A PROBABLE CIS-REGULATORY ELEMENT ON YEAST MITOCHONDRIAL-DNA RESPONSIBLE FOR CAMP-MEDIATED TRANSCRIPTION

Studies from this laboratory have suggested that mitochondrial (mt) tr anscription in yeast (Saccharomyces cerevisiae) is governed by changin g cellular cAMP levels, and that the mechanism of such transcriptional regulation requires cAMP-dependent protein kinase (PKA) activity; the se observations, in turn, suggest a trans-activation process for nucle otide-dependent mt transcriptional control. Here we demonstrate a sequ ence-specific mtDNA-phosphorylated protein interaction, a requisite pa rt of such a control mechanism, using filter-binding and gel mobility shift assays with mt protein extracts and mtDNA from rho(-) strains wh ose retained mt genes show cAMP-sensitive expression. We demonstrate t hat the protein-mt DNA interaction depends on PKA activity, that it sp ecifically involves a tripartite GC-rich sequence element on yeast mtD NA, and that it does not involve mt coding or promoter sequences. Sequ ence analysis indicates that the GC-rich element undergoing protein in teraction is present in ten copies on the yeast mt genome, and that ea ch 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 ev idence and argument strongly suggest that this GC-rich element functio ns as the cis-regulatory sequence involved in cAMP-mediated transcript ional control in yeast mitochondria.