Selective recognition of the Type II PclpP-53 promoter in a chloroplast extract suggests that there may be three distinct phage-type plastid RNA polymerases in higher plants

Abstract

Plastid genes in higher plants are transcribed by two distinct RNA polymerases: the plastid-encoded eubacterial-type multisubunit enzyme (PEP) and the nuclear-encoded single catalytic subunit enzyme (NEP). Type I NEP promoters have been characterized in vitro in an extract prepared from tobacco plants lacking the PEP enzyme and shown to share the core motif with higher plant mitochondrial gene promoters (Liere and Maliga, EMBO J. 18:249, 1999). Here we report on a second in vitro transcription activity from PEP-defective tobacco plants with selective recognition of the PclpP-53 Type II NEP promoter for the proteolytic subunit of the Clp ATP-dependent protease. Study of in vitro transcription from deletion derivatives and scanning mutagenesis identified the ?11/+10 region as important for PclpP-53 promoter activity. The ?11/+10 region includes an A-rich stretch, but lacks a typical YATA Type I core motif. Interestingly, PclpP-53 is transcribed at similar efficiency from linear and super coiled DNA templates unlike Type I promoters, which are more efficiently transcribed from supercoiled templates. The extract utilized in the study will not recognize Type I NEP promoters indicating that tobacco chloroplasts contain two separable NEP activities, the Type I enzyme characterized earlier and the Type II enzyme reported here. Thus, there are possibly three distinct NEP enzymes in higher plants, the Type I and Type II enzymes recognizing the Type I and Type II NEP promoters in tobacco and the Type III enzyme present in spinach recognizing the Pc promoter (Bligny et al., EMBO J. 19: 1851, 2000).