Characterization of the structure and expression of the Euglena gracilis chloroplast rpoB and 23S ribosomal-RNA genes

Abstract

The rpoB gene coding for a β-like subunit (homologous to the E. coli DNA-dependent RNA polymerase β subunit) of the chloroplast DNA-dependent RNA polymerase was located on the chloroplast genome of Euglena gracilis distal to the rrnC ribosomal RNA operon. The complete nucleotide sequence of the gene was determined. The sequence includes 97 bp of the 5S rRNA gene, an intergenic spacer of 1264 bp, the rpoB gene of 4249 bp, 84 bp spacer and 67 bp of the rpoC1 gene. The rpoB gene is of the same polarity as the rRNA operons. The organization of the rpoB and rpoC genes resemble the E. coli rpoB-rpoC and higher plants chloroplast rpoB-rpoC1-rpoC2 operons. The Euglena rpoB gene (1082 codons) encodes a polypeptide with predicted molecular weight of 124,288. The rpoB gene is interrupted by seven Group III introns of 93, 95, 94, 99, 101, 110 and 99 bp, respectively, and a Group II intron of 309 bp. All other known chloroplast rpoB genes lack introns. All the exon-exon junctions were experimentally determined by cDNA cloning and sequencing or direct primer extension RNA sequencing. Transcripts from the rpoB locus were characterized by Northern hybridization. Fully-spliced, monocistronic rpoB mRNAs, as well as rpoB-rpoC1 and rpoB-rpoC1-rpoC2 mRNAs were identified. Unspliced intron-containing transcripts could not be detected in these experiments. The rpoB gene is the first gene in the RNA polymerase rpoB-rpoC1-rpoC2 transcription unit. The three genes are transcribed from a promoter located upstream the rpoB gene. The transcript is processed to mature monocistronic mRNAs. The relative abundance of the mono-, di- and tricistronic mRNAs appear to be similar in RNAs isolated from photoautotrophic, heterotrophic and dark grown cells. The mature 5'- and 3'-ends of the mature rpoB monocistronic transcripts were determined via S1 nuclease mapping and primer extension RNA sequencing. In addition, the sequence of the 23S rRNA from the rrnC operon and the intergenic spacer between the rrnA and rrnB operon were determined. Transcription initiation for the ribosomal RNA transcription unit was determined via Northern analysis and S1 nuclease mapping of chloroplast RNA that was in vitro 5'-end labeled. Two transcription initiation sites were mapped at positions +1 and -50 upstream the 16S rRNA gene. The 3'-ends of the rrnA/rrnB and rrnC 5S rRNA were determined using S1 nuclease protection experiments. The protected fragments were of identical size. The rpoB-C1-C2 DNA sequence has been submitted to EMBL, accession number X17171, and the 23S rRNA DNA sequence was given the number X13310.