An additional promoter within the protein-coding region of thepsbD-psbC gene cluster in tobacco chloroplast DNA

Abstract

Transcription of the psbD-psbC gene cluster in tobacco chloroplasts has been studied. This cluster contains in linear sequence the overlapping genes encoding the D2 and 43 kDa proteins of Photosystem II (psbD and psbC, respectively), and ORF62. Eight major transcripts ranging from 1.5 to 4.4 kb were detected by northern blot analysis. S1 mapping experiments revealed that these multiple transcripts comprise five distinct 5' ends whose precise positions were further determined by primer extension analysis. Two of the five 5' ends were determined to be the transcriptional initiation sites by in vitro capping assays: the main site is located 905 bp upstream from the ATG codon of psbD and the additional site is 194 bp upstream from the first ATG codon of psbC. The latter site and the preceding prokaryotic promoter motif are within the protein-coding region of psbD. The 3' ends of transcripts were determined by S1 mapping.

A novel light-regulated promoter is conserved in cereal and dicot chloroplasts.

“…This mRNA 5' end corresponds to the mRNA 5' end previously localized at position -905 in tobacco (Yao et al, 1989). The light-induced psbD-psbC mRNAs appear to comprise a smaller proportion of the total psbD-psbC mRNA population in dicots than in cereals.…”

“…Their 5'ends mapped to the conserved DNA region at position -900 from thepsbD translational start codon. The 5'ends of these light-induced mRNAs were previously shown to be cappable primary transcripts (Yao et al, 1989). From these results, the 5' ends of the barley and tobacco light-induced mRNAs represent the transcription initiation sites of these mRNAs.…”

“…A weak, previously unmapped signal was observed at position -135 for barley ( Figure 4B). These psfoD proximal mRNA 5' ends had been previously mapped in tobacco (Yao et al, 1989) and spinach (Offerman, 1988). Hence, from these experiments, two classes of dicot psbD-psbC mRNAs were identified, as illustrated in Figure 4A.…”

“…Therefore, we studied the expression of the psbD-psbC operons from three dicots (tobacco, pea, and spinach) to determine if they also had the light response. Tobacco and spinach were chosen because the DNA sequences for the psbD-psbC genes and upstream regions were available (Offerman, 1988;Yao et al, 1989). Pea was also chosen because the genes had previously been mapped, their coding regions sequenced (Rasmussen et al, 1984), and their expression analyzed using RNA dot blots (Woodbury et al, 1989).…”

“…DNA regions involved in three inversions are delineated with dashed boxes. Long horizontal and dashed arrws refer to transcripts, which are subdivided into classes for the psbK-psbl-tmG loci in dicot tobacco (class A and B; Meng et al, 1991), psbD-psbC loci in the dicot tobacco (I, 11, and 111; Yao et al, 1989), and cereal barley (classes 1 to 5 from BerendsSexton et al, 1990b). Low abundance class 6 (dashed lhe) was determined in this study (Figure 4).…”