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. Author manuscript; available in PMC: 2009 Nov 1.
Published in final edited form as: Nat Struct Mol Biol. 2009 Apr 19;16(5):541–549. doi: 10.1038/nsmb.1588

Figure 4.

Figure 4

GST-AtL5 fusion protein binds to P5SM RNA in vitro. (a) In vitro binding analysis for the P5SM RNA (transcribed from nucleotides 603-810 of the AtTFIIIA gene, NCBI gi 42592260) with either GST or GST-AtL5. Radiolabeled RNA was incubated at 25°C in binding buffer in the absence or presence of protein (0-1 μM). RNA-protein complex formation was analyzed by non-denaturing PAGE. Unbound RNA (filled arrowhead) and RNA-protein complex (open arrowhead) are indicated. (b) Representative plot used to determine the apparent KD for the interaction between P5SM RNA and GST-AtL5 protein. Maximal binding observed at 1.25 μM protein was normalized to 1. Graphed line corresponds to the best-fit curve for a two-state binding model with 1:1 stoichiometry and KD of 75 nM. (c) In vitro competition binding analysis. Radiolabeled P5SM RNA mixed with buffer alone or unlabeled RNA (5S rRNA or P5SM, 50-200 nM) was incubated with GST-AtL5 protein (0.6 μM) at 25°C and analyzed by non-denaturing PAGE. The percent bound for each lane is shown below the gel. Other details are as described in a.