Figure 3.

The mtDNA instability phenotype of the rpo41Δ3 mutation does not result from loss of mitochondrial transcription. (A) The sc-mtTFA and sc-mtTFB overexpression plasmids, YEp-mtTFA and YEp-mtTFB, and a control plasmid YEp352 each were introduced into GS125, and the ability of the resulting strains to grow on YPG at 30°C and 37°C is shown labeled as follows: 1, GS125(rpo41Δ3); 2, GS122(RPO41); 3, GS125(rpo41Δ3)+YEp-mtTFA; 4, GS125 (rpo41Δ3)+YEp-mtTFB; 5, GS125(rpo41Δ3)+YEp352. (B) S1 nuclease protection analysis of mitochondrial transcripts from the yeast strain (BS127 HS3324 ΔRPO41) containing either no mtRNA polymerase (lanes 1 and 4), the RPO41-containing plasmid, pGS348 (lanes 2 and 5) or an rpo41Δ3-containing plasmid, pGS348Δ3 (lanes 3 and 6), after growth at 30°C (lanes 1–3) or 37°C (lanes 4–7) for ≈20 generations. Lane 7, same as lane 6, except the sample was pretreated with RNase A. M, HpaII-digested pBR322 markers (length in nucleotides is indicated on the left). (C) Northern analysis of ori5 transcripts in BS127 HS3324 ΔRPO41 strains grown at 37°C: wt, wild-type mtRNA polymerase; Δ3, rpo41Δ3 mtRNA polymerase. The major ori5 RNA transcript is indicated by the bold arrow. Two additional minor transcripts of ≈2 kb and ≈3 kb, corresponding to two and three ori5 repeat lengths, are indicated by thin arrows, as is a subrepeat length transcript that was observed only in the wild-type strain. Indicated on the left is the position where known RNA species migrated.