J. Biol. Chem., Vol. 265, Issue 14, 8259-8267, May, 1990
Two different RNA polymerase II initiation complexes can assemble on the rat brain creatine kinase promoter
MT Mitchell and PA Benfield
Central Research Department, E. I. du Pont de Nemours & Co., Inc., Wilmington, Delaware 19880-0328.
The rat brain creatine kinase (CKB) gene has a structurally complex
promoter. Although it contains a consensus RNA polymerase II TATA box, this
is located at -60 relative to the main transcription start site, an
unprecedented distance for mammalian TATA boxes. Steady-state messenger RNA
for CKB is extremely abundant in brain, yet barely detectable in liver.
Using a nuclear run-on analysis, we show that this difference is due, at
least in part, to a difference in the rate at which CKB is transcribed in
these tissues. We also demonstrate a tissue- specific hypersensitive site
in the promoter region of the CKB gene. In vitro transcription experiments
using nuclear extract isolated from liver, brain, and HeLa cells show a
marked difference in the way these extracts recognize the CKB promoter. Two
different preinitiation complexes can assemble on the CKB promoter, one
which initiated at the same downstream start-site used in normal adult rat
brain and one which initiated 35 base pairs further upstream. Extracts from
brain form exclusively the downstream initiating complex. However, liver
extracts favor formation of the upstream initiating complex, but formation
of this complex can be blocked if brain or HeLa extract is added first. We
demonstrate that the upstream consensus TATA box can function as a TATA box
to mediate upstream initiation, but is not required for downstream
initiation. We have shown previously that brain and HeLa cell nuclear
extracts contain an abundant factor, TARP, which binds to the upstream
consensus TATA box and have proposed that this factor is distinct from the
classical TATA box binding factor TFIID. We propose that binding of TARP to
the upstream consensus TATA box may block formation of the upstream
initiating complex.