Structural Studies of Macromolecular Assemblies 
plexes containing the Cro protein of phage 434 
or the DNA-binding domain of its repressor. We 
are beginning to use computational approaches 
to link observed structural differences among 
these various complexes with the corresponding 
free energies of binding. 
We have also been studying several eukaryotic 
regulatory proteins, initially by preparing crys- 
tals of their DNA-binding domains in complex 
with synthetic binding sites. TflllA, which con- 
trols 5S RNA transcription in Xenopus, represents 
the so-called zinc finger class. The finger is a 
small, 30-residue domain, stabilized by a tightly 
bound zinc ion. A recombinant fragment com- 
prising seven of the nine fingers from TflllA binds 
to a 30-base pair (bp) DNA containing an appro- 
priate part of the total binding site. We have crys- 
tallized this complex. 
GAL4, a regulator of galactose metabolism in 
yeast, has a rather different zinc-containing struc- 
ture. The amino-terminal 65 residues form a do- 
main that binds with dyad symmetry to a 17-bp 
consensus DNA sequence. A first structure of such 
a complex is nearly complete. Each domain con- 
tains two zinc ions in close proximity, liganded 
by six cysteines. 
GCN4, also a yeast regulatory protein, repre- 
sents yet another class of DNA-binding structures. 
It contains a dimerization element, generally 
called a leucine zipper, which forms an a-helical 
coiled coil about 30 residues in length. This seg- 
ment is preceded in the protein sequence by a 
positively charged region, which has little or- 
dered structure in the free protein but which also 
acquires a-helical structure when it binds to 
DNA. We have prepared crystals of the basic re- 
gion/leucine zipper fragment of GCN4, in com- 
plex with a synthetic binding site, and a structure 
determination is in progress. 
Understanding how these various structures 
recognize their DNA sites is only a beginning. The 
specificities of interactions between other do- 
mains of these proteins and additional compo- 
nents of a transcriptional initiation complex pre- 
sent even more challenging puzzles for the 
future. 
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