DNA sequence requirements for the accurate transcription of a protein-coding plastid gene in a plastid in vitro system from mustard (Sinapis alba L.)

Abstract

A nuclease‐treated plastid extract from mustard (Sinapis alba L.) allows efficient transcription of cloned plastid DNA templates. In this in vitro system, the major runoff transcript of the truncated gene for the 32 000 mol. wt. photosystem II protein was accurately initiated from a site close to or identical with the in vivo start site. By using plasmids with deletions in the 5′‐flanking region of this gene as templates, a DNA region required for efficient and selective initiation was detected ˜28‐35 nucleotides upstream of the transcription start site. This region contains the sequence element TTGACA, which matches the consensus sequence for prokaryotic ‘−35′ promoter elements. In the absence of this region, a region ˜13‐27 nucleotides upstream of the start site still enables a basic level of specific transcription. This second region contains the sequence element TATATAA, which matches the consensus sequence for the ‘TATA’ box of genes transcribed by RNA polymerase II (or B). The region between the ‘TATA'‐like element and the transcription start site is not sufficient but may be required for specific transcription of the plastid gene. This latter region contains the sequence element TATACT, which resembles the prokaryotic ‘−10’ (Pribnow) box. Based on the structural and transcriptional features of the 5′ upstream region, a ‘promoter switch’ mechanism is proposed, which may account for the developmentally regulated expression of this plastid gene.

The chloroplast transcription apparatus from mustard (Sinapis alba L.)

“…The mixtures were incubated for 20 min at 30 "C, transcripts prepared [26] and electrophoresed on 8% sequencing gels [56]. Total trichloroacetic-acid-precipitable material was determined as described [57].…”

Transcriptional Analysis of Endogenous and Foreign Genes in Chloroplast Transformants of Chlamydomonas

“…Studies of transcription in vitro using chloroplast RNA polymerase preparations from maize (Crossland et al, 1984;Bradley and Gatenby, 1985), mustard (Link, 1984), ' Current address: Cornell University, Plant Science Center, 139 Biotechnology Building, Ithaca, NY 14853.…”

“…However, it has been observed that removal of the first two, generally very highly conseyved, nucleotides of the spinach tmM2 -35 sequence reduced transcription in vitro by a partially purified spinach chloroplast RNA polymerase by only 60% (Gruissem and Zurawski, 1985a), and a mustard chloroplast psbA gene construct without a -35 sequence (Link, 1984) was transcribed to some extent in vitro. Furthermore, tmR1 and tmS1 of spinach (Gruissem et al, 1986) are transcribed in vitro although they have no recognizable -1 O or -35 sequences.…”

A tRNA Gene Mapping Within the Chloroplast rDNA Cluster is Differentially Expressed During the Development of Daucus carota

“…The significance of multiple promoters of the rDNA cluster is unknown. A developmentally regulated promoter switching was hypothesized (37,41,42). In the attempt to isolate developmentally regulated genes in D.carota, we have cloned the chloroplast rDNA cluster.…”