Mechanisms of Transcriptional Regulation 
simplex virus protein VP 16. By using the VP 16 
activation domain as a ligand for affinity chroma- 
tography, v^e found that VP 16 interacts directly 
with the TBP subunit of TFIID . Mutations in VP 1 6 
that reduce gene activation by VP 1 6 also reduce 
its binding to TBP, and preliminary work has 
identified a mutation in TBP that reduces its abil- 
ity to interact with and respond to VPl6. Interac- 
tion of VP 16 with TBP alters the association of 
TBP with the promoter. Therefore, VP 16 may in- 
fluence the ability of TBP to recruit other general 
factors and RNA polymerase II to the promoter. 
A potent acidic activation domain is also found 
in p53, the product of a human anti-oncogene (a 
tumor-suppressing gene) that is mutated in about 
half of all human cancers. Like VP16, the p53 
activation domain also binds TBP. By using affin- 
ity chromatography to search for other proteins 
that interact with p53, we recently discovered a 
new protein that interacts with the p5 3 and VP 1 6 
activation domains. At least some oncogenic mu- 
tations in p53 prevent binding of this protein to 
the p53 activation domain. The precise role of 
this novel protein in transcription is still under 
investigation. 
Transcriptional Antitermination in E. coli 
The N protein of bacteriophage X prevents ter- 
mination by RNA polymerase during transcrip- 
tion of X operons expressed immediately after in- 
fection of E. coli cells. We have reconstituted 
antitermination by N in vitro in a system contain- 
ing seven purified proteins: E. coli RNA polymer- 
ase, the E. coli transcription termination factor 
Rho, N, and four E. co/? cofactors for antitermina- 
tion (NusA, NusB, SIO, and NusG). By using pro- 
tein affinity chromatography and other methods, 
we have identified many of the protein-protein 
interactions in this system. Three factors, NusA, 
NusG, and SIO, all bind to RNA polymerase. NusA 
is important for termination of transcription at 
some terminators. Since N binds to NusA, NusA is 
also an adapter that couples the antitermination 
factor N to RNA polymerase. In addition, NusB 
binds to SIO and NusG to Rho factor. In fact, 
NusG aids termination by Rho in vitro and is es- 
sential for termination by Rho in vivo. 
The stable association of N with RNA polymer- 
ase requires an N utilization site (nw? site) in the 
transcribed DNA and all four bacterial cofactors. 
The nut site RNA in the growing RNA transcript is 
recognized and bound by the proteins. N recog- 
nizes the boxB component of the nut site RNA, 
while NusB and SIO recognize a better version of 
the box A component of nut site RNA, which is 
found in the antiterminator sequences of bacte- 
rial ribosomal RNA operons. Since mutations in 
RNA polymerase can prevent the formation of a 
stable multiprotein complex and can prevent 
binding of protein to the nut site RNA during 
transcription, we infer that a stable ribonucleo- 
protein complex containing nut site RNA and 
five antitermination proteins assembles on the 
surface of RNA polymerase. This extensively 
modified RNA polymerase can then transcribe 
through kilobases of X DNA containing many tran- 
scriptional terminators. 
The human immunodeficiency viruses (HIV-1 
and HIV-2) produce antitermination factors, 
known as Tat, that recognize regulatory se- 
quences in viral RNA, known as TAR. The human 
host factors involved in antitermination by Tat are 
unknown. Antitermination of HIV transcription 
by Tat may well resemble antitermination of X 
transcription by N in many respects. 
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