Mechanisms of Transcriptional Regulation 
Jack Greenblatt, Ph.D. — International Research Scholar 
Dr. Greenblatt is Professor in the Banting and Best Department of Medical Research and the Department of 
Molecular and Medical Genetics at the University of Toronto. He earned his undergraduate degree in 
physics from McGill University, Montreal, and his Ph.D. degree in biophysics from Harvard University, 
where he studied bacterial gene regulation with Walter Gilbert. He pursued postdoctoral studies with 
Alfred Tissieres at the University of Geneva. He has received the Ayerst Award of the Canadian 
Biochemical Society. 
THE ultimate focus of transcriptional regula- 
tory mechanisms is RNA polymerase, a com- 
plex multisubunit enzyme whose activity is 
guided by interactions with a myriad of regula- 
tory proteins and regulatory sequences in DNA or 
RNA. Much of our research is devoted to the basic 
enzymology of initiation and termination of tran- 
scription and to identifying and characterizing 
some of the key protein-protein interactions in- 
volved. In addition, we are examining how 
model regulatory proteins interact with the basic 
transcriptional apparatus. This work is also sup- 
ported by grants from the Medical Research 
Council of Canada and the National Cancer Insti- 
tute of Canada. 
Initiation of Transcription by Human RNA 
Polymerase II 
A set of general transcription factors (TFILA, B, 
D, E, F, H) is necessary for RNA polymerase II to 
initiate the transcription of protein-coding genes. 
TFIID is the general factor that recognizes TATA 
sequences present in the promoters of many 
genes. After TFIID binds to the DNA, TFIIA and 
TFIIB recognize and bind to the TFIID-DNA com- 
plex. Subsequently the other general factors and 
RNA polymerase II assemble into a multiprotein 
complex at the promoter. We use protein affinity 
chromatography as a technique to identify direct 
protein-protein interactions involved in the as- 
sembly of this complex. 
TFIID has been highly conserved during evolu- 
tion. TFIID molecules from most or all eukar- 
yotes, including fungi, insects, and plants, can 
function in transcription reactions containing 
RNA polymerase II and other general factors of 
human origin. By using yeast TFIID as a ligand for 
affinity chromatography, we have identified 
three human polypeptides that interact with 
TFIID and constitute human TFIIA. Curiously, 
TFIID columns do not retain TFIIB, nor do TFIIB 
columns retain TFIID, suggesting that a confor- 
mational change in TFIID induced by DNA bind- 
ing facilitates its interaction with TFIIB. 
By using RNA polymerase II as a ligand for affinity 
chromatography, we identified human RAP30 and 
RAP74, the small and large subunits of TFIIF. Hu- 
man cDNAs encoding RAP30 and RAP74 have both 
been cloned, the latter in collaboration with 
Zachary Burton (Michigan State University) . Using 
recombinant RAP30, we found that RAP30 is the 
subunit of TFIIF that binds RNA polymerase II. 
RAP30 prevents RNA polymerase II from associat- 
ing with and transcribing nonpromoter sequences 
in DNA, a property also of bacterial a factors. In- 
deed, we found that RAP30 can bind to Escherichia 
coli RNA polymerase and be displaced by the major 
bacterial a factor, known as a""^. 
RAP30 also has a central role in promoter rec- 
ognition by RNA polymerase II. In collaboration 
with Danny Reinberg (University of Medicine and 
Dentistry of New Jersey), we found that RAP30 
can recruit RNA polymerase II to a preinitiation 
complex containing TFIIA, TFIIB, and TFIID. In 
fact, recognition of a promoter containing a TATA 
sequence by RNA polymerase II can be achieved 
with recombinant TFIIB, RAP30, and TBP, the 
TATA sequence-binding subunit of TFIID, all 
produced in E. coli. These three general factors, 
therefore, constitute a minimal set of proteins 
necessary and sufficient for promoter binding by 
RNA polymerase II. However, this preinitiation 
complex containing RNA polymerase II will not 
initiate transcription unless supplied with 
RAP74, TFIIF, TFIIH, and other factors. The roles, 
subunit compositions, and protein-protein inter- 
actions of some of these factors remain to be 
identified. 
Regulation of Initiation 
by RNA Polymerase II 
Many transcriptional activator proteins have 
two domains. One binds to regulatory sequences 
in DNA, and the other, known as an activation 
domain, provides an activating signal to the basic 
transcriptional apparatus. How these activation 
domains function is a fascinating question in reg- 
ulatory biology. It has been the major focus of 
collaborative studies with my colleague, C.James 
Ingles (University of Toronto) . 
Many activation domains are highly acidic. A 
particularly potent one is found in the Herpes 
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