Zn-, Cd-, and Pb-transcription factor IIIA: properties, DNA binding, and comparison with TFIIIA-finger 3 metal complexes.
Properties of the metal ion binding sites of Zn-transcription factor IIIA (TFIIIA) were investigated to understand the potential of this type of zinc finger to undergo reactions that remove Zn(2+) from the protein. Zn-TFIIIA was purified from E. coli containing the cloned sequence for Xenopus laevis oocyte TFIIIA and its stoichiometry of bound Zn(2+) was shown to depend on the details of the isolation process. The average dissociation constant of Zn(2+) in Zn-TFIIIIA was 10(-7). The dissociation constant for Zn-F3, the third finger from the N-terminus of TFIIIA, was 1.0 x 10(-8). The reactivity of Zn-TFIIIA with a series of metal binding ligands, including 2-carboxy-2'-hydroxy-5'-sulfoformazylbenzene (zincon), 4-(2-pyridylazo)-resorcinol (PAR), and 3-ethoxy-2-oxo-butyraldehyde-bis-(N(4)-dimethylthiosemicarbazone) (H(2)KTSM(2)) revealed similar kinetics. The reactivity of PAR with Zn-TFIIIA declined substantially when the protein was bound to the internal control region (ICR) of the 5S ribosomal DNA. Both Cd(2+) and Pb(2+) disrupt TFIIIA binding to its cognate DNA sequence. The Pb(2+) dissociation constant of Pb-F3 was measured as 2.5 x 10(-8). According to NMR spectroscopy, F3 does not fold into a regular conformation in the presence of Pb(2+).
Huang
Meilin
M
Department of Chemistry, University of Wisconsin-Milwaukee, 3210 N. Cramer Street, P.O. Box 413, Milwaukee, WI 53201-0413, USA.
Krepkiy
Dmitriy
D
Hu
Weining
W
Petering
David H
DH
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry; D005260:Female; D066298:In Vitro Techniques; D007700:Kinetics; D007854:Lead / Q000737:chemistry; D008024:Ligands; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D035582:Transcription Factor TFIIIA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis; D015032:Zinc / Q000737:chemistry; D016335:Zinc Fingers / Q000235:genetics
D008024:Ligands; D008670:Metals; D011994:Recombinant Proteins; D035582:Transcription Factor TFIIIA; D029867:Xenopus Proteins; D002104:Cadmium; D007854:Lead; D015032:Zinc