Interaction of wild-type and truncated forms of transcription factor IIIA from Saccharomyces cerevisiae with the 5 S RNA gene.

Transcription factor (TF) IIIA, which contains nine zinc finger motifs, binds to the internal control region of the 5S RNA gene as the first step in the assembly of a multifactor complex that promotes accurate initiation of transcription by RNA polymerase III. We have monitored the interaction of wild-type and truncated forms of yeast TFIIIA with the 5 S RNA gene. The DNase I footprints obtained with full-length TFIIIA and a polypeptide containing the amino-terminal five zinc fingers (TF5) were indistinguishable, extending from nucleotides +64 to +99 of the 5 S RNA gene. This suggests that fingers 6 through 9 of yeast TFIIIA are not in tight association with DNA. The DNase I footprint obtained with a polypeptide containing the amino-terminal four zinc fingers (TF4) was 14 base pairs shorter than that of TF5, extending from nucleotides +78 to +99 on the nontranscribed strand and from nucleotides +79 to +98 on the transcribed strand of the 5 S RNA gene. Protection provided by a polypeptide containing the first three zinc fingers (TF3) was similar to that provided by TF4, with the exception that protection on the nontranscribed strand ended at nucleotide +80, rather than nucleotide +78. Methylation protection analysis indicated that finger 5 makes major groove contacts with guanines +73 and +74. The amino-terminal four zinc fingers make contacts that span the internal control region, which extends from nucleotides +81 to +94 of the 5 S RNA gene, with finger 4 appearing to contact guanine +82. Measurements of the apparent Kd values of the TFIIIA.DNA complexes indicated that the amino-terminal three zinc fingers of TFIIIA have a binding energy that is similar to that of the full-length protein.