THE PLANT CELL, Vol 7, Issue 10 1681-1689, Copyright © 1995 by American Society of Plant Biologists
TATA Box and Initiator Functions in the Accurate Transcription of a Plant Minimal Promoter in Vitro
Q. Zhu, T. Dabi and C. Lamb
Plant Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037
The functional architecture of the proximal region of a rice phenylalanine
ammonia-lyase (PAL) promoter was analyzed by transcription of
PAL-[beta]-glucuronidase (GUS) templates by whole-cell extracts of rice
cell suspension cultures. The promoter 5[prime] truncated to position -35
was sufficient for accurate initiation of basal transcription. Substitution
of the TATTTAA sequence between positions -35 and -28 with GCGGGTT or 2-bp
substitutions to give TCGTTAA and TATGGAA inactivated the minimal promoter.
Moreover, the function of the TATTTAA sequence was dependent on its
position relative to the initiation site; hence, this element is an
authentic TATA box essential for RNA polymerase II-dependent transcription.
Substitutions in the TCCAAG initiator cis element (-3 to +3) at the -1(C to
A or G)and +1(A to C or T) residues caused inaccurate initiation, whereas
mutations at the other residues of this conserved element or sequence
substitutions between the TATA box and initiator had little effect. TATA
box and initiator functions were confirmed by analysis of the effects of
promoter mutations on expression in stably transformed rice cell
suspensions and plants. We concluded that the proximal region of the PAL
promoter has a simple functional architecture involving a TATA box
appropriately positioned upstream of the initiator. Transcription of
derivatives of such minimal promoters by highly active cell extracts should
allow molecular analysis of functional interactions between specific cis
elements and cognate trans factors.
