T7 RNA POLYMERASE-DEPENDENT EXPRESSION OF COXII IN YEAST MITOCHONDRIA

An in vivo expression system has been developed for controlling the tr anscription of individual genes in the mitochondrial genome of Sacchar omyces cerevisiae. The bacteriophage T7 RNA polymerase (T7Pol), fused to the COXIV mitochondrial import peptide and expressed under the cont rol of either the GAL1 or the ADH1 promoter, efficiently transcribes a target gene, T7-COX2, in the mitochondrial genome. Cells bearing the T7-COX2 gene, but lacking wild-type COX2, require T7Pol for respiratio n. Functional expression of T7-COX2 is completely dependent on the COX 2-specific translational activator Pet111p, despite additional nucleot ides at the 5' end of the T7-COX2 transcript. Expression of mitochondr ion-targeted T7Pol at high levels from the GAL1 promoter has no detect able effect on mitochondrial function in rho(+) cells lacking the T7-C OX2 target gene, but in cells with T7-COX2 integrated into the mitocho ndrial genome, an equivalent level of T7Pol expression causes severe r espiratory deficiency. In comparison with wild-type COX2 expression, s teady-state levels of T7-COX2 mRNA increase fivefold when transcriptio n is driven by T7Pol expressed from the ADH1 promoter, yet COXII prote in levels and cellular respiration rates decrease by about 50%. This d iscoordinate expression of mRNA and protein provides additional eviden ce for posttranscriptional control of COX2 expression.