Figure 3. tracrRNA directs pre-crRNA cleavage by RNase III in vitro

a, Schematic representation of

a, Schematic representation of tracrRNA89 corresponding to 89-nt long tracrRNA, and crRNA213 and crRNA148 corresponding to a 213-nt long leader-repeat-spacer1-repeat-spacer2 fragment and a 148-nt long spacer1-repeat-spacer2-repeat-spacer3 fragment, respectively. b, Identification of tracrRNA89 binding sites on crRNA148*. 5 end-labeled crRNA148* (~10 nM) was subjected to lead(II), RNase III and RNase T1 cleavage in the absence (lanes 1, 4, 7) and presence of cold tracrRNA89 (final concentration in lanes 2, 5, and 7: ~50 nM; lanes 3, 6, and 9: ~500 nM). Lane C: untreated crRNA148*; Lane T1: RNase T1 digest of crRNA148* under denaturating conditions; Lane OH: alkaline ladder; cleaved G residues are labeled. Vertical bars: crRNA148 region protected by tracrRNA89. Arrows denote specific RNase III cleavages in the two repeat regions of crRNA148 in the presence of tracrRNA89. c, Identification of crRNA148 and crRNA213 binding sites on tracrRNA89. 5 end-labeled tracrRNA89* (~10 nM) was subjected to RNase T1, lead(II) and RNase III cleavage in the absence (lanes 1, 6, 11) and presence of cold crRNA148 or crRNA213 (final concentration in lanes 2, 4, 7, 9, 12 and 14: ~50 nM; lanes 3, 5, 8, 10, 13 and 15: ~500 nM). Lanes C, T1 and OH, positions of cleaved Gs and vertical bars: as above but referring to tracrRNA89* in the presence of cold crRNA148 or crRNA213.

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CRISPR RNA maturation by trans-encoded small RNA and host ... - PubMed

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