T7 RNA polymerase-dependent expression of COXII in yeast mitochondria.
An in vivo expression system has been developed for controlling the transcription of individual genes in the mitochondrial genome of Saccharomyces cerevisiae. The bacteriophage T7 RNA polymerase (T7Pol), fused to the COXIV mitchondrial import peptide and expressed under the control 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 respiration. Functional expression of T7-COX2 is completely dependent on the COX2-specific translational activator Pet111p, despite additional nucleotides at the 5' end of the T7-COX2 transcript. Expression of mitochondrion-targeted T7Pol at high levels from the GAL1 promoter has no detectable effect on mitochondrial function in rho+ cells lacking the T7-COX2 target gene, but in cells with T7-COX2 integrated into the mitochondrial genome, an equivalent level of T7Pol expression causes severe respiratory deficiency. In comparison with wild-type COX2 expression, steady-state levels of T7-COX2 mRNA increase fivefold when transcription is driven by T7Pol expressed from the ADH1 promoter, yet COXII protein levels and cellular respiration rates decrease by about 50%. This discoordinate expression of mRNA and protein provides additional evidence for posttranscriptional control of COX2 expression.
Pinkham
J L
JL
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst 01003-4505.
Dudley
A M
AM
Mason
T L
TL
D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D016384:Consensus Sequence; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D005822:Genetic Vectors; D016681:Genome, Fungal; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014158:Transcription, Genetic; D014764:Viral Proteins
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D003576:Electron Transport Complex IV; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases