TATA BOX AND INITIATOR FUNCTIONS IN THE ACCURATE TRANSCRIPTION OF A PLANT MINIMAL PROMOTER IN-VITRO

The functional architecture of the proximal region of a rice phenylala nine ammonia-lyase (PAL) promoter was analyzed by transcription of PAL -beta-glucuronidase (GUS) templates by whole-cell extracts of rice cel l suspension cultures. The promoter 5' truncated to position -35 was s ufficient 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 minima l promoter. Moreover, the function of the TATTTAA sequence was depende nt on its position relative to the initiation site; hence, this elemen t is an authentic TATA box essential for RNA polymerase Ii-dependent t ranscription. 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 inac curate initiation, whereas mutations at the other residues of this con served element or sequence substitutions between the TATA box and init iator had little effect. TATA box and initiator functions were confirm ed by analysis of the effects of promoter mutations on expression in s tably transformed rice cell suspensions and plants, We concluded that the proximal region of the PAL promoter has a simple functional archit ecture involving a TATA box appropriately positioned upstream of the i nitiator, Transcription of derivatives of such minimal promoters by hi ghly active cell extracts should allow molecular analysis of functiona l interactions between specific cis elements and cognate trans factors .