Biophysical Studies of Eukaryotic Gene Regulation 
and Molecular Recognition 
Stephen K. Burley, M.D., D.Phil. — Assistant Investigator 
Dr. Burley is also Assistant Professor and Co-Head of the Laboratory of Molecular Biophysics at the 
Rockefeller University. He received a B.Sc. degree in physics from the University of Western Ontario, a 
D.Phil, degree in molecular biophysics from Oxford University, and an M.D. degree from Harvard Medical 
School in the Harvard MIT Joint Program in Health Sciences and Technology. While a medical student, 
he carried out research in protein crystallography with Gregory Petsko. During his clinical training 
at Brigham and Women 's Hospital, he also conducted postdoctoral research in protein crystallography 
with William Lipscomb at Harvard University, where he solved the three-dimensional structure 
of leucine aminopeptidase. 
WE are interested in developing a detailed 
understanding of the physical principles 
that govern the general problem of molecular rec- 
ognition in biological systems. Our approach is 
to use x-ray crystallography and complementary 
biophysical methods to determine and character- 
ize the three-dimensional structure and function 
of biological macromolecules and their com- 
plexes with DNA, proteins, or smaller ligands. 
These structures contain a wealth of atomic detail 
that we can analyze with biochemical, molecular 
genetic, and theoretical methods to provide a 
functional description of the intra- and intermo- 
lecular interactions responsible for stabilizing 
macromolecular complexes. 
In the long term, we hope that our biophysical 
studies and analyses will allow us to exploit the 
powerful formalism of physics to classify system- 
atically the interactions between individual 
atoms that are responsible for molecular recogni- 
tion in biological systems. We believe that such a 
quantitative understanding will ultimately per- 
mit us to harness the machinery of molecular rec- 
ognition and, thereby, make defined interven- 
tions into important biochemical processes such 
as disease states. 
Eukaryotic Gene Regulation 
We are examining the problem of eukaryotic 
gene regulation, with the goal of improving our 
understanding of the structural and physical 
bases of transcriptional control of genes. Three 
distinct classes of proteins are active in transcrip- 
tion, and we are studying representative mem- 
bers of each class. First, we are collaborating with 
Robert Roeder (Rockefeller University) on x-ray 
crystallographic and complementary biophysical 
studies of transcription factor IID (TFIID) and 
other components of the basic transcription ma- 
chinery. These proteins form a stable multipro- 
tein, or preinitiation, complex with DNA se- 
quences found immediately upstream of the 
transcription start site, where they mediate tran- 
scription by RNA polymerase II. We have pu- 
rified, characterized, and crystallized TFIID, 
which begins preinitiation complex formation by 
binding to the TATA consensus sequence. In ad- 
dition, we have started work on TFIIB, the second 
protein to be recruited to the preinitiation 
complex. 
Second, we are also collaborating with Dr. 
Roeder on studies of upstream stimulatory factor 
(USF), a member of the c-mjr-related family of 
DNA-binding proteins that contains both a helix- 
loop-helix motif and a leucine repeat. We have 
used various biophysical methods to purify and 
extensively characterize USF and its mechanisms 
of action. Moreover, we were recently able to 
grow small cocrystals of USF and DNA. During 
transcription, TFIID, the other basic factors, and 
USF bind to DNA in close proximity and interact 
with one another to enhance both DNA binding 
and transcription. After determining the three- 
dimensional structures of each of these proteins 
and their complexes with their respective pro- 
moter DNA elements, we hope to determine the 
structures of some biologically relevant multi- 
protein-DNA complexes. 
Third, we are collaborating with Eseng Lai (Me- 
morial Sloan-Kettering Cancer Center) on struc- 
tural studies of human hepatocyte nuclear factor 
3. This transcriptionally active protein belongs to 
a gene family in mammals that is homologous to 
the Drosophila homeotic gene fork head. These 
diverse proteins share a highly conserved DNA- 
binding region and influence transcription by 
binding to DNA elements, known as enhancer se- 
quences, that are located far from the transcrip- 
tion start site. During the past year, we have co- 
crystallized a member of this family with DNA 
and are proceeding with a three-dimensional 
structure determination. Detailed structural and 
biophysical studies of these three distinct classes 
of participants in eukaryotic transcription should 
provide insights into the precise role molecular 
recognition plays in gene regulation. 
Molecular Recognition 
During the past year, we have also begun 
to study other biological systems that function 
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