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The TATA-binding protein TBP plays a central role in eukaryotic transcription initiation (1, 2). TBP binds to the promoter consensus sequence TATAAA, located approx 30 nucleotides upstream of the transcriptional start site. Once bound, TBP facilitates the assembly of RNA polymerase II and other initiation factors (TFIIA,-B,-E,-F,-G/J, and H) into an active initiation complex (3). [ABSTRACT FROM AUTHOR]

Promoter-specific in vitro transcription by RNA polymerase II is now achievable with cell extracts prepared from a variety of animal and plant sources (1-3). For extracts prepared from mammalian cells, HeLa cells provide many advantages. Large quantities of homogeneous cells can be grown in spinner flasks at a cost below that of many other mammalian tissue-culture cells. A second advantage of HeLa extracts is the low level of RNases, proteases, and other nonspecific inhibitory activities. This results in a long shelf life (indefinitely at −70°C, <24 h at 4°C). The third advantage is that initiation factors from HeLa cells are well characterized, and many of them are available in recombinant form (4-10). [ABSTRACT FROM AUTHOR]