A mutation outside the two zinc fingers of ADR1 can suppress defects in either finger.

Abstract

A second-site mutation that restored DNA binding to ADR1 mutants altered at different positions in the two zinc fingers was identified. This mutation (called IS1) was a conservative change of arginine 91 to lysine in a region amino terminal to the two zinc fingers and known from previous experiments to be necessary for DNA binding. IS1 increased binding to the UAS1 sequence two-to sevenfold for various ADR1 mutants and twofold for wild-type ADR1. The change of arginine 91 to glycine decreased binding twofold, suggesting that this arginine is involved in DNA binding in the wild-type protein. The increase in binding by IS1 did not involve protein-protein interactions between the two ADR1 monomers, nor did it require the presence of the sequences flanking UAS1. However, the effect of IS1 was influenced by the sequence of the first finger, suggesting that interactions between the region amino terminal to the fingers and the fingers themselves could exist. A model for the role of the amino-terminal region based on these results and sequence homologies with other DNA-binding motifs is proposed.Recognition of specific DNA sequences is a critical step in many events of cell life. The combination of genetic analysis and structural studies has allowed the identification and characterization of a large number of DNA-binding domains responsible for recognizing and binding specific DNA sequences (45). These domains generally behave as independent units which can be swapped between different proteins. For each unit, the binding often occurs between an alphahelical structure and the major groove of DNA. The specificity of binding is achieved primarily by the formation of hydrogen bonds between the amino acids and the bases. Interactions with atoms of the sugar phosphate backbone are also important in positioning the domains to allow them to align correctly with the bases. Several structures that have DNA-binding properties have now been identified. Among them are the helix-turn-helix motifs found in prokaryotic proteins, such as the lambda repressor (43), and in eukaryotic homeodomains (27,40); the three classes of zinc fingers (3); the leucine zipper, which is characterized by its dimerization motif (36); and the beta-ribbon motif found in some prokaryotic proteins (48). Structural data are available for each of these classes of . In addition to these highly conserved motifs, an additional element, adjacent to the motif, can contribute to binding by making direct base pair contacts, as in the lambda repressor and in the homeodomains (8, 56). These adjacent regions are more difficult to identify, since they generally contribute less to the binding than the conserved motif and do not have a well-defined secondary structure in either the absence or the presence of DNA (8,37,40,56).In this report, we describe a mutation outside the zinc finger region of ADR1 which enhances the DNA-binding activity of the protein. ADR1 ADH2 promoter, the UAS1 sequence. Even though single molecules of ADR1 bind independently to each half of UAS1,...

Determining the Requirements for Cooperative DNA Binding by Swi5p and Pho2p (Grf10p/Bas2p) at the HO Promoter

“…This additional strand is necessary for complete DNA binding activity of the Swi5p zinc finger protein (26). Similar regions amino-terminal to the zinc fingers of the Drosophila Tramtrak and yeast Adr1p proteins have also been shown to be essential for their complete DNA binding activities (18,46,47). One interesting hypothesis is that Pho2p interacts with Swi5p through this ␤-strand.…”

DNA-Binding Specificity of NGFI-A and Related Zinc Finger Transcription Factors

“…8A). Interestingly, the DNA-binding domains of ADR1 and Ttk extend beyond the amino-terminal limit of their first zinc fingers, and both of these regions contain residues that modulate affinity (5,19). This may be true for some but not all members of the NGFI-A family of proteins as well.…”

Identification of potential target genes for Adr1p through characterization of essential nucleotides in UAS1.

“…It has previously been shown that amino acid residues amino terminal to the zinc fingers of Adrlp influence DNA binding (10,72). Therefore, it is possible that the contacts on the C-rich strand of the binding site could be due to nonfinger contacts.…”

“…Adrlp contains two zinc fingers and a region amino terminal to the fingers, which together are essential for DNA binding (6,10), and functions through UAS1, a perfect 22-bp repeat in the ADH2 promoter (21,61). Each half of the inverted repeat is an independent, functional binding site for one monomer of Adrlp.…”