Differing roles for zinc fingers in DNA recognition: Structure of a six-finger transcription factor IIIA complex

Abstract

The crystal structure of the six NH 2 -terminal zinc fingers of Xenopus laevis transcription factor IIIA (TFIIIA) bound with 31 bp of the 5S rRNA gene promoter has been determined at 3.1 Å resolution. Individual zinc fingers are positioned differently in the major groove and across the minor groove of DNA to span the entire length of the duplex. These results show how TFIIIA can recognize several separated DNA sequences by using fewer fingers than necessary for continuous winding in the major groove.TFIIIA is an essential component of the RNA polymerase III (Pol III) transcription initiation complex for 5S rRNA in Xenopus laevis oocytes (1-3). TFIIIA also participates in the nuclear export (4) and storage of 5S rRNA, with which it forms a stable cytoplasmic 7S particle (5). The DNA-binding site for a single TFIIIA protein extends over 55 bp of the 5S rRNA gene promoter (6, 7). This site lies within the 5S rRNA coding sequence itself. It is effectively a tripartite promoter (8) containing separated ''box A,'' ''intermediate element'' (IE), and ''box C'' sequences ( Fig. 1 A). Similar regulatory elements exist in tRNA gene promoters. Mapping the details of this extensive protein-DNA interaction using chemical, biochemical, and genetic techniques has continued for almost 20 years (1-3, 9, 10). The discovery of nine zinc fingers in TFIIIA (11,12) led to the notion of a transcription factor with repeated modules in its DNA-binding domain (Fig. 1B).Our present knowledge of how zinc fingers bind specifically to DNA comes largely from several x-ray structures (13)(14)(15)(16)(17)(18). In all of these protein-DNA complexes, there are contiguous zinc finger interactions with base pairs in the major groove. In Zif268, for example, three fingers recognize successive, overlapping base pair quartets in the major groove, covering a total of 10 bp. In the DNA complex of a five-finger segment from Gli, the first finger lies outside the major groove and makes no DNA contacts. The remaining fingers wrap in the major groove rather like those of Zif268. An extension of this mode of binding is not sufficient to explain the size of the TFIIIA-binding site, however (see, for example, models proposed in refs. 9 and 10). The NH 2 -terminal six zinc fingers of TFIIIA bound in a complex with 31 bp of the 5S rRNA gene has been reconstituted (19) and crystallized. We report here the structure of the complex at 3.1 Å resolution (Table 1) and describe how its zinc fingers interact with DNA in different ways. An NMR structure of fingers 1-2-3 bound to DNA has recently been published (20, 21). MATERIALS AND METHODSProtein and DNA Oligonucleotides. Recombinant TFIIIA (amino acid residues 1-190) was produced from plasmid pRSET B (Invitrogen) in Escherichia coli BL21(DE3). After sonication the protein was extracted in 7 M urea from cell pellets and purified on Bio-Rex 70 (Bio-Rad) and heparin Sepharose columns. Synthetic oligonucleotides were purified by MonoQ (Pharmacia) chromatography in 7 M urea. Thymines were replaced by 5-iododeoxyurac...

Interprotein metal exchange between transcription factor IIIa and apo-metallothionein

“…Examination of the local molecular environment of the Zn 2+ -binding sites in the threedimensional structures of parts of Zn-TFIIIA bound to cognate portions of the ICR shows that substantial steric hindrance to access to bound Zn 2+ is imposed by DNA [9,12]. Electrostatic repulsion between negatively charged EDTA and the protein-DNA complex which also bears an overall negative charge might contribute to the lack of reactivity of EDTA.…”

“…Zinc-binding is required to achieve stable, folded fingers and specific protein-DNA-binding [1,3,[12][13][14][15][16]. Three-dimensional structures of the first three and six Zn fingers of TFIIIA associated with their DNA-binding sites have been published, showing helical folding of finger domains 1-3 around the major groove of its DNA-binding site, and stretching of fingers 4-6 along the length of the DNA oligomer [9,12].…”

“…All are characterized by a common domain conformation that includes two lengths of antiparallel β structure and an α helix, Zn 2+ bound to a set of two cysteinyl sulfhydryl and histidinyl imidazole nitrogen ligands (C 2 H 2 ), and a mini-hydrophobic core of three non-polar side chains [2][3][4][5][6][7][8][9][10][11][12]. Zinc-binding is required to achieve stable, folded fingers and specific protein-DNA-binding [1,3,[12][13][14][15][16]. Three-dimensional structures of the first three and six Zn fingers of TFIIIA associated with their DNA-binding sites have been published, showing helical folding of finger domains 1-3 around the major groove of its DNA-binding site, and stretching of fingers 4-6 along the length of the DNA oligomer [9,12].…”

The Protein-Binding Potential of C2H2 Zinc Finger Domains

“…Originally crystallized by Pavletich and Pabo (1991), there are now almost 20 structures of Zif268, and its variants, bound to their target DNA sequences (Berman et al, 2000). Non-Canonical (Omichinski et al, 1997) GLI F2 6 Non-Canonical 2GLI (Pavletich and Pabo, 1993) F4 1, 2, 3, 6 Non-Canonical F5 -1, 2, 3, 5, 6 Non-Canonical TFIIIA F1 -1, 2 Non-Canonical 1TF3, 1TF6 (Nolte et al, 1998;Wuttke et al, 1997) F2 2, 3, 6 Non -Canonical F3 2, 3, 6, 10 Canonical F5 -1, 2, 3, 6 Non-Canonical TTK F1 2, 3, 6 Non-Canonical 2DRP (Fairall et al, 1993) F2 -1, 2, 3 Canonical YY1 F1 6 Non-Canonical 1UBD (Houbaviy et al, 1996) F2 -1, 2, 3, 6 Non -Canonical 1UBD F3 -1, 2, 3 Canonical F4 -1, 2, 3 Non-Canonical WT1 F2 -1, 6 Non-Canonical (Stoll et al, 2007) F3 -1, 2, 3 Non-Canonical F4 -1, 6 Non-Canonical Zif268 F1 -1, 2, 6 Canonical 1AAY (Pavletich and Pabo, 1991) F2 -1, 2, 3 Canonical F3 -1, 2, 6 Canonical As shown in figure 1.3, Zif268's C2H2 domains interact with DNA by inserting the N-terminal portion of the α-helix into the DNA's major groove (Pavletich and Pabo, 1991). Key residues include the arginine immediately preceding the start of the α-helix, henceforth referred to as position -1, as well as the glutamic acid and the arginine in positions 3 and 6, respectively, of the α-helix (Figure 1.3).…”

Zn-, Cd-, and Pb-transcription factor IIIA: properties, DNA binding, and comparison with TFIIIA-finger 3 metal complexes

“…Thus, in this and another paper, the behavior of the third finger (F3) of TFIIIA was also examined [31]. F3 is centrally involved in the binding interactions between Zn-TFIIIA and the ICR and, thus, may be thought of as a representative Zn-finger domain for comparison with the ensemble properties seen in the holoprotein [32,33].…”