A probable cis-regulatory element on yeast mitochondrial DNA responsible for cAMP-mediated transcription

Studies from this laboratory have suggested that mitochondrial (mt) transcription in yeast (Saccharomyces cerevisiae) is governed by changing cellular cAMP levels, and that the mechanism of such transcriptional regulation requires cAMP-dependent protein kinase (PKA) activity; these observations, in turn, suggest a trans-activation process for nucleotide-dependent mt transcriptional control. Here we demonstrate a sequence-specific mtDNA-phosphorylated protein interaction, a requisite part of such a control mechanism, using filter-binding and gel mobility shift assays with mt protein extracts and mtDNA from rho- strains whose retained mt genes show cAMP-sensitive expression. We demonstrate that the protein-mt DNA interaction depends on PKA activity, that it specifically involves a tripartite GC-rich sequence element on yeast mtDNA, and that it does not involve mt coding or promoter sequences. Sequence analysis indicates that the GC-rich element undergoing protein interaction is present in ten copies on the yeast mt genome, and that each copy is located 5' to a strong mt promoter; the elements appear in both orientations relative to, and at varying distances upstream from, the putatively associated mt promoter elements. The mt element shows no sequence homology to relevant nuclear cis-elements examined and is unrelated to published vertebrate mt cis-elements. Several lines of evidence and argument strongly suggest that this GC-rich element functions as the cis-regulatory sequence involved in cAMP-mediated transcriptional control in yeast mitochondria.