-hLa to generate a theoretical saturation binding curve. The data from the saturation binding curve were transformed (inset). The K_D was estimated to be 38 nM.