CHEMICAL MECHANISMS IN CELLULAR REGULATION 
Paul B. Sigler, M.D., Ph.D., Investigator 
Dr. Sigler's research is centered on the stereo- 
chemical mechanisms of specific macromoiecular 
interactions involved in cellular regulation. Two 
main regulatory systems are emphasized: 1) the 
regulation of gene expression and 2) transmem- 
brane signaling. In all cases the experimental core 
of the project is to determine the high-resolution 
crystal structure of the relevant macromolecules, 
both alone and in specific functional complexes. 
Biochemical, physicochemical, and directed muta- 
tional studies are used to test the mechanistic infer- 
ences drawn from the structural work. The long- 
range goal is to describe the detailed chemistry of 
these interactions in dynamic terms. 
Regulation of gene expression will be explored in 
three areas, with the initial focus on the specific af- 
finity of regulatory proteins for their DNA targets. 
The following systems are under study: 1) tran- 
scription in prokaryotes: trp repressor, arg repres- 
sor; 2) transcription in eukaryotes: the CCAAT/en- 
hancer binding protein and other leucine zipper 
proteins, steroid receptors, GAL4, bovine papilloma 
virus E2, and the TATA-binding factor; 3) initiation 
of translation in eukaryotes: the structure and func- 
tion of yeast tRNA.'^". 
Research on transmembrane signaling is focused 
on the action of phospholipases that release sec- 
ond messengers and arachidonate, the essential 
biosynthetic precursor of the mediators of inflam- 
mation. The immediate aim is to establish struc- 
ture-function relationships in phospholipases A^, 
both alone and in complexes with their essential 
CO factor, Ca^^, and phosphonate transition-state 
analogues. 
L Transcriptional Regulation: The Chemistry of Spe- 
cific Protein-DNA Interactions. 
A well-defined problem in transcriptional regula- 
tion is the physicochemical basis for the unusual af- 
finity of regulatory proteins for their DNA targets. 
High-resolution crystallographic studies of the li- 
gand-activated trp repressor have provided insight 
into this process and are being extended to the 
study of binding of the argR protein to tandem Arg 
boxes. In the past year. Dr. Sigler and his colleagues 
have also begun to study the interactions of a series 
of eukaryotic transcription factors with their DNA 
elements. 
A. trp repressor-operator complex. The crystal 
structure of the trp repressor-operator complex 
o 
was first solved and refined to 2.4 A resolution and 
o 
later extended to 1.9 A with imaging-plate data 
from the Photon Factory (Japan's national syn- 
chrontron light source). Each of the four repre- 
sentations of the unique structure in the asym- 
metric unit shows the same specifying features in 
vivid detail. Since they are present in all four copies 
of the unique structure, they must be intrinsic 
to the chemistry of the interface and not an artifact 
of the crystal lattice. The results were startling. 
1) Every hydrogen-bondable group on the surface 
of the repressor facing the DNA makes a hydrogen- 
bonded interaction with the operator, either di- 
rectly or indirectly through solvent with either 
the phosphates or the functional groups of 
the major groove. There was a surprising paucity 
of direct interactions (only one per half repres- 
ser/operator) between protein side chains and the 
bases. There are at least 18 well-defined water mol- 
ecules that mediate contacts to the bases and back- 
bone. 2) Specificity apparently arises from two 
sources: a) a sequence-dependent deformation of 
the DNA that permits an extensive (2,900 A ) con- 
tact surface to form, presumably without signifi- 
cantly raising the internal energy of the DNA, and 
b) highly polarized water-mediated hydrogen bonds 
between the peptide N-Hs at the tip of the helix- 
turn-helix and the major-groove functional groups 
of the most mutationally sensitive base pairs of the 
operator. 
To check the inferences drawn from the struc- 
ture, Dr. Sigler and his colleagues will grow crystals 
in which the repressor and/or operator is changed 
either genetically or chemically to enhance or di- 
minish affinity. These models should serve as a basis 
for developing calculations that predict sequence- 
specific protein-DNA affinity. 
B. Transcriptional regulation in eukaryotes. An at- 
tempt is being made to crystallize and determine 
the structures of a series of eukaryotic transcription 
factors, both alone and in complexes with their 
DNA elements. Each factor is representative of a 
class of proteins bearing a characteristic DNA-bind- 
ing motif 
In collaboration with Dr. Keith Yamamoto (Uni- 
versity of California at San Francisco), large 
amounts of the DNA-binding domain of the rat 
glucocorticoid receptor have been expressed. This 
Continued 
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