Characterization of P. sativum chloroplast psbA transcripts produced in vivo, in vitro and in E. coli

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.