Identification of Two Essential Sequence Elements in the Nonconsensus Type II PatpB-290 Plastid Promoter by Using Plastid Transcription Extracts from Cultured Tobacco BY-2 Cells

In higher plants, plastid genes are transcribed by at least two types of DNA-dependent RNA polymerases. One of them is the well-known plastid-encoded prokaryotic type of polymerase that recognizes sigma(70)-type promoters consisting of -35 and -10 consensus elements. The other recently recognized RNA polymerase has been found to be encoded entirely in the nucleus, and it recognizes a completely different set of promoters, designated previously as nonconsensus type II (NCII) promoters. Here, we report the development of an in vitro transcription system using nonphotosynthetic plastids of cultured tobacco BY-2 cells. This system preferentially and accurately initiates transcription from NCII promoters. The conditions for in vitro transcription were optimized by using the tobacco PatpB-290 promoter, which has been found to be the most highly expressed NCII promoter in vivo. Analysis of in vitro transcription initiation in a series of PatpB-290 5' deletion constructs revealed that sequences upstream of nucleotide -41 do not influence the transcriptional activity of this promoter. A 43-bp region (nucleotides -35 to +8) was further analyzed by introducing single or multiple nucleotide substitutions into two regions (box I and box II) of high sequence conservation. We report here that the ATAGAA sequence comprising box II and the -11 to +4 region (relative to transcription initiation) in box I significantly influence the activity of this NCII promoter.