Transcription and intercistronic RNA processing of polycistronic operons of Euglena gracilis chloroplast.

Abstract

The Euglena chloroplast genome contains 3 sets of genes for ribosomal RNAs 16S, 23S and 5S, and an additional 16S gene; 31 tRNA genes; 25 protein coding genes for transcription and translation; 27 protein coding genes for photosynthesis; and 15 open reading frames. The majority of these genes are transcribed as polycistronic operons. Using primer extension RNA sequencing, in vitro capping and S1 nuclease protection, I have determined 6 transcription start sites for operons of protein coding genes. These are found upstream of chlI, psbD, psbA, rpl20, psbI and rpoB. Sequence upstream of the chlI, rpl20 and rpoB genes resembles the bacterial -10 and -35 promoter consensus (TATAAT and TTGACA, respectively). Sequence upstream of psbD and psbI is divergent from the bacterial consensus possessing a T-hexamer in the -35 position. The psbA promoter also has a -10 element and an AT-rich stretch of nucleotides between -14 to -42. Additional promoters are located upstream of psbK and each 16S copy. These promoters may be sufficient to drive transcription of the Euglena chloroplast genome. Transcription from the 8 known Euglena chloroplast promoters produces large polycistronic RNAs which are processed in the intercistronic regions to smaller rRNAs, tRNAs and mRNAs. In order to understand the mechanisms and function of intercistronic processing, I defined the processing sites of the psaA operon between the psaA and psaB genes, and the psaB and psbE genes using S1 nuclease protection and primer extension RNA sequencing. Using RNA blot hybridization I also determined that intercistronic processing of the psaA operon or the products of intercistronic processing are developmentally regulated. An unusual intercistronic processing event was determined by genomic and cDNA sequencing of the rps4-rps11 dicistronic operon. These genes are not processed to monocistronic mRNAs, however, an intron located in the intercistronic region of rps4 and rps11 is spliced. This intron lies between the Shine-Dalgarno sequence and the AUG for rps11. Removal of the intron would restore the ribosome binding site for translation of the rps11 gene.