Yx. Lin et al., Optimization of a versatile in vitro transcription assay for the expression of multiple start site TATA-less promoters, BIOCHEM, 40(43), 2001, pp. 12959-12966
Previous work from our laboratory has allowed for the subdivision of RNA po
lymerase II TATA-less promoters into two classes: those that initiate at a
single start site (SSS) and those that initiate at multiple start sites (MS
S). MSS promoters are defined by the lack of a TATA box and the presence of
a transcription initiation window and a downstream MED-1 element (GCTCCC/G
) [Ince, T. A., and Scotto, K. W. (1995) J. Biol. Chem. 270, 30249-30252].
Further insight into the mechanisms regulating TATA-less MSS promoters has
been hampered by the lack of an in vitro transcription assay in which multi
ple start sites can be reproduced. In the present study, we describe the de
velopment of a versatile in vitro transcription system optimized for the ex
pression of MSS promoters, termed the multiple promoter comparison (MPC) as
say. By alteration of assay parameters including template length, cation an
d nucleotide concentrations, and RNA isolation method, the accurate and rob
ust transcription of two MSS promoters, pgp1 (hamster P-glycoprotein class
I homologue) and HPRT (human hypoxanthine phosphoribosyltransferase), was a
ccomplished. Moreover, both TATA-containing and TATA-less single start site
promoters were also transcribed in the MPC assay, making this the first ge
neral in vitro transcription system for the simultaneous analysis of all th
ree classes of RNA polymerase II genes.