Relative contributions of the zinc fingers of transcription factor IIIA to the energetics of DNA binding.
We have expressed and purified a series of recombinant zinc finger polypeptides derived from the cDNA for the Xenopus 5 S gene-specific transcription factor TFIIIA. Dissociation constants for the interaction of each of the truncated polypeptides with the 5 S gene promoter have been measured using gel mobility shift assays. DNase I footprinting and proteolysis experiments provide additional insights into the interactions of individual fingers within complexes of the truncated proteins. These results are discussed in terms of recently proposed models for the TFIIIA-DNA interaction. The effects of mutations in two of the strongly binding proteins, zf1-3 and zf1-7, on DNA binding affinity have been investigated. Mutations have been made both in putative DNA-contact residues and in the linker regions between zinc fingers. The observed decreases in binding affinity cannot be explained simply in terms of loss of protein-DNA contacts. Our results support a model in which DNA binding is accomplished through sets of interacting zinc fingers that make different energetic contributions to the overall binding of the protein and different contacts with the DNA.
Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037.
K R
KR
Clemens
P
P
Zhang
X
X
Liao
S J
SJ
McBryant
P E
PE
Wright
J M
JM
Gottesfeld
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004275:DNA, Ribosomal / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012341:RNA, Ribosomal, 5S / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D013816:Thermodynamics; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus / Q000235:genetics; D016335:Zinc Fingers / Q000235:genetics
D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D012341:RNA, Ribosomal, 5S; D011994:Recombinant Proteins; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors