Structure and transcript processing of a Euglena gracilis chloroplast operon encoding genes rps2, atpI, atpH, atpF, atpA and rps18.

Abstract

A 9.8 kbp region of the Euglena gracilis chloroplast genome has been cloned, sequenced and analyzed. This region contains six genes, rps2, rps18, atpI, atpH, atpF and atpA which encode ribosomal proteins S2 and S18 and ATP synthase subunits CFₒIV, CFₒIII, CFₒI and CF₁α, respectively. The linear order of these genes, 5'-rps2-atpI-atpH-atpF-atpA-rps18-3', is similar to that of land plant chloroplasts. These six genes are co-transcribed with two tRNA genes which are 5' to rps2. A fully spliced, 5.5 kb transcript containing all six genes accumulates. The spliced hexa-cistronic transcript is processed by intercistronic cleavage to mono-cistronic mRNAs. The 5' ends of the accumulated mono-cistronic transcripts map to single-stranded regions of the most stable secondary structure for each intercistronic sequence. There is no evidence for initiation of transcription in this region of the Euglena gracilis chloroplast genome. This Euglena chloroplast operon is interrupted by 17 introns. Nine of the introns are group III and seven are group II. All of the group III introns have potential secondary structures near their 3' ends which resemble domain VI of group II introns. The remaining intron is a complex twintron excised as four group III introns. This intron is comprised of two group III introns within the internal intron of a group III twintron. Two of the internal introns are excised from multiple splice sites. Two of the internal introns interrupt the domain VI-like structure of the host group III intron. The 16S rRNA sequence of Euglena chloroplasts is phylogenetically related to the 16S rRNA sequence of chromophyte chloroplasts, while the Euglena derived atpA amino acid sequence is more closely related to atpA sequences of chlorophyte chloroplasts than to atpA sequences of chromophyte chloroplasts. Too few chloroplast ribosomal protein sequences are available in the databases to perform meaningful phylogenetic analysis of rps2 or rps18. Although clustering of rps2 with the ATP synthase genes in chloroplasts of chlorophytes, rhodophytes, chromophytes and euglenophytes, but not prokaryotes, is evidence that chloroplasts are of mono-phyletic origin.