dimensional structure and function of the TATA-box 
binding protein (TBP), formerly known as recombi- 
nant transcription factor IID. TBP, which is required 
for transcription of eukaryotic genes by all three 
RNA polymerases, has been highly conserved in evo- 
lution. This universal transcription factor does not 
act alone in higher organisms. Instead, it functions 
as part of a large multiprotein assembly, consisting 
of TBP and various TBP-associated factors (TAFs) . 
In class II gene transcription, the TBP-TAF com- 
plex binds to the TATA consensus sequence and di- 
rects accretion of a set of general transcription fac- 
tors to form the preinitiation complex, which 
stabilizes interactions between RNA polymerase II 
and the promoter. The role played by TBP in tran- 
scription by the other two RNA polymerases is not as 
well characterized. However, it is believed that TBP 
functions with a discrete non-overlapping set of 
TAFs that are polymerase specific. The immediate 
challenge in understanding the problem of tran- 
scriptional regulation of eukaryotic gene expression 
is to explain how TBP can have such extreme func- 
tional versatility. 
Dr. Burley and his co-workers have overexpressed 
and purified large amounts of TBP from various 
eukaryotes, including Homo sapiens, Arabidop- 
sis thaliana, Drosophila melanogaster, Saccha- 
romyces cerevisiae, and Schizosaccharomyces 
pombe. Native crystals of TBP from S. cerevisiae und 
A. thaliana (both isoforms) have been obtained. 
During the past year Dimitar Nikolov solved the 
three-dimensional structure of TBP isoform 2 from 
A. thaliana at 2.58-A resolution, using the multiple 
isomorphous replacement method. The structure is 
a novel, highly symmetric, saddle-shaped a//? pro- 
tein that sits astride the DNA and presents its convex 
surface for interaction with TAFs and other tran- 
scriptionally active proteins. Nikolov continues his 
attempts at crystallization and structure determina- 
tion with the remaining TBPs. 
Cocrystals of some of the TBPs complexed with 
duplex oligonucleotides bearing the TATA consen- 
sus sequence have also been obtained, and 
determination of the structure of the transcription 
factor-DNA complex is under way. Finally, these 
crystallographic studies are being complemented 
with detailed biophysical comparisons of these 
structurally and functionally related proteins and 
their complexes with DNA. 
Crystal Structure of Human Hepatocyte 
Nuclear Factor-3 
In collaboration with Dr. Eseng Lai (Memorial 
Sloan-Kettering Cancer Center) , Dr. Burley and his 
co-workers are engaged in structural and functional 
studies of human hepatocyte nuclear factor-3 (HNF- 
3). This group of transcriptionally active proteins 
belongs to a gene family in mammals that is homolo- 
gous to the Drosophila homeotic gene fork head. 
These diverse proteins share a highly conserved 
DNA-binding region, which is thought to represent 
an entirely new type of DNA-binding motif. Dr. Bur- 
ley's group overexpressed and purified the DNA- 
binding domain of two members of the HNF-3 
family. 
During the past year Dr. Kirk L. Clark has obtained 
cocrystals of HNF-37 with a duplex oligonucleotide 
bearing the DNA recognition sequence. He has mea- 
sured x-ray diffraction data at 2.8-A resolution from 
native and three heavy-atom derivative crystals and 
will soon complete the determination of the multi- 
ple isomorphous replacement structure. Thereafter, 
the structure of HNF-3« bound to its DNA recogni- 
tion sequence can be solved by molecular replace- 
ment using previously obtained crystals. 
Dr. Burley is also Assistant Professor and Co- 
Head of the Laboratory of Molecular Biophysics at 
the Rockefeller University. 
Articles 
Burley, S.K., David, P R., Sweet, R.M., Taylor, A., 
and Lipscomb, W.N. 1992. Structure determina- 
tion and refinement of bovine lens leucine amino- 
peptidase and its complex with bestatin. / Mol 
Biol 224:113-140. 
David, P.R., and Burley, S.K. 1991. A method for 
equilibrating protein crystals with heavy atom re- 
agents. /^pp/ Crystallog 24:1073-1074. 
STRUCTURAL BIOLOGY 467 
