Structure and expression of a Euglena gracilis chloroplast transcription unit encoding 11 ribosomal protein genes, a tRNA gene and a 2.8 kb intergenic region

Abstract

The structure and expression of a novel Euglena gracilis chloroplast ribosomal protein operon was studied by gene mapping, molecular cloning, nucleotide sequencing primer extension and Northern analyses. The nucleotide sequence (12,240 bp) was determined for 100% of both strands encoding the 12 genes, rpl23 - rpl2 - rps19 - rpl22 - rps3-(2.8 kb region)- rpl16 - rpl14 - rpl5 - rps8 - rpl36 - trnI - rps14. The gene organization resembles the S10 and spc ribosomal protein operons of E. coli. The rpl5 gene was a new chloroplast gene not previously reported for any chloroplast genome nor described as a nuclear gene. The presence of numerous introns and an unusual 2.8 kb rps3-rpl16 intercistronic region were additional features that were unparalleled in other chloroplast DNAs. At least 15 introns were identified in the genes. Evidence is presented from primer extension analysis of chloroplast RNA for the correct in vivo splicing of six of the introns. Two introns within rps8 flanked an 8 bp exon, the smallest exon yet characterized in a chloroplast genome. Four introns shared structural properties with group II organelle introns. The remaining 11 introns were defined as new category of organelle intron, now designated "group III." The presence of additional introns in several intercistronic regions is proposed. Conserved regions in the predicted polypeptides were identified from the alignments with related proteins from other chloroplasts and bacteria. Evidence from Northern hybridization experiments with gene-specific probes supported the interpretation that 11 ribosomal protein genes, the 2.8 kb rps3-rpl16 intercistronic region and trnI were co-transcribed and encoded in a single operon. The co-transcription of genes coding for proteins and a tRNA is a novel finding for a chloroplast operon. Several stable polycistronic transcripts were identified, including a common 8.3 kb pre-mRNA. Stepwise processing pathways proposed for the mRNAs are described. Most mRNAs appeared to be fully spliced. The 5$\sp\prime$ ends of mRNAs for the first gene in the operon, rpl23, were mapped by primer extension. Plastid mRNAs from dark and light grown Euglena were analyzed on Northern blots.