FIG. 2.

Effect of MnCl₂, ATP, and α-ketoglutarate (α-KG) on uridylylation. (A) Uridylylation of GlnB (0.5 μM) with 0, 0.3, 0.6, 3.0, 6.0, or 16 mM MnCl₂ together with 2 mM ATP, 60 μM α-ketoglutarate, 0.13 μM GlnD, and 0.5 mM UTP supplemented with [α-³²P]UTP. (B) Effect of the absence of ATP or α-ketoglutarate on uridylylation of GlnJ (0.5 μM). The assays also contained 3 mM MnCl₂, 0.13 μM GlnD, and 0.5 mM UTP supplemented with [α-³²P]UTP. Uridylylation was assayed by incorporation of [α-³²P]UMP as described in Materials and Methods. Samples were withdrawn from the reaction mixtures after 20 min of incubation and stopped by addition of SDS cocktail.