Mechanism of transcription initiation by the yeast mitochondrial RNA polymerase

Abstract

Mitochondria are the major supplier of cellular energy in the form of ATP. Defects in normal ATP production due to dysfunctions in mitochondrial gene expression are responsible for many mitochondrial and aging related disorders. Mitochondria carry their own DNA genome which is transcribed by relatively simple transcriptional machinery consisting of the mitochondrial RNAP (mtRNAP) and one or more transcription factors. The mtRNAPs are remarkably similar in sequence and structure to single-subunit bacteriophage T7 RNAP but they require accessory transcription factors for promoter-specific initiation. Comparison of the mechanisms of T7 RNAP and mtRNAP provides a framework to better understand how mtRNAP and the transcription factors work together to facilitate promoter selection, DNA melting, initiating nucleotide binding, and promoter clearance. This review focuses primarily on the mechanistic characterization of transcription initiation by the yeast Saccharomyces cerevisiae mtRNAP (Rpo41) and its transcription factor (Mtf1) drawing insights from the homologous T7 and the human mitochondrial transcription systems. We discuss regulatory mechanisms of mitochondrial transcription and the idea that the mtRNAP acts as the in vivo ATP “sensor” to regulate gene expression.

Reduced Histone Expression or a Defect in Chromatin Assembly Induces Respiration

“…However, the mechanism by which the chromatin structure in the nucleus affects the expression of genes carried by mitochondrial DNA is less obvious. A major mechanism that regulates the activity of the mitochondrial RNA polymerase Rpo41p/ Mtf1p in vitro is the concentration of ATP (70,71). To test whether this mechanism regulates the transcription of COX1, COX2, and COX3, three genes carried by mitochondrial DNA, we measured the corresponding transcript levels in spt10⌬ cells.…”

“…However, how does a defect in the chromatin structure of nuclear genes affect the expression of genes carried by mitochondrial DNA? In vitro, the transcription of genes carried by mitochondrial DNA is regulated by the ATP concentration, and different genes carried by mitochondrial DNA differ in their responsiveness to the ATP level (70,71). However, our results indicate that the increased expression of genes carried by mitochondrial DNA (COX1, COX2, and COX3) in spt10⌬ cells requires the nuclear factors Hap4p and Rtg1p and is driven by the increased expression of the RPO41 and MTF1 genes, encoding mitochondrial RNA polymerase and its associated factor, respectively.…”

Yeast PPR proteins, watchdogs of mitochondrial gene expression

“…The mitochondrial RNA polymerase is of the viral T7 type (reviewed in ref. 22), it is a multidomain polypeptide that contains two to several PPR domains, depending on the organism, located in an N-terminal extension of non-viral origin. The structure of the human mitochondrial RNA polymerase (mtRNAP or POLRMT) has been determined and this is the first crystal structure of a PPR protein.…”

Fluorescent Methods to Study Transcription Initiation and Transition into Elongation

“…In T7 phage, the single-subunit T7 RNAP recognizes the consensus −17 to +6 promoter sequence without requiring any transcription factors. In yeast ( Saccharomyces cerevisiae ) mitochondria, transcription is carried out by a single-subunit class of enzymes related to T7 RNAP, but the core Rpo41 polymerase requires the transcription factor Mtf1 for specific transcription [17]. T7 RNAP discriminates against the non-promoter sequences at the initial binding step, showing ∼10 5 -fold tighter affinity for promoter relative to non-promoter sequences [18].…”

“…Single-molecule time traces showed that Rpo41 alone can bend the promoter DNA to the same degree as Rpo41–Mtf1, however, with a very low frequency. Thus, the low E FRET of the Rpo41-promoter complex observed in ensemble studies is not due to a small bending angle, but a consequence of the rare bending events [49, 17]. The single-molecule FRET time traces measured the DNA bending rates; k bending by Rpo41 alone is 0.0052 s −1 and by Rpo41–Mtf1 is 2.8 s −1 .…”