Cloning and transcription analysis of the ndh(A-I-G-E) gene cluster and the ndhD gene of the cyanobacterium Synechocystis sp. PCC6803

The plastid DNA of higher plants contains eleven reading frames that are homologous to subunits of the mitochondrial NADH-ubiquinone oxidoreductase (complex I). The genes are expressed, but a plastid NAD(P)H dehydrogenase has not yet been isolated and the function of the enzyme in plastid metabolism is unknown. Cyanobacteria also contain a NADH dehydrogenase that is homologous to the mitochondrial complex I. The enzyme is sensitive to rotenone and is located on the cytoplasmic and the thylakoid membrane. We report here the sequence of five subunits (ndhA, -I, G, -E and -D) of the NADH dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC6803. As in plastid DNA, the genes ndh(A-I-G-E) are clustered and probably constitute an operon. The ndhD gene is associated with a gene encoding an iron-sulphur protein of photosystem I (psaC) as in plastid DNA. In contrast to the situation in plastids, psaC and ndhD are not cotranscribed but transcribed from opposite strands. The deduced amino acid sequence of the cyanobacterial polypeptides is more similar to the corresponding plastid (40-68% identity) than to the corresponding mitochondrial subunits (17-39% identity). Thus, the cyanobacterial NADH-dehydrogenase provides a prokaryotic model system which is more suitable to genetic analysis than the enzyme of plastids.     

Both RNA editing and RNA cleavage are required for translation of tobacco chloroplast ndhD mRNA: a possible regulatory mechanism for the expression of a chloroplast operon consisting of functionally unrelated genes.

Tobacco chloroplast genes encoding a photosystem I component (psaC) and a NADH dehydrogenase subunit (ndhD) are transcribed as a dicistronic pre-mRNA which is then cleaved into short mRNAs. An RNA protection assay revealed that the cleavage occurs at multiple sites in the intercistronic region. There are two possible initiation codons in the tobacco ndhD mRNA: the upstream AUG and the AUG created by RNA editing from the in-frame ACG located 25 nt downstream. Using the chloroplast in vitro translation system, we found that translation begins only from the edited AUG. The extent of ACG to AUG editing is partial and depends on developmental and environmental conditions. In addition, the in vitro assay showed that the psaC/ndhD dicistronic mRNA is not functional and that the intercistronic cleavage is a prerequisite for both ndhD and psaC translation. Using a series of mutant mRNAs, we showed that an intramolecular interaction between an 8 nt sequence in the psaC coding region and its complementary 8 nt sequence in the 5' ndhD UTR is the negative element for translation of the dicistronic mRNA. A possible mechanism in which the differential expression of the chloroplast operon consists of functionally unrelated genes is discussed.