activity in partially purified chloroplast soluble protein fraction

National Agriculture Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki, 305-8666 Japan

     The differentiation of proplastids into chloroplasts is accompanied by a drastic change in transcriptional mechanisms. Particular photosynthesis genes encoded in the plastid genome are specifically expressed during or after this conversion. Biochemical studies have proved the existence of two types of RNA polymerase (RNAP) in the chioroplast. One is nuclear encoded T7 bacteriophage type RNAP which has been presumed to function in every type of plastid for transcribing housekeeping genes like ribosomal components and tRNAs (Hedtke et a!. 1997). Another is the chioroplast-specific enzyme sharing extensive similarities with the bacterial a2BB'o RNAP. Genes corresponding to the bacterial counter parts of the core enzyme, a2BB', have been found in the plastid genome (Hiratsuka et al. 1989) and recently, genes encoding the a factor, which is necessary for the transcriptional initiation, have been reported to be encoded in the nuclear genomes of higher plants (Tanaka et a!. 1997, Tozawa et al. 1998). The rice sigA gene shows shoot-specific and light-dependent expression in seedlings and its amino-terminal region shows features of potential plastid localization signals (Tozawa et a!. 1998). Transient expression assay using GFP have revealed that amino-terminal region of SigA homolog of Arabidopsis thaliana functions as a chloroplast targeting signal (Isono eta!. 1998). Therefore, contribution of SigA to chloroplast-specific gene expression has been proposed. To confirm its localization and correlation with the activity of the bacterial type RNAP, immunoblot analysis and in vitro transcription assays were performed.

     Recombinant SigA was prepared using the pQE vector kit (Qiagen) and rabbit antiserum against this protein was prepared. Intact chioroplasts were isolated and purified from 10 day seedlings of rice cv. Nipponbare, grown at 28°C under continuous light condition, by percol gradient centrifugation (Orozco et at. 1985). Approximately 2 x i09 chloroplasts were obtained which were lysed in the high-salt buffer containing 1M NaC1. They were then separated into two fractions: a soluble fraction including mainly stromal proteins and an insoluble fraction including mainly proteins associated with thylakoid membranes. Polypeptide composition pattern of those fractions are apparently different (Fig. 1A). For in vitro transcription assay, -35/-10 type promoter of the rice psbA gene was used. RNA polymerase activity was calculated based on the amount of 32P-labelled UTP incorporated into RNA. Immunoblot analysis revealed that SigA is located in the soluble fraction of the chloroplast (Fig. 1B), which is also associated with the activity of bacterial RNAP (Fig. lB and 2). This transcriptional activity was completely inhibited by supplemented tagetitoxin (Fig. 2), the best known inhibitor of plastidial and bacterial RNAP (Mathews eta!. 1990). SigA protein conserves domains of promoter recognition, promoter melting, and of core RNAP binding, those which have been assigned in bacterial sigma factors (Tozawa eta!. 1998). Therefore, these observations suggest crucial contribution of SigA, plastidial sigma factor coexisting with the bacterial RNAP, to the process of transcriptional initiation in the chioroplasts.