Proteins encoded by a complex chloroplast transcription unit are each translated from both monocistronic and polycistronic mRNAs.

Abstract

Chloroplast genes are typically organized into polycistronic transcription units that give rise to complex sets of overlapping RNAs through a series of processing steps. The functional significance of this complicated mode of expression is unknown. To determine whether processing of the primary transcript is required to create translatable mRNAs, the translational properties of the RNAs derived from the maize psbB gene cluster (containing the psbB, psbH, petB and petD genes) were examined. Almost all of the approximately 20 RNAs derived from this region co‐sediment with polysomes in sucrose gradients, suggesting that at least one coding region on most transcripts is translated. To determine which sequences are translated on each polycistronic RNA, antibodies to psbB, petB or petD proteins were used to immunoselect polysomes engaged in the synthesis of each protein. Northern and S1 nuclease analyses of the immunoselected RNAs revealed that (i) potential start codons within the petB and petD introns are not functional in translation; (ii) all transcripts containing spliced petB or petD sequences are translated to give these proteins, regardless of upstream or downstream sequences; (iii) psbB is translated from all transcripts encoding it. It is concluded that intercistronic processing is not required for translation of these RNAs, although certain processing steps may enhance translational efficiency.

BIOGENESIS FACTOR REQUIRED FOR ATP SYNTHASE 3 Facilitates Assembly of the Chloroplast ATP Synthase Complex

“…RNA gel-blot analysis was performed using the DIG Easy Hyb system (Roche). Polysome association analysis was performed as described by Barkan (1988). The signals were visualized with a LuminoGraph WSE-6100 (ATTO).…”

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

“…Likewise, details of the homologous recombination process and the deletion of mismatched nucleotides were evident using heterologous flanking sequences (Ruhlman et al, 2010). The translation of native polycistrons without the need for processing to monocistrons has been demonstrated (Barkan, 1988;Zoschke and Barkan, 2015), but the similarity of this process using heterologous polycistrons engineered via the chloroplast genome offered even more direct evidence for this process (De Cosa et al, 2001;Quesada-Vargas et al, 2005). The insertion of replication origins into chloroplast vectors offered further insight into minimal sequences required to study this process (Daniell et al, 1990).…”

hcf5, a Nuclear Photosynthetic Electron Transport Mutant of Arabidopsis thaliana with a Pleiotropic Effect on Chloroplast Gene Expression

“…Like other plants, Arabidopsis has a polycistronic psbBpsbH-petB-petD transcript that undergoes a complex series of processing events (Tanaka et al, 1987;Barkan, 1988;Westhoff and Herrmann, 1988). Figure 7 shows RNA blots from mutant and wild-type siblings hybridized with the probes shown as double-headed arrows in the map.…”