Figure 3.

Filter binding assays indicate two allosterically regulated nucleotide binding sites. Binding of ³²P-labeled bantam to DmAgo1 in a and DmAgo2ΔQ in b in the presence of non-labeled RNAs of varying lengths (5, 15, 23 and 30 nts). Competition between labeled and non-labeled RNAs was performed by titration of the non-labeled species and represented by the ratio of bound ³²P-bantam in the presence and absence of competing RNA species. Influence of different nucleotide substrates on the ³²P-bantam binding to DmAgo1 in c and DmAgo2ΔQ in d. Nucleotide substrates were titrated and the amount of the bound ³²P-bantam was followed. Values represent the ratio of bound ³²P-bantam in the presence and absence of indicated nucleotide substrate. e, Binding of bantam duplex (miRNA*) to DmAgo1 and DmAgo2ΔQ stimulates only binding of DmAgo1 to ³H-labeled m⁷GpppG mRNA. f, Influence of different nucleotide substrates on binding of ³H-m⁷GpppG mRNA to DmAgo1. Error bars represent the standard error from at least three experiments.