PETD MESSENGER-RNA MATURATION IN CHLAMYDOMONAS-REINHARDTII CHLOROPLASTS - ROLE OF 5' ENDONUCLEOLYTIC PROCESSING

Complex processing of primary transcripts occurs during the expression of higher-plant chloroplast genes. In Chlamydomonas reinhardtii, most chloroplast genes appear to possess their own promoters, rather than being transcribed as part of multicistronic operons. By generating spe cific deletion mutants, we show that petD, which encodes subunit IV of the cytochrome b(6)/f complex, has an RNA processing site that is req uired for accumulation of monocistronic petD mRNA in petD promoter del etion mutants; in such mutants, transcription of petD originates from the upstream petA promoter. The 5' ends of transcripts initiated at th e petD promoter are probably also generated by processing, since the 5 ' end of monocistronic petD mRNA is the same in wild-type strains as i t is in the petD promoter mutants. The location and function of the pr ocessing site were further examined by inserting petD-uidA fusion gene s into the chloroplast genome (uidA, is an Escherichia coli gene that encodes beta-glucuronidase). When a promoterless petD-uidA fusion gene was inserted downstream of petA, a monocistronic uidA transcript accu mulated, which was apparently initiated at the petA promoter and was p rocessed at a site corresponding precisely to the petD mRNA 5' end. Wh en a construct including only sequences downstream of +25 relative to the mature mRNA 5' end,vas inserted into the same site, a dicistronic petA-uidA transcript accumulated but no monocistronic uidA transcript could be detected, suggesting that a processing site lies at least par tially within the region from -1 to +25. beta-Glucuronidase activity w as not detected in transformants that accumulated only the dicistronic petA-uidA transcript. suggesting that the first 25 bp of the 5' untra nslated region are required for translation initiation, One explanatio n for this translational defect is that Chlamydomonas chloroplasts can not translate the second coding region of some dicistronic messages.