Investigation of yeast mitochondrial transcription initiation factor Mtf1p
Being the first step in gene expression, transcription is a tightly regulated process in the cell cycle. In humans, mitochondrial transcription defects have been associated with numerous diseases. In order to gain a greater understanding of mitochondrial transcription initiation in higher eukaryotes, the mitochondrial transcription initiation factor, Mtf1p, from Saccharomyces cerevisiae was studied. This protein, which has homologues in all other eukaryotes, is known to interact with Rpo41p, the core yeast mitochondrial RNA polymerase, prior to promoter-DNA binding. Four mutants were created, Mtf1p Y54F, C192A, C192F, and C192M, and then expressed in Escherichia coli cells. Initially, two mutants (C192F and C192M) were successfully expressed with an N-terminal GST-tag, and Mtf1p C192F was used to optimize an overall purification scheme. Gel filtration analysis revealed that after purification, Mtf1p C192F is in an aggregated state, and as a result, Mtf1p C192F was N-terminally His-tagged in an alternative vector. However, expression was of the His-tagged protein was again unsuccessful. At the time this thesis was written, experiments to express the four Mtf1p mutants are ongoing.