2756 Nucleic Acids Research, 2005, Vol. 33, No. 8 Figure 4. Analysis of rRNA expression and processing. (A) Diagram of the rRNA operon and location of probes (A–F) used for RNA gel blot analyses, and sizes of transcripts (in kb) observed by the gel blot analysis shown in (B). (B) RNA gel blot analysis. One microgram of total RNA from WT or rnr1-3 was separated in 1.2% agarose–formaldehyde gels, and blots were probed as indicated above each blot, with transcripts identified shown to the right. 28S rRNA is shown in the ethidium bromide-stained membrane below each experiment to confirm equal loading. The transcripts marked by asterisks for probe D are mature 23S rRNA processed at hidden breaks (see text). Figure 5. Mapping of 16S rRNA 50 and 30 ends by cRT–PCR. The 50 and 30 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 and 30 ends of the mature 16S rRNA sequence are underlined. clone contained a 12 nt extension. Thus, while the lack of RNR1 causes only a modest increase in the accumulation of the 1.7 kb pre-16S RNA, the mutation prevents the ﬁnal 30 processing of the mature transcript. The processing of 23S RNA at the hidden breaks complic- ates its analysis by cRT–PCR. Maturation of 23S 30 ends was, therefore, analyzed by RNase protection, as shown in Figure 6. RNA from WT or from rnr1 (or yeast tRNA as a negative control) was hybridized to a 190 nt antisense RNA probe (Figure 6A), digested with RNase T1 and RNase A, and ana- lyzed in a 6% denaturing polyacrylamide gel (Figure 6B). In RNA samples from WT plants, the probe protected an 86 nt fragment that represents the mature 23S RNA and, to a lesser extent, an 184 nt band that represents the dicistronic 23S– 4.5S species, as shown below. No mature 23S 30 ends were observed in the rnr1 mutant, but these plants did accumulate incompletely processed 23S 30 ends with 10–15 nt extensions. Thus, 30 end maturation of both 16S and 23S rRNAs is affected in rnr1. The small sizes of 4.5S and 5S rRNAs allow their analysis by high-resolution RNA gel blots in addition to RNase pro- tection. As shown in Figure 7A, although monocistronic 4.5S rRNA accumulates to a much lower level in rnr1 plants than in WT, no defect in its 30 processing was observed, which would have been apparent as a slightly longer species. The 30 end processing of 4.5S rRNA was also analyzed by RNase protection (Figure 7B and C). In WT plants, 4.5S rRNA accumulates both as a mature species of 97–99 nt, and as a weaker band of 270 nt, which represents an 170 nt 30 extension. In the mutant, a signiﬁcantly higher proportion of 4.5S rRNA accumulated as the precursor species, but a low level of mature 4.5S rRNA also accumulated, as was observed by RNA gel blot. While RNase protection clearly showed the accumulation of precursor 4.5S transcripts in the mutant (Figure 7C), RNA gel blot analysis suggested that these did not represent monocis- tronic pre-4.5S rRNAs, as such a species of 270 nt would have easily been observed (Figure 4B, probe E; Figure 7A). It was likely, therefore, that the 4.5S precursors were derived entirely from dicistronic 23S–4.5S species rather than from