Mechanisms of Gene Regulation in Animal Cells 
DNA-binding proteins, such as the prototype hu- 
man factor Spl, direct transcriptional interac- 
tions. To address this critical issue, our group re- 
cently fractionated and isolated the multiple 
components necessary to reconstitute transcrip- 
tion. In the process of dissecting the general tran- 
scriptional apparatus, we discovered two previ- 
ously undetected components that serve as the 
functional bridge between upstream trans-activa- 
tors and the initiation complex. These novel fac- 
tors appear to be part of the missing link that di- 
rects promoter-selective transcription in animal 
cells, and it is likely that they will be members of 
a diverse and essential class of regulatory 
proteins. 
Indeed, this past year has seen significant pro- 
gress in the purification and characterization of 
transcriptional "coactivators" and TATA-binding 
protein (TBP) -associated factors (TAFs). In partic- 
ular, several TAFs and coactivators have been pu- 
rified from both Drosophila and human cells. In 
addition, the genes encoding this novel class of 
transcription factors have recently been cloned. 
The structure and function of these multisubunit 
complexes should prove to be very revealing. 
One of the most exciting and unexpected find- 
ings this year was the discovery that the TBP, a 
general transcription factor thought to be only 
responsible for RNA polymerase II transcription 
of mRNA, is also an integral subunit of a TBP-TAF 
complex responsible for recognition of the RNA 
polymerase I promoter. Most interestingly, the 
RNA polymerase I complex carries out all the 
functions ascribed to the species-specific tran- 
scription factor SLl , and the subunit composition 
reveals the presence of TBP and three novel TAFs, 
apparently adapted uniquely to direct RNA poly- 
merase I transcription. This surprising finding 
provides a unifying mechanism of transcription 
initiation. 
Transcription of Developmentally 
Regulated Genes 
One of our long-term interests is the mecha- 
nisms underlying regulation and expression in 
the development of higher organisms. We have 
begun to address this issue in two ways: first, by 
initiating a series of in vitro experiments aimed 
at dissecting the transcriptional regulation of 
Drosophila genes, including the alcohol dehy- 
drogenase, Ultrabithorax, Antennapedia, dopa 
decarboxylase, and hunchback genes. A major 
advance was the development of in vitro tran- 
scription reactions from staged Drosophila em- 
bryos that accurately initiate RNA synthesis and 
recapitulate the temporal program of transcrip- 
tion displayed by these tissue-specific and devel- 
opmentally regulated genes. 
A second approach has been to investigate the 
regulatory mechanism of RNA polymerase initia- 
tion factors in vivo. Various systems have been 
adapted to introduce altered genes back into cells 
or whole organisms to study their patterns of 
expression. 
These in vitro and in vivo studies have re- 
cently led to two exciting results. First, a negative 
regulator of a developmentally important gene 
has been identified by direct biochemical means, 
and its mode of operation can now be dissected. 
The finding of specific transcriptional repressors 
is of particular importance because it is thought 
that an interplay of positive activators and nega- 
tive regulators is seminal to the spatially re- 
stricted patterns of expression observed during 
embryogenesis. 
Also arising from Drosophila studies are the 
identification and subsequent biochemical char- 
acterization of a transcription factor that appears 
to govern the expression of genes in cells of the 
central nervous system. The gene encoding this 
neurogenic-specific activator has recently been 
isolated, and its structure is expected to reveal 
interesting information. Advantages in the use of 
fruit flies include the ability to probe the develop- 
mental and tissue-specific function of this neuro- 
genic regulator in a rapid and highly informative 
manner not readily applicable to mammalian 
cells. These studies are expected to yield new 
insights concerning the tissue-specific distribu- 
tion and temporal timing of expression during 
development. 
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