Transcription Factor Interactions 
Steven Lanier McKnight, Ph.D. — Investigator 
Dr. McKnight is also a staff member of the Department of Embryology in the Carnegie Institution of 
Washington, Baltimore, and an adjunct faculty member in the Departments of Biology and of Molecular 
Genetics at the Johns Hopkins University. He earned his Ph.D. degree in biology from the University of 
Virginia and, except for four years with the Fred Hutchinson Cancer Research Center in Seattle, has been 
with the Carnegie Institution ever since. 
FOR the past several years my colleagues and I 
have studied a mammalian transcription fac- 
tor termed CCAAT/enhancer-binding protein (C/ 
EBP) . This protein is capable of binding to DNA 
in a sequence-specific manner and thereby regu- 
lating gene expression. By studying the detailed 
properties of C/EBP, v^e have sought to develop a 
better understanding of how genes are regulated 
in mammalian cells. 
These efforts have been revi^arded by a surpris- 
ing discovery. The mechanism by which C/EBP 
binds DNA is common to that used by many other 
sequence-specific DNA-binding proteins, includ- 
ing the products of two important proto-onco- 
genes. The observations that led to this discovery 
were as follows. 
After sequencing the gene encoding C/EBP, we 
fed its conceptually translated amino acid se- 
quence into a computer database of known pro- 
tein sequences. Much to our delight, a 60-amino 
acid segment of C/EBP proved to be sequence 
related to the products of the Fos and Jun proto- 
oncogenes. Earlier studies on C/EBP had shown 
that this same region of the protein was responsi- 
ble for its ability to bind DNA. In other words, the 
region of C/EBP that was related in amino acid 
sequence to the FOS and JUN proteins was its 
DNA-binding domain. 
Next we began to focus attention on the amino 
acids that evolution has conserved among the 
three proteins. We reasoned that the related se- 
quences might reveal the underpinnings of a 
structural motif that facilitates specific interac- 
tion with DNA. The amino acid sequences shared 
by the three proteins occurred in two patches. 
One patch contained a conserved set of basic 
amino acids — arginines and lysines. On the car- 
boxyl-terminal side of this "basic region," each 
protein exhibited leucine residues spaced every 
seven amino acids. 
Bill Landschulz, then an M.D./Ph.D. student in 
the laboratory, noticed that neither the basic re- 
gion nor leucine repeat region contained proline 
or glycine residues. Since prolines and glycines 
tend to be incompatible with a-helical structure, 
we reasoned that much of the DNA-binding do- 
main of C/EBP, as well as FOS and JUN, might be 
a-helical. This prediction of a-helical structure 
provided an attractive role for the repeating leu- 
cine residues. Since the repeating period of a- 
helices is 3.5 amino acids per helical turn, and 
since the leucines were spaced at a heptad inter- 
val, the putative helix projected a continuous 
array of leucine residues from one helical face. 
What might be the role of an a-helix that dis- 
plays repeating leucines along one of its sides? 
Chemists have long known that leucines are un- 
usually hydrophobic. Rather than being exposed 
to an aqueous or hydrophilic environment, hy- 
drophobic amino acids prefer to interact with 
other hydrophobic compounds, most often 
within the internal fold of a protein. Following 
this lead we hypothesized that the a-helical re- 
gion displaying a repeated array of leucines might 
represent a dimerization interface, allowing two 
polypeptide chains to coalesce along the helical 
face that contained the repeating array of leu- 
cines. We termed this hypothetical structure the 
"leucine zipper." 
Elegant experiments by Peter Kim (HHMI) and 
his colleagues at the Whitehead Institute con- 
firmed the general tenets of the zipper hypothesis 
and further established that zippered helices as- 
sociate with each other in a parallel orientation. 
During the time that we were developing the 
zipper idea, research from several other laborato- 
ries demonstrated that the FOS and JUN polypep- 
tides were capable of stable association. Recog- 
nizing this fact. Bill Landschulz, Peter Johnson, 
and I predicted that FOS and JUN would associate 
as dimers by virtue of their respective leucine 
zippers. Supportive evidence has been obtained 
by Robert Tjian (HHMI, University of California, 
Berkeley), Edward Ziff" (HHMI, New York Univer- 
sity Medical School), and Daniel Nathans (HHMI, 
the Johns Hopkins University Medical School). 
If the leucine zipper region of each of these 
proteins is indeed responsible for allowing pro- 
tein dimers to form, what is the role of the basic 
region? Knowing that the substrate for binding by 
these proteins is DNA — a negatively charged 
polymer — we reasoned that the basic region 
might facilitate direct contact with DNA. Such 
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