Subject Area

Biochemistry

Description

Mitochondria are important organelles within eukaryotic cells especially for their role in metabolism and ATP production by the oxidative phosphorylation (OXPHOS) pathway. In human cells there are approximately 80 protein subunits that make up the OXPHOS pathway, thirteen of which are encoded by the mitochondrial genome (mtDNA). Mitochondria house all the transcription and translation machinery (i.e., mitochondrial RNA polymerase, mitochondrial ribosome, tRNAs, etc.) required to produce those thirteen mtDNA encoded subunits. In vitro mitochondrial transcription is a method that utilizes recombinantly purified proteins and linear mitochondrial DNA templates to investigate transcription regulation of the organelle. To visualize the products of in vitro transcription, it is still common practice to utilize radioactive nucleotides or staining with ethidium bromide. These conditions can be undesirable due to safety hazards, expense, interference with electrophoresis, and time demands. As an alternative, fluorescent dyes have been developed for DNA and RNA tagging. This work establishes a procedure for post-staining of T7 RNA polymerase in vitro transcription products run via denaturing gel electrophoresis and stained with Gel Red and SYBR Gold dyes. Our current studies focus on applying this procedure to the in vitro mitochondrial transcription assay.

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May 17th, 12:00 AM May 17th, 12:00 AM

Fluorescent Visualization of In Vitro Mitochondrial DNA Transcription

Mitochondria are important organelles within eukaryotic cells especially for their role in metabolism and ATP production by the oxidative phosphorylation (OXPHOS) pathway. In human cells there are approximately 80 protein subunits that make up the OXPHOS pathway, thirteen of which are encoded by the mitochondrial genome (mtDNA). Mitochondria house all the transcription and translation machinery (i.e., mitochondrial RNA polymerase, mitochondrial ribosome, tRNAs, etc.) required to produce those thirteen mtDNA encoded subunits. In vitro mitochondrial transcription is a method that utilizes recombinantly purified proteins and linear mitochondrial DNA templates to investigate transcription regulation of the organelle. To visualize the products of in vitro transcription, it is still common practice to utilize radioactive nucleotides or staining with ethidium bromide. These conditions can be undesirable due to safety hazards, expense, interference with electrophoresis, and time demands. As an alternative, fluorescent dyes have been developed for DNA and RNA tagging. This work establishes a procedure for post-staining of T7 RNA polymerase in vitro transcription products run via denaturing gel electrophoresis and stained with Gel Red and SYBR Gold dyes. Our current studies focus on applying this procedure to the in vitro mitochondrial transcription assay.

 

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