Identification and characterization of CIS-element on yeast mitochondrial DNA which is responsible for cAMP-mediated transcriptional regulation
Authors:   , Iqbal Jabed 
Published: July 2024

The research undertaken in this Thesis continues study of the transcriptional control system required for cAMP-mediated mitochondrial gene expression in yeast. It focuses specifically on the identification and functional characterization of a cis-element on the mitochondrial genome that is required for nucleotide-mediated gene expression. To demonstrate the possible presence of a cis-regulatory element on the yeast mitochondrial genome, we initially set out to identify a sequence-specific protein-DNA interaction dependent on PKA activity, a requisite part of any trans-activation-based transcriptional control mechanism. We showed that the protein-mitochondrial DNA interaction depends on PKA activity, that it specifically involves a tripartite GC-rich DNA sequence element on the yeast organellar genome, and that it does not involve mitochondrial coding or promoter sequences. Of the ten recognized strong promoters on the yeast mitochondrial genome, the tripartite element is associated with seven. We then demonstrated, that the downstream-most portion of the tripartite mitochondrial sequence element is the primary, although probably not the sole, site of interaction with a PKA-phosphorylated protein. This downstream-most portion is a 30 base pair sequence showing an imperfect internal inverted repeat. We further demonstrated that alteration of various portions of the 30 base pair protein-binding site abolished interaction with the as yet unidentified phosphorylated protein. In order to assess whether the mitochondrial sequence element undergoing interaction with a PKA-phosphorylated protein shows any DNA sequence homology with other known nuclear and/or mitochondrial cis-regulatory sequences from yeast or other organisms, we performed computer analyses with all such elements included in GeneBank. The mitochondrial sequence element identified in these initial studies appears to be unique. Several lines of evidence and argument are provided which strongly suggest that this GC-rich element functions as the cis-regulatory sequence involved in cAMP-mediated transcriptional control in yeast mitochondria. Demonstration of the transcriptional relevance of the mitochondrial sequence element identified in the above studies had to derive from both in vivo and in vitro transcription assays. We therefore developed such an in vitro assay, based on the use of Brij-35 lysates of purified mitochondria from a p- yeast strain in which the nuclear gene NUC1 had been insertionally-inactivated. This nuclear gene is known to encode the primary ribonuclease active in yeast mitochondria, and inactivation of the gene removes such activity from organellar lysates prepared from the nuc1 strain. We demonstrated that Brij lysates of mitochondria from the nuc1 petite strain incorporate radioactive precursors into mitochondrial RNA at a high rate, and that PKA activity as well as mitochondrial RNA polymerase activity are fully functional in such lysates. It was further demonstrated that addition of exogenous template DNA of specified structure allows production of transcripts from those templates. The role of the tripartite mitochondrial DNA sequence as a transcriptional regulatory element was then examined using both this new in vitro transcription assay and in vivo transcript analyses. We demonstrated that in a standard wild-type yeast strain, raising overall cellular cAMP levels six-fold by substitution of an "activated" (ie., oncogenic) allele of the nuclear gene RAS2 leads to no difference in OXl2 transcript levels. We then prepared a petite mutant strain of yeast whose mitochondrial genome retains the OXl2 coding sequence and its strong promoter but no other mitochondrial genes. (Abstract shortened by UMI.).