Initiation of Transcription by RNA 
Polymerase II 
The general initiation factors assemble in an or- 
dered pathway with RNA polymerase II at promoters 
for protein-coding genes. The TATA box usually 
present in such promoters is recognized by TBP, the 
DNA-binding subunit of the general factor TFIID. 
Using TBP affinity chromatography, Dr. Greenblatt's 
laboratory showed that TBP interacts with the hu- 
man general factor TFIIA and that TFIIA has three 
subunits: A35, A21, and A12. Site-specific protein- 
DNA crosslinking was used to establish the align- 
ments of TBP, A3 5, and A2 1 along promoter DNA. 
The general factor TFIIF has two subunits, RAP30 
and RAP74. Dr. Greenblatt and his colleagues had 
previously cloned human cDNAs encoding RAP30, 
and this year, in collaboration with Dr. Zachary Bur- 
ton (Michigan State University) , they cloned human 
cDNAs encoding RAP74. RAP30 behaves in several 
ways like a bacterial a factor: it binds to E. colt RNA 
polymerase in competition with (t^°; it prevents non- 
specific binding of RNA polymerase II to DNA; and it 
recruits RNA polymerase II to a preformed preinitia- 
tion complex containing TFIID and TFIIB. Recombi- 
nant TBP, TFIIB, and RAP30 were found to be suffi- 
cient for promoter recognition by RNA polymer- 
ase II. 
Activation of Transcription by RNA 
Polymerase II 
Activator proteins usually have two critical do- 
mains. One binds to DNA and the other interacts 
with the transcription apparatus. In collaboration 
with the laboratory of Dr. C. James Ingles, Dr. 
Greenblatt's group showed previously that the 
acidic activation domain of the Herpes simplex 
virus activator VP16 interacts with TBP. Similarly, 
the acidic activation domain of the human anti- 
oncogenic protein p53 binds to TBP, and this inter- 
action is prevented by at least some oncogenic mu- 
tations in p53. Recent data indicate that even Spl , a 
ubiquitous human activator whose activation do- 
mains are glutamine-rich rather than acidic, binds to 
TBP, suggesting that TBP may be a universal target 
for activator proteins. However, other recent data in 
the Greenblatt laboratory indicate that activation 
domains may also contact the general factor TFIIH 
and a 100-kDa protein whose role in transcription is 
unknown. The mechanism that activates transcrip- 
tional initiation is clearly complex and is currently 
the subject of intensive investigation. 
Dr. Greenblatt is Professor in the Banting and 
Best Department of Medical Research and the De- 
partment of Molecular and Medical Genetics at 
the University of Toronto, Faculty of Medicine. 
Articles 
Buratowski, S., Sopta, M., Greenblatt, J., and Sharp, 
P.A. 1 99 1 . RNA polymerase Il-associated proteins 
are required for a DNA conformation change in 
the transcription initiation complex. Proc Natl 
Acad Sci USA 88:7509-7513- 
Coulombe, B., Killeen, M., Liljelund, P., Honda, B., 
Xiao, H., Ingles, C.J., and Greenblatt, J. 1992. 
Identification of three mammalian proteins that 
bind to the yeast TATA box protein TFIID. Gene 
Expr 2:99-1 10. 
Finkelstein, A., Kostrub, C.F., Li, J., Chavez, D.P., 
Wang, B.Q., Fang, S.M., Greenblatt, J., and Bur- 
ton, Z.F. 1992. A cDNA encoding RAP74, a gen- 
eral initiation factor for transcription by RNA poly- 
merase II. Nature 555:464-467 . 
Flores, O., Lu, H., Killeen, M., Greenblatt, J., Bur- 
ton, Z.F., and Reinberg, D. 1991. The small sub- 
unit of transcription factor IIF recruits RNA poly- 
merase II into the preinitiation complex. Proc 
Natl Acad Sci USA 88:9999-10003. 
Formosa, T., Barry, J., Alberts, B.M., and Green- 
blatt, J. 1 99 1 . Using protein affinity chromatogra- 
phy to probe the structure of protein machines. 
Methods Enzymol 208:24-45. 
Greenblatt, J. 1991. RNA polymerase-associated 
transcription factors. Trends Biochem Sci 16: 
408-411. 
Greenblatt, J. 1991. Roles of TFIID in transcrip- 
tional initiation by RNA polymerase II. Cell 
66:1067-1070. 
Killeen, M.T., and Greenblatt, J. 1992. The general 
transcription factor RAP30 binds to RNA polymer- 
ase II and prevents it from binding nonspecifi- 
cally to DNA. Mol Cell Biol 12:30-37. 
Li, J., Horwitz, R., McCracken, S., and Greenblatt, 
J. 1992. NusG, a nt-w Escherichia co/? elongation 
factor involved in transcriptional antitermination 
by the N protein of phage X. / Biol Chem 
267:6012-6019. 
Linn, T., and Greenblatt, J. 1992. The NusA and 
NusG proteins of Escherichia coli increase the in 
vitro readthrough frequency of a transcriptional 
attenuator preceding the gene for the |8 subunit of 
RNA polymerase./ 5/0/ Chem 267:1449-1454. 
Mason, S.W., and Greenblatt, J. 1991 . Assembly of 
transcription elongation complexes containing 
the N protein of phage X and the Escherichia coli 
elongation factors NusA, NusB, NusG, and SIO. 
Genes Dev 5:1504-1512. 
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