Abstract
The primary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from the marine diatom Cylindrotheca sp. strain N1 has been determined. Unlike higher plants and green algae, the genes encoding the large and the small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase are chloroplast-encoded and closely associated (Hwang and Tabita, 1989). The rbcL and rbcS genes in strain N1 are cotranscribed and are separated by an intergenic region of 46 nucleotide base pairs. Ribosome binding sites and a potential promoter sequence were highly homologous to previously determined chloroplast sequences. Comparison of the deduced primary structure of the diatom large and small subunits indicated significant homology to previously determined sequences from bacteria; there was much less homology to large and small subunits from cyanobacteria, green algae, and higher plants. Although high levels of recombinant diatom large subunits could be expressed in Escherichia coli, the protein synthesized was primarily insoluble and incapable of forming an active hexadecameric enzyme. Edman degradation studies indicated that the amino terminus of the large subunit isolated from strain N1 was blocked, suggesting that the mechanism responsible for processing and subsequent assembly of large and small subunits resembles the situation found with other eucaryotic ribulose-1,5-bisphosphate carboxylase/oxygenase proteins, despite the distinctive procaryotic gene arrangement and sequence homology.
Highlights
The primary structure of ribulose-1,5-b:sphosphate carboxylase/oxygenase from the marine diatom Cylindrotheca sp. strain N1 has been determined
Edman degradation studies indicated that the amino terminus of the large subunit isolated from strain N1 was blocked, suggesting that the mechanism responsible for processing and subsequent assembly of large and small subunits resembles the situation found with other eucaryotic ribulose-1,5-bisphosphatecarboxylaseloxygenase proteins, despite the distinctiveprocaryotic gene arrangement and sequence homology
Thesmall subunit sequence from two other chlorophyll c-containing organisms, Olisthodiscus luteus(Boezar et al, 1989) and Cryptomonas (Douglas and Durnford, 1989), indicates that the small subunit resembled the sequencefrom chemolithoautotrophic (AndersenandCaton, 1987) andphotosynthetic bacteria’ more than sequences obtained from plants and green algae
Summary
C l y Phe Tyr Asp ThrLeuArp Leu ThrThrLeu Asp ValAsn Leu ProTyr Gly Leu PhePhe Clu Met Ser Trp A l a Ser Leu Arg Arg C y 0 Wet Pro ValAla Ser Gly Gly I l e His C y s Gly C l n Met. Rbu-P, Northern Blot Analysis-Total RNA was isolated according to the carboxylase large and small subunit genes were expressed in Esche- procedures of Maniatis et al (1982). 0.4 ml of the infected cells were transferred into 10 ml of precipitation with ethanol, the RNA was resuspended in H20; 10 pg. The cells of RNA was separated on a 1.2% agarose gel containing 6%. In A, the 2.1-kb BamHI-EcoRI fragment of plasmidpVT223, containing both rbcL and rbcSwas nick-translated and labeled with R2Pand used as a probe for DNARNA hybridization.XIHindIIIDNAwasused as asize marker as indicated by the numbers obtained from a fluorescence photo (not shown). In an independent experimen(t B ) ,the RNA transcript was identified using a ‘?P-labeled internal DNA fragment of rbcL or the entire rbcS as probes. SDS-PAGE was performed as previously described (Hwang and Tabita1, 989)
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