Figure 5.

An RNA chain terminator rapidly inhibits nascent RNA formation in isolated mitochondria but affects mtDNA replication much more slowly. (A) 2D-AGE analysis of rat mtDNA labeled for 120 min with [α³²P]-dUTP after pre-incubation without (control) or with 20 µM cordycepin triphosphate for 20 min. The intactness of the mtRIs is confirmed in Supplementary Figure S5C. (B) 2D-AGE analysis of rat mtDNA labeled for 5 min with [α³²P]-dATP after pre-incubation without (control) or with 20 µM cordycepin triphosphate for 55 or 115 min. (C) Quantitation of the signals from the linear BlpI fragments of mtDNA (panel B and equivalent samples), during 3′ dATP treatment. Note the slow but gradual decrease in [α³²P]-dATP incorporation into mtDNA. [α³²P]-dATP incorporation was standardized to the level of steady-state mtRIs, based on Southern hybridization. (D) Steady-state levels, relative to zero time point, of mitochondrial rRNA (12S and 16S) and mRNA (COX2, ND1, ND3, ND5 and CYTB), after the indicated times of incubation with 20 µM cordycepin triphosphate. Measurements were based on Q-PCR values normalized to those for mtDNA.