FIG. 3.

U addition to different RNA substrates using glycerol gradient-purified complex I. (A) Predominately one stable U is added to gRNAs. 5′-end-labeled gA6-[14] and gA6-[48] were incubated with the designated NTP at 1 mM. Arrows point to the single-U addition product, and circles designate circularization of the gRNA by RNA ligase present in the extract. A minus sign indicates buffer without enzyme. (B) A poly(A) RNA is a substrate for both a single U addition and a poly(U) tail. The nucleotides added are indicated at the bottom. The arrow points to the single-U addition product, and the bracket shows the 10- to 15-nucleotide poly(U) tail. A minus sign represents buffer without enzyme. The values on the right are numbers of nucleotides. (C) A 5′-end-labeled gA6-[14] gRNA that already contains a single 3′ U is not a substrate for another U. The plus and minus symbols refer to with- and without-enzyme conditions, respectively. All products were analyzed by 8% denaturing PAGE and visualized by autoradiography.