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. 2018 Jul 2;7:e36734. doi: 10.7554/eLife.36734

Figure 5. Effect of mismatched targets on cOA production.

Oligonucleotides containing mismatches ranging along the length of the target RNA were used to test both backbone cleavage and cOA synthesis activity by Csm. Backbone cleavage is abolished when two mismatches flank the cleavage site, but is otherwise active. cOA synthesis is not observed for substrates MM0.5 and MM1, which introduce two consecutive mismatches close to the 3’ end of the target RNA. Bands marked with an asterisk are artefactual bands present in the RNA substrate. For all targets, lane c shows the reaction at the zero time point and the three further time points correspond to incubation for 5, 10 and 20 min. Further substrates are shown in Figure 5—figure supplement 1. (B) Table summarising backbone cleavage and cOA production for the mismatch substrates studied in Figure 5A and Figure 5—figure supplement 1. Backbone cleavage sites are indicated with a triangle and mismatches with an ‘X’. The target RNA is represented by the grey line.

Figure 5.

Figure 5—figure supplement 1. Backbone cleavage and cOA production for target RNAs with altered 5’ and 3’ ends.

Figure 5—figure supplement 1.

(A) Backbone cleavage for Target RNA wild-type, Match (with eight nt match to 5’ handle of crRNA) and 3’-blunt (with no 3’ extension). All three targets are cleaved by the Csm complex. Control c1 and c2 represent incubation of target RNA without Csm for 30 min at 0 or 70°C, respectively. Lanes – and + represent assays in the presence or absence of 1 mM ATP, respectively. For the 3’-blunt target, time points of 5 and 30 min are shown. (B) cOA production is seen for the WT but not the Match or 3’-blunt targets. A control target (5’ -blunt) lacking the 5 nt 5’ overhang present in the WT target behaves like the WT target. This serves as a control for the Match target, which also lacks these 5 nt. Lane c is run in the absence of Csm protein. The time points are 5 and 30 min for WT and 3’-blunt, and 5, 10, 30 min for the 5’-blunt substrate. All oligonucleotide sequences are shown in Table 1. (C) Target RNAs carrying mismatches at sites M1.5, M2.5 and M4 all support backbone cleavage and cOA production. Details as for Figure 5.