Nucleic Acids Research, 2005, Vol. 33, No. 8 2761 Figure 11. Sucrose density gradient fractions from WT and rnr1 were analyzed by RNA gel blot with the probes indicated to the right of each panel. The 12 lanes from left to right indicate the gradient fractions, with 1 representing the top of the gradient and 12 representing the bottom of the gradient. An ethidium bromide-stained membrane for each sample is included; equal proportions of each fraction were loaded. The positions of ribosomal (<80S) and polysomal fractions were determined by running puromycin-treated samples in parallel to experimental samples, as indicated at the bottom. Chloroplast 5S rRNA maturation requires two exonucleolytic processing steps 5S rRNA maturation pathways vary widely, even among bacteria. In E.coli, the 9S precursor is cleaved 3 nt upstream of the 5S rRNA 50 end and 3 nt downstream of its 30 end by RNase E (41), incorporated into ribosomes, and its 30 end is trimmed primarily by RNase T (42). However, not all organ- isms require RNase E for rRNA processing. Bacillus subtilis and many other gram-positive bacteria generate mature 5S rRNA directly by double-stranded cleavage of the 5S ‘molecular stalk’ by RNase M5 (43,44). The Arabidopsis nuclear genome encodes an RNase E-like protein that is predicted to be chloroplast targeted (At2g04270), but whether this enzyme is important in rRNA processing is unknown. As in E.coli, chloroplast 5S rRNA appears to be generated by single-stranded endonucleolytic cleavages upstream and downstream of the mature 5S sequence, although the precursor is much longer. Based on our results, the chloroplast pre-5S rRNA 30 end is generated by endonucleolytic cleavage near the 50 end of tRNAArg, possibly by RNase P, to generate the 350 nt precursor sometimes observed in rnr1 plants. This precursor is trimmed through sequential exonucleolytic steps, the ﬁrst gen- erating transcripts with 10–15 nt 30 extensions, and the second generating the mature 30 end. One or both of these steps appear to be catalyzed by RNR1, but because some mature 5S rRNA accumulates in rnr1 mutants, another less active exonuclease, perhaps PNPase, can act in its place. An interesting observation is that while 16S, 23S and 23S–4.5S precursors accumulated signiﬁcantly in rnr1, the 350 nt 5S rRNA precursor did not, and was often difﬁcult to observe. We hypothesize, therefore, that it is actively degraded, possibly through the polyadenylation-dependent RNA turnover pathway. Polyadenylation of pre-5S rRNA in an E.coli RNase T PH D BN mutant strain has been observed previously (45). RNR1 may require accessory proteins for processivity in vivo Similar to puriﬁed E.coli RNase II and RNase R, recombinant RNR1 is a non-speciﬁc exoribonuclease. The question, then, is what drives RNRs1 substrate speciﬁcity in vivo. One probab- ility is that RNR1 activity, similar to that of E.coli RNase II (46), is modulated by RNA secondary structure in vivo, much as it is in vitro (14,19). Secondary structure increases the stability of mRNAs, such as the trpT terminator, and in general those containing repetitive extragenic palindrome sequences in bacteria (47–49), or 30 stem–loop structures in chloroplasts (1). In this view, RNR1 would digest the unstructured exten- sions downstream of the mature rRNA 30 ends, but stall when it reached their structured regions. The speciﬁcity of RNR1 in rRNA processing is also emphasized by the fact that the mat- uration of trnR appeared to be RNR1-independent, or the role of RNR1 is fully redundant in its processing. However, rnr1 mutants were less efﬁcient at processing long, highly structured pre-16S, pre-23S–4.5S and to a lesser