Characterization of P. sativum chloroplast psbA transcripts produced in vivo, in vitro and in E. coli.
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We have analyzed the region of the chloroplast genome from P. sativum which encodes the 5'-end of psbA. S1 nuclease mapping and primer extension analysis of chloroplast RNA revealed psbA transcripts with 5'-termini 92, 93 and 68 nucleotides upstream from the psbA open reading frame. The psbA transcripts with 5'-ends 92-93 nucleotides upstream from the psbA open reading frame can be labeled with alpha-(32)P-GTP by guanylyltransferase. DNA sequences 10 and 35 bp upstream from the longest psbA transcript showed homology to -10 and -35 consensus promoter sequences in E. coli. Truncated psbA constructs which contain the putative psbA promoter sequences were shown to promote transcription in E. coli from a site similar to that used by chloroplast RNA polymerase in vivo. Accurate transcription of psbA constructs was also observed in a homologous in vitro transcription extract from chloroplasts. Sequence analysis of the region upstream from the psbA transcripts revealed a putative 3'-exon of a tRNA-Lys (240 bp upstream) and an unidentified open reading frame (URF, 485 bp upstream). The 3'-end of the URF mRNA was located approximately 240 bp from the 5'-end of the longest psbA transcript indicating that the URF and tRNA-Lys sequence are cotranscribed. Comparison of P. sativum and N. tabacum DNA sequences at the 5'-end of psbA revealed homology between sequences coding for psbA mRNA including 40 bp upstream from the longest psbA transcript. A second region of homology which includes the tRNA-Lys sequence was also located. In contrast the intergenic DNA exhibited extensive divergence in size and sequence. re]19850627 rv]19851211 ac]19851216.
