MECHANISM OF TRANSCRIPTIONAL REGULATION IN ANIMAL CELLS 
Robert TjiAN, Ph.D., Investigator 
Dr. Tjian has focused his research on the specific 
protein-DNA and protein-protein interactions that 
regulate the synthesis of RNA in animal cells. The 
recent molecular cloning and structure-function 
characterization of DNA-binding transcription fac- 
tors carried out by Dr. Tjian and his colleagues have 
helped identify novel protein structural motifs re- 
sponsible for DNA recognition, protein contact, and 
transcriptional activation. These studies have, in 
turn, allowed Dr. Tjian to address the mechanism of 
action of promoter-specific transcription factors. 
In particular, he has obtained evidence for a new 
class of transcription factors, called coactivators, 
that seem to function as an intermediary between 
upstream sequence-specific regulatory elements 
and the basal-level transcriptional apparatus. This 
line of investigation, in turn, led Dr. Tjian and his 
colleagues to the discovery that the TATA-binding 
protein (TBP) plays a central role in assembling an 
initiation complex, not only at RNA polymerase II 
(pol II) promoters but also at RNA pol I transcrip- 
tion. Their finding that TBP is associated with differ- 
ent promoter-selective factors called TAFs (TBP- 
associated factors) revealed an underlying and 
unifying principle that appears to govern transcrip- 
tion by all three classes of RNA polymerase. This 
unexpected discovery significantly altered current 
views concerning the role of TBP and the mecha- 
nism that mediates promoter specificity and tran- 
scriptional regulation in animal cells. 
Isolating and Characterizing Basal 
Transcription Factors 
The TATA-binding protein, TBP, plays a central 
role in the initiation of eukaryotic mRNA synthesis. 
Dr. Tjian recently isolated the human and Drosoph- 
ila cDNA clones for this factor. He and his collabora- 
tors have also succeeded in isolating cDNAs encod- 
ing a second essential basal transcription factor, 
TFIIE. Human cDNA clones for both the 56- and 34- 
kDa subunits of TFIIE were isolated. Using recombi- 
nant proteins purified from Escherichia coli, they 
found that both these subunits are required to form 
a stable preinitiation complex on a basal promoter 
with TBP, TFIIA, TFIIB, TFIIF, and RNA pol II. Re- 
constituted transcription reactions establish that 
both the 56- and 34-kDa subunits of TFIIE are essen- 
tial for basal-level as well as Sp 1 -activated transcrip- 
tion in vitro. 
Analysis of their predicted amino acid sequences 
reveal several intriguing structural motifs. The 56- 
kDa subunit sequence includes a zinc finger homol- 
ogy that may interact with DNA, an amphipathic a 
helix that may define a protein-protein interaction 
domain, and a protein kinase consensus sequence. 
The 34-kDa subunit contains a region that resembles 
an ATP-binding motif. These similarities may pro- 
vide insights into the role of TFIIE in transcription 
initiation. 
Upstream Activation by Transcription 
Factor Spl 
The process of transcriptional activation in eu- 
karyotes by site-specific DNA-binding proteins is a 
key step in gene regulation. When the appropriate 
trans-activators are assembled at the promoter, they 
are thought to help direct RNA polymerase to initi- 
ate transcription. The mechanism by which se- 
quence-specific transcriptional regulators act to in- 
fluence rates of mRNA initiation remains largely 
unknown. Dr. Tjian and his colleagues examined the 
properties of four distinct activator domains of the 
human transcription factor Spl. In vivo transient 
cotransfection assays with Spl showed that tem- 
plates bearing multiple Spl sites activated tran- 
scription with a high degree of synergism. However, 
there was no evidence of cooperative binding of Sp 1 
to adjacent sites. 
Using deletion mutants of Spl, the group deter- 
mined that the glutamine-rich activation domains A 
and B and the previously uncharacterized carboxyl- 
terminal domain D were all required for Spl to acti- 
vate transcription synergistically. Gel-shift, DNase 
footprinting, and chemical crosslinking experi- 
ments revealed a strong correlation between the 
ability of Spl mutants to form homomultimeric 
complexes and to activate transcription synergisti- 
cally when bound to multiple sites. The group also 
examined the process of superactivation, in which a 
molecule of Sp 1 tethered to DNA via its zinc fingers 
could be transcriptionally enhanced by interacting 
directly with fingerless Spl molecules. The domains 
involved in superactivation appeared to be a subset 
of those necessary to achieve synergistic activation. 
These findings suggested that different domains of 
Sp 1 carry out distinct functions and that the forma- 
tion of multimeric complexes may direct synergism 
and superactivation. 
In an effort to understand how Spl could activate 
a TATA-less promoter, Dr. Tjian and his colleagues 
previously addressed the factor requirement at a 
promoter containing only Spl -binding sites and an 
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