Subject Area

Biochemistry and Molecular Biology

Description

Mitochondria are important organelles in most eukaryotic cells due to their essential role in metabolism. Within the matrix of the organelle, they also have their own genome (mtDNA) which encodes many of the protein subunits for complexes within the oxidative phosphorylation (OXPHOS) pathway. The remaining OXPHOS protein complex subunits are encoded in the nuclear genome, translated in the cytosol and imported into mitochondria. Transcriptional coordination between these two genomes to produce functional OXPHOS complexes is critical to maintaining cellular homeostasis. This process likely requires regulatory proteins/transcription factors that determine the expression of these genes. Retinoid X receptor alpha (RXRα) is a protein that has many functions in human cells and is a ligand activated nuclear receptor. It can also translocate to the mitochondria where its purpose is unknown. The goal of this project is to determine the role of RxRα as a mitochondrial transcription factor. We have expressed and purified the RXRα protein from bacteria for use in an in vitro mitochondrial transcription assay. Using an electrophoretic mobility shift assay (EMSA) we have also shown the purified protein binds to DNA with canonical binding sequences (DR1 repeats) and spacing as well as non-canonical spacing and have concluded RXRα binds as a dimer with a single nucleotide space between the binding sites being optimal. We have applied these results to assess changes in mitochondrial transcriptional output based on RXRa binding. Our aim is to investigate RxRa binding the mtDNA using atomic force microscopy (AFM). In doing so we will better understand the mechanisms that are involved in mtDNA transcription. If RxRα is a transcription factor that is another protein that we can regulate to increase or decrease transcription. Being able to increase or decrease transcription in mitochondria is important as mitochondrial dysfunction causes a wide array of diseases and plays a part in aging.

Download

Share

COinS
 

Is RxR alpha a mitochondrial transcription factor?

Mitochondria are important organelles in most eukaryotic cells due to their essential role in metabolism. Within the matrix of the organelle, they also have their own genome (mtDNA) which encodes many of the protein subunits for complexes within the oxidative phosphorylation (OXPHOS) pathway. The remaining OXPHOS protein complex subunits are encoded in the nuclear genome, translated in the cytosol and imported into mitochondria. Transcriptional coordination between these two genomes to produce functional OXPHOS complexes is critical to maintaining cellular homeostasis. This process likely requires regulatory proteins/transcription factors that determine the expression of these genes. Retinoid X receptor alpha (RXRα) is a protein that has many functions in human cells and is a ligand activated nuclear receptor. It can also translocate to the mitochondria where its purpose is unknown. The goal of this project is to determine the role of RxRα as a mitochondrial transcription factor. We have expressed and purified the RXRα protein from bacteria for use in an in vitro mitochondrial transcription assay. Using an electrophoretic mobility shift assay (EMSA) we have also shown the purified protein binds to DNA with canonical binding sequences (DR1 repeats) and spacing as well as non-canonical spacing and have concluded RXRα binds as a dimer with a single nucleotide space between the binding sites being optimal. We have applied these results to assess changes in mitochondrial transcriptional output based on RXRa binding. Our aim is to investigate RxRa binding the mtDNA using atomic force microscopy (AFM). In doing so we will better understand the mechanisms that are involved in mtDNA transcription. If RxRα is a transcription factor that is another protein that we can regulate to increase or decrease transcription. Being able to increase or decrease transcription in mitochondria is important as mitochondrial dysfunction causes a wide array of diseases and plays a part in aging.