Two small open reading frames are co-transcribed with the pea chloroplast genes for the polypeptides of cytochrome b-559

Abstract

The genes encoding the 9 kDa and 4 kDa polypeptides of cytochrome b-559 have been located in pea chloroplast DNA by coupled transcription-translation of cloned restriction fragments of chloroplast DNA in a cell-free extract of Escherichia coli and by nucleotide sequence analysis. The genes (psbE and psbF) are located approximately 1.0 kbp downstream of the gene for cytochrome f and are transcribed in the opposite direction, similar to the arrangement in the chloroplast genomes of other higher plants. Nucleotide sequence analysis of this region revealed four open reading frames encoding hydrophobic proteins of 83 (psbE), 39 (psbF), 38 and 40 amino acid residues, which are co-transcribed as a single major RNA of 1.1 kb. The 5' and 3' ends of this RNA have been located by primer extension and S1 nuclease mapping. The 5' end of the RNA is located 140 bp upstream of the initiating ATG codon of psbE and is preceded by typical chloroplast promoter sequences. The 3' end of the RNA is located approximately 515 bp downstream of the TAA stop codon of psbF close to a stable stem-loop structure.

Plastid Transcriptomics and Translatomics of Tomato Fruit Development and Chloroplast-to-Chromoplast Differentiation: Chromoplast Gene Expression Largely Serves the Production of a Single Protein

“…Although most polycistronic precursor transcripts are posttranscriptionally processed into monocistronic or oligocistronic units (presumably by specific endonucleolytic cleavage) (Westhoff and Herrmann, 1988;Sugita and Sugiura, 1996;Herrin and Nickelsen, 2004), some mRNAs remain largely polycistronic. Examples of these are the psbE operon transcript comprising four small genes for polypeptides of photosystem II (psbE, psbF, psbL, and psbJ) (Carrillo et al, 1986;Willey and Gray, 1989) and the psaA/psaB transcript (Meng et al, 1988), which is also not regularly cleaved by intercistronic processing. Thus, the polysome association data do not allow us to distinguish between an altered translation of psaA or psaB or both.…”

An open reading frame encoding a putative haem-binding polypeptide is contranscribed with the pea chloroplast gene for apocytochrome f

“…The RNA samples were electrophoresed in 1.1~o agarose gels and transferred to nitrocellulose (BA85 Schleicher and Schtill) according to Thomas [ 39]. Prehybridisation and hybridisation with 32p_ labelled probes obtained by second-strand synthesis of M13 single-stranded DNA using a pentadecamer sequencing primer [20] were carried out as described by Willey and Gray [48].…”

Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons

“…In at least some cases, cutting of the polycistronic precursor RNA into monocistronic units is required to facilitate efficient translation of all cistrons, as is supported by the analysis of nuclear mutants defective in distinct intercistronic processing events and by in vitro translation studies (29)(30)(31)(32). Some polycistronic transcripts in plastids clearly remain unprocessed and are translated efficiently [e.g., the psbE operon comprising four small genes for polypeptides of photosystem II and the dicistronic psaA/B mRNA encoding the two reaction center subunits of photosystem I (33,34)], but the rules determining the dependency of translation on intercistronic RNA processing are currently unknown. Thus which (trans)genes in synthetic operons will be expressed efficiently from unprocessed polycistronic transcripts and which transgenes will require intercistronic processing for efficient translation remains unpredictable, and previous attempts to stack transgenes in operons have produced mixed results (e.g., [35][36][37].…”