We have characterized two maize cDNAs, rpoTm and rpoTp, that encode putative T7-like RNA polymerases. In vivo cellular localization experiments using transient expression of the green fluorescent protein suggest that their encoded proteins are targeted exclusively to mitochondria and plastids, respectively. An antibody raised against the C terminus of the rpoTp gene product identified mitochondrial polypeptides of approximately 100 kD. Their presence was correlated with RNA polymerase activity, and the antibody inhibited mitochondrial in vitro transcription activity. Together, these results strongly suggest that the product of rpoTm is involved in maize mitochondrial transcription. By contrast, immunoblot analysis and an antibody-linked polymerase assay indicated that rpoTp specifies a plastid RNA polymerase component. A quantitative reverse transcription-polymerase chain reaction assay was used to study the transcription of rpoTp and rpoTm in different tissues and under different environmental conditions. Although both genes were constitutively expressed, rpoTm transcripts were generally more prevalent in nonphotosynthetic tissues, whereas an increase in rpoTp transcripts paralleled chloroplast development. We suggest that these two genes encode constitutive components of the organelle transcription machinery but that their expression is nonetheless subject to modulation during plant development.

The plastid genome is known to be transcribed by a plastid-encoded prokaryotic-type RNA polymerase (PEP) and by a nucleus-encoded phage-type RNA polymerase (NEP). The spinach plastid rrn operon promoter region harbours three different, overlapping promoters. Two of them are of the prokaryotic type. The third promoter is a non-consensus-type NEP promoter. We separated three different transcriptional activities from spinach chloroplasts: PEP, the phagetype RNA polymerase NEP-1, and a third, hitherto undescribed transcriptional activity (NEP-2). NEP-2 speci®cally transcribes the rrn operon in the presence of the transcription factor CDF2. CDF2 was previously shown to recruit PEP to the rrn promoter to repress transcription. Together, our results suggest the existence of a third RNA polymerase in plastids and a mechanism of rDNA transcriptional regulation that is based on the interaction of the transcription factor CDF2 with two different transcriptional systems.

We have characterized two maize cDNAs, rpoTm and rpoTp, that encode putative T7-like RNA polymerases. In vivo cellular localization experiments using transient expression of the green fluorescent protein suggest that their encoded proteins are targeted exclusively to mitochondria and plastids, respectively. An antibody raised against the C terminus of the rpoTp gene product identified mitochondrial polypeptides of approximately 100 kD. Their presence was correlated with RNA polymerase activity, and the antibody inhibited mitochondrial in vitro transcription activity. Together, these results strongly suggest that the product of rpoTm is involved in maize mitochondrial transcription. By contrast, immunoblot analysis and an antibody-linked polymerase assay indicated that rpoTp specifies a plastid RNA polymerase component. A quantitative reverse transcription-polymerase chain reaction assay was used to study the transcription of rpoTp and rpoTm in different tissues and under different environmental conditions. Although both genes were constitutively expressed, rpoTm transcripts were generally more prevalent in nonphotosynthetic tissues, whereas an increase in rpoTp transcripts paralleled chloroplast development. We suggest that these two genes encode constitutive components of the organelle transcription machinery but that their expression is nonetheless subject to modulation during plant development.

The spinach rrn operon is used as a model system to study transcriptional regulation in higher plant photosynthetic and non-photosynthetic plastids. We performed capping experiments to determine whether P1, PC, or P2 promoters are employed for rrn transcription start sites in cotyledon and root tissues. By using a new method of analysis of capped RNA we demonstrate for the first time that 1) in both organs the rrn operon is expressed in a constitutive manner by cotranscription with the preceding tRNA(GAC)Val gene, and 2) the PC transcription start site is used only in cotyledons and leaves, i.e. we demonstrate the organ-specific usage of a plastid promoter. Both start sites, PC and that of the tRNA(GAC)Val cotranscript, lack Escherichia coli-like consensus sequences. The cotranscript is initiated 457 base pairs upstream of the tRNA(GAC)Val gene. The PCspecific DNA-binding factor, CDF2, is not detectable in root tissues confirming its regulatory role in PC-initiated rrn expression and the organ specificity of PC expression. Furthermore, our results show that rrn operon expression patterns differ in spinach and tobacco indicating species-specific transcriptional regulation of plant plastid gene expression.In accordance with the hypothesis that plastids are of endosymbiotic origin most plastid genes are organized into polycistronic transcription units reminiscent of bacterial operons. Plastid rRNA operons show the typical procaryotic gene order of 16 S, 23 S, and 5 S rDNA. These genes are transcribed as large precursor RNAs that are subsequently processed into the various mature rRNA species (1, 2).The promoter regions of plastid rrn operons harbor Escherichia coli-like "-10" and "-35" consensus sequences, like most of the plastid transcription units. These E. coli-like consensus sequences serve as promoter structures (3-5) or as regulatory elements (6, 7). However, the interpretation of results on studies of transcriptional regulation in plastids is complicated by the existence of different types of RNA polymerases. One is nuclear encoded (8 -11) and the other one is encoded on the plastid genome (12-15). The plastid-encoded enzyme can be considered as "E. coli-like" with respect to its subunit composition (16, 17) and promoter usage (6, 18 -20). The composition of the nuclear-encoded RNA polymerase and the promoter structures that are used by this enzyme are not yet clear although several potential transcription start sites for this enzyme have been mapped (5,21,22).In spinach, the rrn operon upstream region contains three different promoter elements (P1, PC, P2), and transcription is thought to be regulated by the transcription factor CDF2 (23). CDF2 acts as a repressor of rRNA transcription by the E. coli-like plastid RNA polymerase and probably as an activator of rRNA transcription by the nuclear-encoded RNA polymerase (6), i.e. rRNA transcription could be regulated exclusively by CDF2. On the other hand, up to now correct initiation at the putative PC start site could not be demonstrated in vitro raising the ...