Figure 3.

B. subtilis NrnA possesses a dinucleotide substrate length preference with a slight preference for 5′ purine residues. (A, B) DRaCALA, as described elsewhere (35), was used to measure binding of different radiolabeled dinucleotides to the catalytically inactive mutant NrnA_Bs D80N D156N. (C) For cleavage assays, 1 μM of the indicated dinucleotide substrate was incubated with trace amounts of ³²P-radiolabeled RNA and 100 nM of NrnA Products were removed at time intervals and resolved by denaturing PAGE. Quantification of the normalized radioactive intensity of the initial substrate was plotted as the average and SD of three independent experiments. (D) Substrate binding competitions were conducted by DRaCALA. 1 μM of purified NrnA_Bs D80N D156N was bound to ∼1 nM of ³²P labeled AA, and subsequently incubated with either 10 or 100 μM of the indicated unlabeled competitor molecules. The fraction bound was normalized to the maximum binding exhibited by NrnA_Bs D80N D156N to ³²P-radiolabeled AA. (D, E) Kinetic analysis of B. subtilis NrnA was performed using a previously published continuous kinetic assay (39), with slight modifications. The continuous coupled enzyme assay depended on the liberation of AMP from the RNA substrate by NrnA_Bs, followed by the conversion of AMP to ATP by PPDK_Cs, which was then used by luciferase to generate a pulse of light. Since the hydrolysis of AA results in the generation of 2x AMP molecules, the initial rates for AA hydrolysis were divided by 2. Substrate degradation was restricted to 10% or less, and a blank containing all reaction materials except for NrnA_Bs was included as a control for background luminescence. (B–F) All data was plotted as the average and SD of three independent experiments.