SOLUTION STRUCTURE OF THE FIRST 3 ZINC FINGERS OF TFIIIA BOUND TO THECOGNATE DNA-SEQUENCE - DETERMINANTS OF AFFINITY AND SEQUENCE SPECIFICITY

The high resolution solution structure of a protein containing the thr ee amino-terminal zinc fingers of Xenopus laevis transcription factor IIIA (TFIIIA) bound to its cognate DNA duplex was determined by nuclea r magnetic resonance spectroscopy. The protein, which is designated zf 1-3, binds with all three fingers in the DNA major groove, with a numb er of amino acids making base-specific contacts. The DNA structure is close to B-form. Although the mode of interaction of ef1-3 with DNA is similar to that of zif268 and other structurally characterized zinc f inger complexes, the TFIIIA complex exhibits several novel features. E ach zinc finger contacts four to five base-pairs and the repertoire of known base contact residues is extended to include a tryptophan at po sition +2 of the helix (finger 1) and arginine at position +10 (finger 3). Sequence-specific base contacts are made over virtually the entir e length of the finger 3 helix. Lysine and histidine side-chains invol ved in base recognition are dynamically disordered in the solution str ucture; in the case of lysine, in particular, this could significantly decrease the entropic cost of DNA binding. The TGEKP(N) linker sequen ces, which are highly flexible in the unbound protein, adopt ordered c onformations on DNA binding. The linkers appear to play an active stru ctural role in stabilization of the protein-DNA complex. Substantial p rotein-protein contact surfaces are formed between adjacent fingers. A s a consequence of these protein-protein interactions, the orientation of finger 1 in the major groove differs from that of the other finger s. Contributions to high affinity binding by zf1-3 come from both dire ct protein-DNA contacts and from indirect protein-protein interactions associated with structural organization of the linkers and formation of well-packed interfaces between adjacent zinc fingers in the DNA com plex. The structures provide a molecular level explanation for the lar ge body of footprinting and mutagenesis data available for the TFIIIA- DNA complex. (C) 1997 Academic Press Limited.