Evidence for Nuclear Control of the Expression of the atpA and atpB Chloroplast Genes in Chlamydomonas

We have found that sequences in the 5' leader of the Chlamydomonas chloroplast rbcL gene, when fused 5' to foreign genes, destabilize transcripts of these chimeric genes in the chloroplast of transgenic Chlamydomonas but that 5' sequences ofthe rbcL structural gene prevent this destabilization. Transcripts of the chloroplast rbcL gene are about equally abundant at all times in Chlinydomonas reinhardij growing on an alternating 12-h light/12-h dark cycle. However, Chlamydomonas chloroplast transformants, harboring chimeric genes containing the same rbcL promoter with 63 or 92 bp of the rbcL 5' leader sequence fused upstream of the Escherichia coli uidA (P-glucuronidase, GUS) gene, accumulated GUS transcripts only in the dark. Transcripts disappeared rapidly upon Mumination of the cells. The same phenomenon was exhibited by transcripts of chimeric genes in which the GUS gene coding sequence was replaced by other unrelated genes. The precipitous light-induced drop in GUS transcript abundance was found to be due to an "16-fold increase in the rate of degradation of GUS transcripts in light rather than to a decrease in the rate of transcription of the GUS gene. Transcripts of a chimeric rbcL-GUS construct in which the leader sequence of the rbcL gene was replaced by 103 bp of the leader sequence of the atpB gene were stable in illuminated cells. The destabilizing effect of the rbcL 5' leader sequence was reversed by adding 257 bp of the 5' coding region of the rbcL gene. The results show that chloroplast transcript levels in illuminated Chiamydomonas cells-and perhaps in other cases-can be determined, at least to some extent, by sequences and interactions of sequences transcribed from the 5' ends of genes.

Section: Discussionmentioning

confidence: 99%

5' sequences are important positive and negative determinants of the longevity of Chlamydomonas chloroplast gene transcripts.

Salvador

Klein

Bogorad

1993

“…The green alga Chlamydomonas reinhardtii is an excellent system to study such regulatory pathways. Mutations in nuclear genes that alter the expression of chloroplast-encoded components of photosystem I, photosystem II, ATP synthase, and the photosynthetic electron transport chain have been identified (1)(2)(3)(4)(5). These genes presumably encode proteins that regulate the splicing, stability, or translation of specific chloroplast RNAs.…”

Section: Introductionmentioning

confidence: 99%

In vivo analysis of Chlamydomonas chloroplast petD gene expression using stable transformation of beta-glucuronidase translational fusions.

Sakamoto

Kindle

Stern

1993

104

We have used the Escherichia coli fi-glucuronidase (uidA) gene as a reporter gene to localize the promoter and analyze the function ofthe 5' untranslated region (UTR) of the Chlamydomonas chloroplast petD gene. Using particle bombardment, petD-uidA transcriptional and translational fusion genes were introduced into the chloroplast genome in the large inverted repeat flanking the atpB gene. In transformants carrying a petD-uidA transcriptional fusion, uidA mRNA accumulated but was not translated. However, in a translational fusion that included the entire petD 5' UTR, uidA mRNA accumulated and a high level of P-glucuronidase activity was detected. When ==70% of the petD 5' UTR was deleted from the translational fusion, uid4 mRNA accumulation and P-glucuronidase activity decreased 4-to 6-fold and 8-fold, respectively. Run-on transcription assays demonstrated that all strains transcribe the uidA gene at equivalent rates. Our results show that sequences essential for translation reside in the petD 5' UTR and also that sequences within the 5' UTR directly or indirectly affect mRNA stability. The expression of P-glucuronidase under the control of chloroplast transcriptional and translational signals will facilitate further studies of chloroplast gene regulatory mechanisms.

“…11 and 12). Some chloroplast-encoded subunits display a much lower rate of synthesis in the absence of their assembly partners, as detailed in Table 1 (6,(13)(14)(15)(16)(17). Thus, chloroplast protein synthesis is to some extent hierarchical.…”

mentioning

confidence: 99%

Translation of cytochrome f is autoregulated through the 5′ untranslated region of petA mRNA in Chlamydomonas chloroplasts

Choquet

Stern²,

Kuras³

et al. 1998

120

116

A process that we refer to as control by epistasy of synthesis (CES process) occurs during chloroplast protein biogenesis in Chlamydomonas reinhardtii : the synthesis of some chloroplast-encoded subunits, the CES subunits, is strongly attenuated when some other subunits from the same complex, the dominant subunits, are missing. Herein we investigate the molecular basis of the CES process for the biogenesis of the cytochrome b 6 f complex and show that negative autoregulation of cytochrome f translation occurs in the absence of other complex subunits. This autoregulation is mediated by an interaction, either direct or indirect, between the 5′ untranslated region of petA mRNA, which encodes cytochrome f , and the C-terminal domain of the unassembled protein. This model for the regulation of cytochrome f translation explains both the decreased rate of cytochrome f synthesis in vivo in the absence of its assembly partners and its increase in synthesis when significant accumulation of the C-terminal domain of the protein is prevented. When expressed from a chimeric mRNA containing the atpA 5′ untranslated region, cytochrome f no longer showed an assembly-dependent regulation of translation. Conversely, the level of antibiotic resistance conferred by a chimeric petA - aadA - rbcL gene was shown to depend on the state of assembly of cytochrome b 6 f complexes and on the accumulation of the C-terminal domain of cytochrome f . We discuss the possible ubiquity of the CES process in organellar protein biogenesis.