Structure of the TFIIIA-5 S DNA complex.

The missing-nucleoside experiment, a recently developed approach for determining the positions along a DNA molecule that make energetically important contacts with protein, has been used to investigate the structure of the complex of transcription factor IIIA with a somatic 5 S RNA gene from Xenopus borealis. We detect three distinct regions of the 5 S promoter that are contacted by TFIIIA, corresponding to the A-box, intermediate element and C-box regions previously identified by mutagenesis experiments. The advantage of the missing-nucleoside experiment over mutagenesis is that additional information, directly related to the structure of the complex, is obtained. Of most importance is that contacts to each strand of DNA are determined independently, and can be assigned unambiguously as interactions with TFIIIA. Throughout the binding site the strongest contacts are made with the non-coding strand of the 5 S gene. The two groups of contacts at either end of the binding site (boxes A and C) are comprised of sets of approximately ten contiguous nucleosides for which the contacts are reflected, without stagger, from one strand to the other. In contrast, contacts in the center of the promoter (the intermediate element) are staggered about five base-pairs in the 5' direction with respect to each strand. These results, when analyzed in conjunction with the hydroxyl-radical footprint of the complex, support a model in which TFIIIA wraps around the DNA in the major groove of the helix for one turn at the two ends of the complex in boxes A and C, and lies on one side of the DNA helix in the center of the complex at the intermediate element.