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      Molecular genetic analysis of chloroplast gene promoters dependent on SIG2, a nucleus-encoded sigma factor for the plastid-encoded RNA polymerase, in Arabidopsis thaliana.

      Nucleic Acids Research
      Arabidopsis, cytology, enzymology, genetics, Arabidopsis Proteins, Base Sequence, Cell Nucleus, Chloroplasts, DNA, metabolism, DNA-Binding Proteins, DNA-Directed RNA Polymerases, Genes, Plant, Genetic Complementation Test, Molecular Sequence Data, Mutation, Nuclease Protection Assays, Plastids, Promoter Regions, Genetic, Protein Binding, RNA, Plant, RNA, Transfer, Sigma Factor, Transcription Initiation Site, Transcription, Genetic, Transgenes

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          Abstract

          Most photosynthesis-related genes in mature chloroplasts are transcribed by a eubacterial-type RNA polymerase (PEP) whose core subunits are encoded by the plastid genome. It has been shown previously that six putative nuclear genes (SIG1 to SIG6) encode promoter-specificity factors for PEP in Arabidopsis thaliana, and we isolated a T-DNA insertion line of SIG2 (sig2-1 mutant) that manifests aberrant chloroplast development. With the use of S1 nuclease protection and primer extension analyses, we have now characterized the SIG2-dependent chloroplast promoters in A.thaliana. The amounts of transcripts derived from one of the multiple psbD promoters (psbD -256) and from the promoters of two tRNA genes (trnE-UUC and trnV-UAC) were markedly and specifically decreased in the sig2-1 mutant. The abundance of these transcripts was restored to wild-type levels by introduction into the mutant of a SIG2 transgene. The recombinant SIG2 protein mixed with Escherichia coli core RNA polymerase could bind to a DNA fragment that contains the SIG2-dependent psbD -256, trnE-UUC or trnV-UAC promoter. Sequences similar to those of the -35 and -10 promoter elements of E.coli were identified in the regions of the SIG2-dependent chloroplast genes upstream of the transcription initiation sites.

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