Zinc finger structure of a ribosomal gene-specific transcription factor

Xenopus transcription factor IIIA (TFIIIA) or TFIIIA mutants with internal deletions were expressed in E.coli, isolated from E.coli cell extracts, and identified by SDS PAGE and immunoblotting with rabbit antiserum against native TFIIIA. Specific DNA binding of intact or internally deleted TFIIIA was compared by analyzing their abilities to protect the internal control region (ICR) of the Xenopus 5S ribosomal RNA gene from DNase I digestion. Intact protein bound specifically to the entire ICR (+96 to +43). One TFIIIA deletion mutant, expressed from cDNA lacking the coding sequence for the putative fourth zinc finger protected the ICR from DNase I digestion from nucleotide positions +96 to +78. A second TFIIIA mutant resulting from fusion of putative zinc fingers 7 and 8 protected the 5S gene ICR from positions +96 to +63. The regions of the protein comprising the N-terminal 3 fingers and N-terminal six fingers appear to be in contact with approximately 18 and 33 bp of DNA respectively on the 3' side of the 5S gene ICR.