Figure 8. Ordered processing of the 23S–4.5S rRNA. (A) Analysis of 4.5S precursor rRNA accumulation. One microgram of total RNA from either WT or from rnr1-3 was separated in a 1.2% agarose–formaldehyde gel, and probed with the oligonucleotides indicated in the diagram above. 28S rRNA accumu- lation on the ethidium bromide-stained membrane is shown below each blot to confirm equal loading. (B) Mapping 23S–4.5S precursor 50 ends by cRT–PCR. The 50 ends are shown by solid arrowheads for WT and by open arrowheads for rnr1, with numbers of corresponding clones obtained shown at each position. The 50 end of the mature 23S rRNA is underlined. (C) Mapping of 23S–4.5S precursor 30 ends by cRT–PCR, labeled as in (B). The mature 4.5S and 5S rRNA 30 ends are underlined. Nucleic Acids Research, 2005, Vol. 33, No. 8 2759 organelles in rnr1 plants, suggesting that indeed RNR1 is present both in mitochondria and in chloroplasts. In the longer term, careful immunoblot analysis should resolve these remaining ambiguities. Chloroplast 23S rRNA maturation requires several processing steps Chloroplast rrn operon processing differs somewhat from that of Escherichia coli. In E.coli, 16S and 23S precursor RNAs are excised from a 30S precursor by RNase III, which cleaves the double-stranded stem formed by base pairing of their 50 and 30 ends. This is followed by endonucleolytic 50 end maturation by RNase E, which for 23S RNA generates a 30 overhang that is subsequently processed exonucleolytically to the mature 30 end by RNase T, and to some extent by RNase PH (27). The 30 end processing of 23S RNA occurs on the ribosome, as RNase T-deﬁcient cells accumulate ribosomes with immature 23S RNA (27). Based on its homologous position in the rrn operon, chloro- plast 4.5S rRNA is derived from the fragmentation of the 23S rRNA (28,29), which is accompanied by a 99 nt insertion in Arabidopsis. Endonucleolytic processing within this sequence results in the formation of a mature 4.5S rRNA and a slightly shorter 23S RNA, in comparison with those of most bacteria (29). Therefore, E.coli 23S RNA 30 processing can be com- pared with chloroplast 23S–4.5S rRNA 30 processing. In chloroplasts, there is no sequence complementarity between the 23S 50 end and the 4.5S rRNA 30 end that would suggest base pairing and endonucleolytic excision from the 7.4 kb precursor by an endonuclease like RNase III. In contrast, the 23S–4.5S intermediate is generated by endonucleolytic cleavage 78 nt upstream of the mature 23S 50 end, and 170 nt downstream of the 4.5S rRNA 30 end. This is followed by 30 maturation of the 4.5S sequence in a single step, which either precedes or occurs concomitantly with 23S 50 processing, since cRT–PCR has shown that dicistronic intermediates with mature 4.5S rRNA 30 ends also have mature 23S 50 ends. Our data are also consistent with the hypothesis that 4.5S rRNA 30 maturation must precede excision of the 4.5S rRNA from the dicistronic intermediate (3,4). This is reminiscent of the ordered mitochondrial tRNA processing, although in those cases 30 end maturation is dependent on 50 maturation (30,31). The 50 end maturation of 23S rRNA is probably endonuc- leolytic, as there is no evidence for a 50–30 exoribonuclease in plant chloroplasts (32), although ample evidence for this activ- ity exists for Chlamydomonas chloroplasts (33,34). Based on sedimentation of the dicistronic intermediate with polysomes in sucrose density gradients, this processing occurs on ribosomes, as has been proposed previously for the Brassica napus 23S–4.5S intermediate and for E.coli 23S rRNA (4,35,36). Maturation of the 4.5S rRNA 30 end Processing of the 4.5S rRNA 30 end occurs in a single step, and based on polysome analysis, both this and ﬁnal matura- tion of 23S rRNA also occur on ribosomes. In rnr1 mutants, accumulation of translation-competent ribosomes is limited, presumably due to limiting levels of mature 16S or 5S rRNA or both. This is also thought to be true of maize hcf 7 and