Characterization of the structure and expression of the Euglena gracilis chloroplast rpoC1 and rpoC2 gene loci.
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AbstractIn order to expand our understanding of the expression of chloroplast genes, the structure and expression of the Euglena gracilis rpoC1 and rpoC2 loci were studied. The rpoC1 and rpoC2 gene products are similar to the amino- and carboxyl-terminal regions of the $\beta\sp\prime$ subunit of E. coli RNA polymerase. The nucleotide sequence (7,270 bp) was determined for 100% of both strands encoding these two genes. The rpoC1 and rpoC2 genes are located downstream and in the same polarity as the rpoB gene. The organization of the Euglena rpoB-rpoC1-rpoC2 genes is conserved in plant chloroplasts and is similar to the E. coli rpoB-rpoC operon. The Euglena rpoC1 gene (586 codons) encodes a polypeptide with a predicted molecular weight of 68,043. The rpoC1 gene is interrupted by one group II intron of 349 bp, seven group III introns of 107, 100, 119, 97, 110, 102 and 103 bp, and three atypical introns of 210, 213 and 198 bp. The Euglena rpoC2 gene (830 codons) encodes a polypeptide with a predicted molecular weight of 94,628. The rpoC2 gene is interrupted by two group II introns of 580 and 514 bp, respectively. All of the exon-exon junctions were experimentally determined via cDNA cloning and sequencing analysis. Multiple protein alignments of the rpoC1 and rpoC2 gene products with related proteins from bacteria and chloroplasts were used to identify conserved regions. Transcripts from the rpoC1 and rpoC2 loci were characterized via Northern analysis. The rpoB, rpoC1 and rpoC2 genes are cotranscribed. Fully spliced tri-, di- and monocistronic transcripts were detected with hybridization probes specific for each gene. The relative abundance of the rpoC1 and rpoC2 transcripts is similar in RNA from dark- and light-grown Euglena. The mature 5'-ends of the rpoC1 and rpoC2 genes were mapped by primer extension. The 3'-end of the mature rpoC2 transcript was localized via an S1 nuclease protection assay. The rpoC1 and rpoC2 gene products were also compared to the largest subunits of RNA polymerases from archaebacteria and eukaryotes. The evolution of the Euglena genes is discussed.