EXPRESSION OF A RECODED NUCLEAR GENE INSERTED INTO YEAST MITOCHONDRIAL-DNA IS LIMITED BY MESSENGER-RNA-SPECIFIC TRANSLATIONAL ACTIVATION

Genetic code differences prevent expression of nuclear genes within Sa ccharomyces cerevisiae mitochondria. To bridge this gap a synthetic ge ne, ARG8(m), designed to specify an arginine biosynthetic enzyme when expressed inside mitochondria, has been inserted into yeast mtDNA in p lace of the COX3 structural gene. This mitochondrial cox3::ARG8(m) gen e fully complements a nuclear arg8 deletion at the level of cell growt h, and it is dependent for expression upon nuclear genes that encode s ubunits of the COX3 mRNA-specific translational activator. Thus, cox3: :ARG8(m) serves as a mitochondrial reporter gene. Measurement of cox3: :ARG8(m) expression at the levels of steady-state protein and enzymati c activity reveals that glucose repression operates within mitochondri a. The levels of this reporter vary among strains whose nuclear genoty pes lead to under- and overexpression of translational activator subun its, in particular Pet494p, indicating that mRNA-specific translationa l activation is a rate-limiting step in this organellar system. Wherea s the steady-state level of cox3::ARG8(m) mRNA was also glucose repres sed in an otherwise wild-type strain, absence of translational activat ion led to essentially repressed mRNA levels even under derepressing g rowth conditions. Thus, the mRNA is stabilized by translational activa tion, and variation in its level may be largely due to modulation of t ranslation.