Two different RNA polymerase II initiation complexes can assemble on the rat brain creatine kinase promoter.
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.
Mitchell
M T
MT
Central Research Department, E. I. du Pont de Nemours & Co., Inc., Wilmington, Delaware 19880-0328.
Benfield
P A
PA
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D003402:Creatine Kinase / Q000235:genetics*; D004262:DNA Restriction Enzymes; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D012333:RNA, Messenger; D014157:Transcription Factors; D003402:Creatine Kinase; D012319:RNA Polymerase II; D004262:DNA Restriction Enzymes