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 5 S RNA gene as the firs t 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 TFI IIA 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 TFI IIA are not in tight association with DNA. The DNase I footprint obtai ned with a polypeptide containing the amino-terminal four zinc fingers (TF4) was 14 base pairs shorter than that of TF5, extending from nucl eotides +78 to +99 on the nontranscribed strand and from nucleotides 79 to +98 on the transcribed strand of the 5 S RNA gene. Protection pr ovided by a polypeptide containing the first three zinc fingers (TF3) was similar to that provided by TF4, with the exception that protectio n on the nontranscribed strand ended at nucleotide +80, rather than nu cleotide +78. Methylation protection analysis indicated that finger 5 makes major groove contacts with guanines +73 and +74. The amino-termi nal four zinc fingers make contacts that span the internal control reg ion, which extends from nucleotides +81 to +94 of the 5 S RNA gene, wi th finger 4 appearing to contact guanine +82. Measurements of the appa rent K-d values of the TFIIIA DNA complexes indicated that the amino-t erminal three zinc fingers of TFIIIA have a binding energy that is sim ilar to that of the full-length protein.