Use of yeast nuclear DNA sequences to define the mitochondrial RNA polymerase promoter in vitro.

Abstract

We have extended an earlier observation that the TATA box for the nuclear GALIO gene serves as a promoter for the mitochondrial RNA polymerase in in vitro transcription reactions (C. S. Winkley, M. J. Keller, and J. A. Jaehning, J. Biol. Chem. 260:14214-14223, 1985). In this work, we demonstrate that other nuclear genes also have upstream sequences that function in vitro as mitochondrial RNA polymerase promoters. These genes include the GAL7 and MEL] genes, which are regulated in concert with the GALIO gene, the sigma repetitive element, and the 2,um plasmid origin of replication. We used in vitro transcription reactions to test a large number of nuclear DNA sequences that contain critical mitochondrial promoter sequences as defined by Biswas et al. (T. K. Biswas, J. C. Edwards, M. Rabinowitz, and G. S. Getz, J. Biol. Chem. 262:13690-13696, 1987). The results of these experiments allowed us to extend the definition of essential promoter elements. This A T A aAA extended sequence, -CTAAACGTrG-, was frequently found in the upstream regulatory regions of nuclear genes. On the basis of these observations, we hypothesized that either (i) a catalytic RNA polymerase related to the mitochondrial enzyme functions in the nucleus of the yeast cell or (ii) a DNA sequence recognition factor is shared by the two genetic compartments. By using cells deficient in the catalytic core of the mitochondrial RNA polymerase (rpo4l -) and sensitive assays for transcripts initiating from the nuclear promoter sequences, we have conclusively ruled out a role for the catalytic RNA polymerase in synthesizing transcripts from all of the nuclear sequences analyzed. The possibility that a DNA sequence recognition factor functions in both the nucleus and the mitochondria remains to be tested.The nuclear and mitochondrial compartments must communicate and coordinate their respective biosynthetic reactions to ensure balanced cell growth. Mitochondria are themselves composed primarily of proteins encoded in the nucleus (14); the regulated expression of these nuclear gene products involves many diverse elements, but in many cases the genes are subject to repression by glucose (17,22). Transcription of the genes encoded in the mitochondrial DNA is dependent on the mitochondrial RNA polymerase, a multicomponent enzyme which is itself encoded in the nucleus (47,48). The regulation of this enzyme by glucose may control the rate of mitochondrial transcript synthesis in a simple way (47).The catalytic core of the mitochondrial RNA polymerase is encoded by the RP041 gene (24, 47), which bears striking similarity to RNA polymerases from bacteriophages T3 and T7 (29). Although the core is transcriptionally active in vitro on nonspecific templates such as poly[d(AT)], it requires the addition of a specificity factor to initiate selectively at mitochondrial promoter sequences (37,44,48). The promoter for the mitochondrial RNA polymerase was initially described as a simple nonanucleotide sequence, -ATATAAGTA-(12, 34). Transcripts isolated from mitochondria an...

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