petD mRNA 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 specific deletion mutants, we show that petD, which encodes subunit IV of the cytochrome b₆lf complex, has an RNA processing site that is required for accumulation of monocistronic petD mRNA in petD promoter deletion mutants; in such mutants, transcription of petD originates from the upstream petA promoter. The 5' ends of transcripts initiated at the petD promoter are probably also generated by processing, since the 5' end of monocistronic petD mRNA is the same in wild-type strains as it is in the petD promoter mutants. The location and function of the processing site were further examined by inserting petD-uidA fusion genes into the chloroplast genome (uidA is an Escherichia coli gene that encodes β-glucuronidase). When a promoterless petD-uidA fusion gene was inserted downstream of petA, a monocistronic uidA transcript accumulated, which was apparently initiated at the petA promoter and was processed at a site corresponding precisely to the petD mRNA 5' end. When a construct including only sequences downstream of +25 relative to the mature mRNA 5' end was 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 partially within the region from -1 to +25. β-Glucuronidase activity was not detected in transformants that accumulated only the dicistronic petA-uidA transcript, suggesting that the first 25 bp of the 5' untranslated region are required for translation initiation. One explanation for this translational defect is that Chlamydomonas chloroplasts cannot translate the second coding region of some dicistronic messages.