Untersuchung des Mechanismus der direktionalen Transkriptionstermination für den humanen mitochondriellen Transkriptionskomplex auf Einzelmolekülebene
Biochemie
Zusammenfassung der Projektergebnisse
The proposal aimed at characterizing the mechanism of transcription termination in human mitochondria. Mitochondria are the powerhouse of the cell as they produce adenosine triphosphate (ATP), which fuels most of cellular reactions. Mitochondria contain mitochondrial DNA (mtDNA) in a form of a ~16.6 kbp long plasmid in human. mtDNA is composed of a heavy and a light strand, i.e. HS and LS, and both are transcribed starting from either the heavy strand promoter (HSP) or the light strand promoter (LSP) by the mitochondrial RNA polymerase (mtRNAP). The mitochondria transcription termination factor 1 (MTERF1) is a protein factor that binds to mtDNA at the end of the LS open reading frame to terminate mtRNAP coming from the LSP, but not from the HSP. The goal of this proposal was to discover the mechanism by which MTERF1 terminates directionally mtRNAP transcription activity. We employed singlemolecule magnetic tweezers to show that directional mtDNA strand displacement was sufficient to activate MTERF1 and block further opening temporarily. We show that mtDNA strand displacement was halted by MTERF1 binding when opened from the LSP direction, but not from the HSP direction. This difference was abolished when employing MTERF1 mutants with specific interactions with both DNA strands. We have also established a novel assay to characterize the equilibrium dissociation constant for MTERF1 from the mtDNA termination site. Last, we have developed a novel high-throughput fluorescence microscope, enabling the observation of thousands of fluorescently labelled molecules in parallel and in real-time. These developments will be used to carry on this project.
Projektbezogene Publikationen (Auswahl)
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Single-Molecule Magnetic Tweezers Characterization of MTERF1 as a Directional Roadblock. Biophysical Journal, 118(3), 73a.
Ostrofet, Eugeniu; Stal, Papini Flavia; Johnson, Britney; Arnold, Jamie J.; Cameron, Craig E. & Dulin, David
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A high-throughput correlative magnetic tweezers-TIRF assay to investigate protein-nucleic acids interactions. Biophysical Journal, 123(3), 286a.
Feiz, M. Sadegh; Wubulikasimu, Yibulayin; Cnossen, Jelmer; Papini, Flavia Stal; Bera, Subhas C.; Quack, Salina & Dulin, David
