J. Biol. Chem., Vol. 263, Issue 21, 10272-10277, Jul, 1988

Xenopus transcription factor IIIA binds to the flanking regions of the 5 S RNA gene intragenic control region in a unique and highly ordered state

WT Windsor, TC Lee, TJ Daly and CW Wu
Department of Pharmacological Sciences, State University of New York, Stony Brook 11794.

The interaction of Xenopus transcription factor IIIA (TFIIIA) with the intragenic control region (ICR) of the 5 S RNA gene was studied by footprinting techniques under conditions which elicited a unique DNase I digestion pattern. Although a typical full footprint at the ICR was apparent at a 5 nM TFIIIA concentration, higher concentrations (greater than or equal to 50 nM) resulted in a decrease in the size of the footprint (on the 5' side of the ICR) concomitant with the appearance of an alternating protection pattern (APP) located both in the 3'- and 5'-flanking regions of the ICR with a periodicity of approximately 10 base pairs. This periodicity indicates that TFIIIA binding occurs on only one side of the DNA helix. The minimum size of the highly ordered and apparently cooperative APP effect was determined to be at least 250 base pairs in length and could be abolished through competition with nonspecific DNA. However, binding of TFIIIA to nonspecific DNA alone was not sufficient to generate the APP effect at any of the TFIIIA concentrations studied. Removal of the C-terminal domain of the protein by either tryptic or papain digestion resulted in the abolition of the APP effect at all concentrations studied, indicating the necessity of the protein-protein interactions for this effect. A nucleation site, most likely at or near the ICR, is proposed to exist through which TFIIIA specifically interacts and orients the binding of additional protein molecules in a cooperative and highly ordered manner to the flanking DNA sequences on either side of the ICR. The APP effect near the ICR may play a role in the initiation and stabilization of 5 S RNA gene transcription.
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