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. 2016 Feb 15;44(7):3373–3389. doi: 10.1093/nar/gkw073

Figure 7.

Figure 7.

(A) Details of 16S processing stalk. The sequence for the rrnW stalk is shown. Mature 16S rRNA sequences are in blue. Numbering is the distance from the mature 5′ or 3′ ends of 16S rRNA. Sites of previously known endonuclease cleavage are indicated by red arrows. RNase J1 processing of 16S rRNA 5′ end is indicated by the green hooked arrow. Site of endonuclease cleavage discovered in this study is indicated by the purple arrowhead. Sequence downstream of the stalk (in green) is shown for rrnW and other operons (top) and for rrnA and rrnO (bottom). For rrnW and other operons, the 16S sequence is followed by the 23S sequence. The underlined sequence is the single-stranded region between the 16S and 23S processing stalks, which may be the target of RNase Y endonuclease cleavage. For the rrnA and rrnO operons, the 16S sequence is followed by two tRNAs (cf. Figure 6A), whose 5′ end is matured by RNase P cleavage. (B) Northern blot analysis of 16S rRNA 3′ fragment, using an oligonucleotide probe complementary to a sequence immediately upstream of the RNase III cleavage site (III-2) in the 16S stalk. Relevant genotype of strains indicated above each lane. Strains used for experiments in Figure 7 are the ‘CCB’ strains listed in Table 1. Migration of precursor 16S rRNA and processed fragments indicated on the left. Migration of unlabeled RNA markers (nts) shown on the right. (C) Primer extension assay for 5′-end mapping of the 65-nts fragment in the indicated ribonuclease mutant strains. Control sequencing lanes of an rDNA fragment are the leftmost lanes. For ease of reading, the sequence readout is written as the reverse complement. Major 5′ end indicated by arrowhead on right. Strong stops at A1518 and A1519 are due to methylation of these residues by KsgA (67). (D) 5′-RACE mapping of the 65-nts fragment. The 65-nts fragment was ligated to 5S rRNA present in total RNA and reverse transcriptase-PCR was performed across the junction. Boldface sequence is from the 65-nts fragment 5′ end. In different operons, the eighth residue is either A or T.