Inhibition of in vitro gRNA-independent U-insertions into pre-edited cytochrome b mRNA by NADP(H). The pNB2 substrate RNA was incubated with mitochondrial TL extract with [a-32P]UTP as described in Methods. Different amounts of dinucleotides were added to the reaction mixtures prior to addition of the [a-32P]UTP. The labeled intact RNA was gel-isolated, annealed to an oligomer and digested with Rnase H. The 3' and 5' fragments (indicated by arrows) were separated by electrophoresis and the relative amount of label in the 5' fragments measured by PhosphorImager densitometry. Lane 1, control - no dinucleotide addition. Lanes 2-9, 0.5 mM and 5 mM of indicated dinucleotides added.
Inhibition of in vitro gRNA-independent and gRNA-dependent U-insertions into site 1 of pre-edited ND7x mRNA by NADP(H). The indirect primer extension assay of Byrne et al. (10) was used. The two first lanes show the ladder of gRNA-independent U-insertions obtained with the ND7.1x mRNA substrate in the absence (lane 1) or in the presence (lane 2) of 5 mM NADP and the two last lanes show the gRNA-dependent U-insertions obtained with the ND7.2x mRNA substrate with added gND7x[+3] gRNA in the absence (lane 3) or in the presence (lane 4) of 5 mM NADP. The position of the +3 primer extension band is indicated on the left. The +1 band in all the lanes is an artifact that occurs in the absence of treatment with mitochondrial lysate.