Two forms of RPO41-dependent RNA polymerase. Regulation of the RNA polymerase by glucose repression may control yeast mitochondrial gene expression.

We have identified two chromatographically separable forms of mitochondrial RNA polymerase from Saccharomyces cerevisiae which utilize different DNA templates. One form is only active in a nonselective assay utilizing a poly[d(A-T)] template. The other form selectively initiates from a mitochondrial promoter consensus sequence. Both enzymes can be extracted from yeast mitochondria and all components are encoded by nuclear genes. The possibility that these two activities represent core and holoenzyme forms of the multicomponent mitochondrial RNA polymerase is supported by our observation that both enzymes are absent from a strain bearing a disrupted copy of the RPO41 gene (Greenleaf, A. L., Kelly, J. L., and Lehman, I. R. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 3391-3399). The two enzyme activities are differentially regulated by carbon source; the nonselective enzyme is repressed during growth on glucose relative to the selective enzyme. The 5-fold increase in RNA polymerase activity on a nonrepressing carbon source correlates with the increased level of transcript production from mitochondrial DNA. These results suggest that the mitochondrial RNA polymerase and, in consequence, mitochondrial transcription are regulated by carbon catabolite control.

A multicomponent mitochondrial RNA polymerase from Saccharomyces cerevisiae.

Using a whole cell extract from Saccharomyces cerevisiae (bakers' yeast) we have been able to detect a selective RNA polymerase activity originally identified in purified yeast mitochondria (Levens, D., Morimoto, R., and Rabinowitz, M. (1981) J. Biol. Chem. 256, 1466-1473). We have shown that in in vitro transcription reactions this activity recognizes a consensus mitochondrial promoter sequence ATA-TAAGTA (Osinga, K. A., DeHaan, M., Christianson, T., and Tabak, H. F. (1982) Nucleic Acids Res. 10, 7993-8006) in the upstream region of the nuclear GAL10 gene as well as promoters from yeast mitochondrial DNA. Using these promoter-containing templates for in vitro assays, we have chromatographically separated the mitochondrial specific RNA polymerase activity from the three nuclear RNA polymerases (I, II, and III). Further characterization has revealed that this preparation has distinctive properties on two different types of DNA templates, poly[d(AT)] and cloned DNA containing mitochondrial promoters. Salt and divalent cation optima and substrate saturation kinetics are different for the two types of templates. Using promoter-containing DNA as an assay template, we have chromatographically dissociated the RNA polymerase activity into two nonfunctional components. Selective transcription of the GAL10 template is restored when the two components are recombined. It is possible that the RNA polymerase active on poly[d(AT)] is a nonspecific component of the selective transcription apparatus or that two distinct RNA polymerases are present in the preparation.