Regulation of Gene Expression in Eukaryotes:- DNA-Binding Transactivators Have a Modular Structure
DNA-binding transactivators typically have a distinct structural domain for specific DNA binding and one or more additional domains for transcriptional activation or for interaction with other regulatory proteins. Inter action of two regulatory proteins is often mediated by domains containing leucine zippers or helix loop-helix motifs . We consider here three distinct types of structural domains used in activation by DNA-binding transactivators (Fig. 1a): Gal4p, Sp1, and CTF1.
Gal4p contains a zinc fingerlike structure in its DNA-binding domain, near the amino terminus; this domain has six Cys residues that coordinate two Zn2+. The protein functions as a homodimer (with dimerization mediated by interactions between two coiled coils) and binds to UASG, a palindromic DNA sequence about 17 bp long. Gal4p has a separate activation domain with many acidic amino acid residues. Experiments that substitute a variety of different peptide sequences for the acidic activation domain of Gal4p suggest that the acidic nature of this domain is critical to its function, although its precise amino acid sequence can vary considerably. Sp1 (Mr 80,000) is a DNA-binding transactivator for a large number of genes in higher eukaryotes. Its DNA binding site, the GC box (consensus sequence GGGCGG), is usually quite near the TATA box. The DNA-binding domain of the Sp1 protein is near its carboxyl terminus and contains three zinc fingers. Two other domains in Sp1 function in activation, and are no table in that 25% of their amino acid residues are Gln. A wide variety of other activator proteins also have these glutamine-rich domains. CCAAT-binding transcription factor 1 (CTF1) be longs to a family of DNA-binding transactivators that bind a sequence called the CCAAT site (its consensus sequence is TGGN6GCCAA, where N is any nucleotide). The DNA-binding domain of CTF1 contains many basic amino acid residues, and the binding region is probably arranged as an helix. This protein has neither a helix turn-helix nor a zinc finger motif; its DNA-binding mechanism is not yet clear. CTF1 has a proline-rich activation domain, with Pro accounting for more than 20% of the amino acid residues. The discrete activation and DNA-binding domains of regulatory proteins often act completely independently, as has been demonstrated in “domain-swapping” experiments. Genetic engineering techniques (Chapter 9) can join the proline-rich activation domain of CTF1 to the DNA-binding domain of Sp1 to create a protein that, like normal Sp1, binds to GC boxes on the DNA and activates transcription at a nearby promoter (as in Fig. 1b). The DNA-binding domain of Gal4p has similarly been replaced experimentally with the DNA binding domain of the prokaryotic LexA repressor. This chimeric protein neither binds at UASG nor activates the yeast GAL genes (as would normal Gal4p) unless the UASG sequence in the DNA is replaced by the LexA recognition site.
FIGURE 1 DNA-binding transactivators. (a) Typical DNA-binding transactivators such as CTF1, Gal4p, and Sp1 have a DNA-binding domain and an activation domain. The nature of the activation domain is indicated by symbols: acidic; Q Q Q, glutamine-rich; P PP, proline-rich. Some or all of these proteins may activate tran scription by interacting with intermediary complexes such as TFIID or mediator. Note that the binding sites illustrated here are not generally found together near a single gene. (b) A chimeric protein containing the DNA-binding domain of Sp1 and the activation domain of CTF1 activates transcription if a GC box is present.
