RESUMO
Studies of the biogenesis of the photosynthetic protein complexes in the unicellular green alga Chlamydomonas reinhardtii have pointed to the importance of the concerted expression of nuclear and chloroplast genomes. The accumulation of chloroplast- and nuclear-encoded subunits is concerted, most often as a result of the rapid proteolytic disposal of unassembled subunits, but the rate of synthesis of some chloroplast-encoded subunits from photosynthetic protein complexes, designed as CES proteins (Controlled by Epistasy of Synthesis), is regulated by the availability of their assembly partners from the same complex. Cytochrome f, a major subunit of the cytochrome b(6)f complex is a model protein for the study of the CES process. In the absence of subunit IV, another subunit of the cytochrome b(6)f complex, its synthesis is decreased by 90%. This results from a negative autoregulation of cytochrome f translation initiation, mediated by a regulatory motif carried by the C-terminal domain of the unassembled protein [Choquet, Stern, Wostrikoff, Kuras, Girard-Bascou and Wollman (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4380-4385]. Using site-directed mutagenesis, we have characterized this regulatory motif. We discuss the possible implications regarding the mechanism of the CES process for cytochrome f expression. We have studied the possible generalization of this mechanism to other CES proteins.
Assuntos
Chlamydomonas reinhardtii/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica , Complexo de Proteína do Fotossistema I , Biossíntese de Proteínas , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Citocromos/química , Citocromos/genética , Citocromos f , Mutagênese Sítio-Dirigida , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Subunidades Proteicas , Deleção de SequênciaRESUMO
RIP (repeat-induced point mutation) is a silencing process discovered in Neurospora crassa and so far clearly established only in this species as a currently occurring process. RIP acts premeiotically on duplicated sequences, resulting in C-G to T-A mutations, with a striking preference for CpA/TpG dinucleotides. In Podospora anserina, an RIP-like event was observed after several rounds of sexual reproduction in a strain with a 40 kb tandem duplication resulting from homologous integration of a cosmid in the mating-type region. The 9 kb sequenced show 106 C-G to T-A transitions, with 80% of the replaced cytosines located in CpA dinucleotides. This led to the alteration of at least six genes, two of which were unidentified. This RIP-like event extended to single-copy genes between the two members of the repeat. The overall data show that the silencing process is strikingly similar to a light form of RIP, unaccompanied by C-methylation. Interestingly, the N. crassa zeta-eta sequence, which acts as a potent de novo C-methylation RIP signal in this species, is weakly methylated when introduced into P. anserina. These results demonstrate that RIP, at least in light forms, can occur beyond N. crassa.
Assuntos
Ascomicetos/genética , Genoma Fúngico , Mutação Puntual , Sequências de Repetição em Tandem , Metilação de DNA , FenótipoRESUMO
The chloroplast atpB gene of Chlamydomonas reinhardtii, which encodes the beta subunit of the ATP synthase, contains three in-frame ATGs that are candidate translation initiation codons. An earlier study revealed that the N terminus of the assembled beta subunit maps at the +2 position with respect to the second in-frame methionine codon (Fiedler et al. 1995). Using chloroplast transformation, we have examined the possibility that either of the two additional in-frame ATG codons is competent for translation initiation. We provide evidence that translation of atpB is initiated exclusively at the second ATG codon. We conclude that the beta subunit is not synthesized with an N-terminal leader before its assembly into a functional ATP synthase complex.
Assuntos
Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/genética , Códon de Iniciação , Genes de Protozoários , Complexos Multienzimáticos/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Regiões 5' não Traduzidas , Complexos de ATP Sintetase , Sequência de Aminoácidos , Animais , Sequência de Bases , Cloroplastos/enzimologia , Cloroplastos/genética , Primers do DNA/genética , Precursores Enzimáticos/genética , Expressão Gênica , Genes Reporter , Dados de Sequência Molecular , Mutação , Biossíntese de Proteínas , Subunidades Proteicas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Most chloroplast genes in vascular plants are organized into polycistronic transcription units, which generate a complex pattern of mono-, di-, and polycistronic transcripts. In contrast, most Chlamydomonas reinhardtii chloroplast transcripts characterized to date have been monocistronic. This paper describes the atpA gene cluster in the C. reinhardtii chloroplast genome, which includes the atpA, psbI, cemA, and atpH genes, encoding the alpha-subunit of the coupling-factor-1 (CF1) ATP synthase, a small photosystem II polypeptide, a chloroplast envelope membrane protein, and subunit III of the CF0 ATP synthase, respectively. We show that promoters precede the atpA, psbI, and atpH genes, but not the cemA gene, and that cemA mRNA is present only as part of di-, tri-, or tetracistronic transcripts. Deletions introduced into the gene cluster reveal, first, that CF1-alpha can be translated from di- or polycistronic transcripts, and, second, that substantial reductions in mRNA quantity have minimal effects on protein synthesis rates. We suggest that posttranscriptional mRNA processing is common in C. reinhardtii chloroplasts, permitting the expression of multiple genes from a single promoter.
