Importance of minor groove binding zinc fingers within the transcription factor IIIA-DNA complex.
The gene-specific transcription factor IIIA (TFIIIA) binds to the internal promoter element of the 5 S rRNA gene through nine zinc fingers which make specific DNA contacts. Seven of the nine TFIIIA zinc fingers participate in major groove DNA contacts while two fingers, 4 and 6, have been proposed to bind in or across the minor groove. Pyrrole-imidazole polyamides are minor groove binding ligands that recognize predetermined DNA sequences with affinity and specificity comparable to natural DNA-binding proteins. We have examined the DNA binding activity of nine finger TFIIIA and shorter recombinant analogs in the presence of polyamides that bind six base-pair sequences (Kd = 0.03 to 1.7 nM) in the minor groove of the binding site for zinc finger 4. DNase I footprint titrations demonstrate that the polyamides and a recombinant protein containing the three amino-terminal zinc fingers of TFIIIA (zf1-3) co-occupy the TFIIIA binding site, in agreement with the known location of zf1-3 in the major groove. In contrast, the polyamides block the specific interaction of TFIIIA or zf1-4 with the 5 S RNA gene, supporting a model for minor groove occupancy by zinc finger 4. Minor groove binding polyamides targeted to specific DNA sequences may provide a novel chemical approach to probing multidomain protein-DNA interactions.
Neely
L
L
Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037, USA.
Trauger
J W
JW
Baird
E E
EE
Dervan
P B
PB
Gottesfeld
J M
JM
D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D006147:Guanine / Q000737:chemistry / Q000378:metabolism; D009757:Nylons / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D009757:Nylons; D011994:Recombinant Proteins; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D006147:Guanine; D004247:DNA; D003850:Deoxyribonuclease I