Xenbase Image ID: 138451

Figure 1. Nocturnin Exhibits Deadenylase Activity In Vitro in a Magnesium-Dependent Manner (A) A diagram of G52 DNA shows resulting constructs G52 A+ (189 nt) and G52 A(100)N(31) (217 nt) when linearized with BamHI and HindIII restriction enzymes, respectively. After restriction digests, radiolabeled runoff transcripts are generated from the SP6 promoter in the presence of [α-32P]-UTP. G52 A+ and G52 A(100)N(31) contain a 100 nucleotide poly(A) tail followed by either 3 or 31 non-adenylate sequence, respectively. (B) Nocturnin deadenylates a synthetic mRNA substrate in a time-dependent, EDTA-sensitive manner. Purified nocturnin protein (GST-noc, 1 μg) was incubated with radiolabeled poly(A)+ G52 mRNA, and deadenylation was measured at time points up to 30 min (lanes 1–6). A decrease of activity was observed with the addition of EDTA (lane 7). Arrows indicate poly(A)+ and poly(A)− sizes, and estimated transcript size is based on comparison with a marker of 189 nt and 75 nt (M, lane 8) and an RNA ladder (not shown). (C) HIS-PARN (0.08 μg) was tested by using the same assay conditions and serves as a positive control. (D) Deadenylase activity was abolished when a point mutation was generated in the putative magnesium binding domain of nocturnin (E152A). Equal amounts of WT and E152A protein (1 μg) were added for direct comparison. Image published in: Baggs JE and Green CB (2003) Copyright © 2003. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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