Fig. 4.

The var PEs form a specific DNA–protein complex. (A) EMSA of extracts using a radiolabeled (³²P) DNA ligand containing the PE of the var 5′UTR (Ups1) shows specific DNA–protein complex formation when incubated with a nuclear extract. Specific competition assays were performed with increasing concentrations of unlabeled DNA ligands (2×, 10×, and 25× of the labeled ligand, respectively) containing the PEs found in a var 5′UTR (Ups1), in a var intron (Int1), and in the PbDT-3′UTR (PbDT-3′). The DNA sequence from the hrp2-3′ UTR was used as nonspecific competitor (hrp2-3′). Blank, no protein was added; CE, cytoplasmic extract; NE, nuclear extract. (B) Phosphorimaging quantification of the EMSA data presented in A. The percentage of complex formation measured is presented relative to the value measured in the absence of competitive ligand, which was considered to be 100%. (C) Competition EMSA of radiolabeled Ups1 ligand with various mutated nonlabeled Ups1 ligands. The various mutated ligands are named “mut1-3,” “mut3-5,” and so forth, relative to the replacement of the original PE sequence with cytosine bases. The PE sequence is marked in bold. Base-pair exchange is underlined in the PE and is marked in gray in the flanking regions. Blank represents the original sequence of the PE and its flanking regions. (D) Phosphorimaging quantification of the EMSA data presented in C was performed, and the relative DNA-binding percentage was calculated as in B. (E) Schematic description of the importance of each nucleotide position in the PE for formation of the DNA–protein complex. Nucleotides shown in larger type have a great influence on binding.