Dynamik und Einflussfaktoren der RNA Polymerase I Transktiptionsinitiation
Biochemie
Zusammenfassung der Projektergebnisse
The proposal aimed at characterizing the mechanism of transcription initiation by yeast RNA polymerase 1 (Pol I). Pol I is responsible for synthesizing most of the ribosomal RNA in the host cell, which represents up to 60% of all RNA synthesized in the cell. Therefore, Pol I transcription initiation, meaning the phase where Pol I recognizes the start of the promoter, is extremely regulated by the cell. Furthermore, Pol I activity is up regulated in many cancers, making it an excellent target for anti-cancer drugs. However, how Pol I transcription initiation is regulated is largely unknown. The Pol I pre-initiation complex (PIC) is multi-protein complex constituted of Pol I associated with several co-factors that enables Pol I to recognize the ribosomal DNA promoter. The yeast complex, which is functionally conserved in respect of the human one, is made of the upstream activation factor (UAF), the core factor (CF), the tata binding protein (TBP), Rrn3 and Pol I. How the assembly occurs and results in a transcriptionally active PIC is unknown, neither are the determinants of transcription initiation. Here, we proposed to use single-molecule biophysics assays to investigate this mechanism. First, we established a high-throughput magnetic tweezers assay to monitor transcription initiation, and specifically the dynamics of the transcription bubble opening-closing that regulates transcription initiation. We were able to observe the formation of the transcription bubble for Pol I PIC. In parallel, we then set to establish the methodology to process, analyze and model transcription bubble dynamics, using the model system for cellular transcription of E. coli RNA polymerase as a large statistic is easily achievable with this system. Our data are consistent with an intermediate and a stable transcription bubble state, the latter being a dead-end state. We also developed a correlative magnetic tweezers-fluorescence microscope to monitor in real-time the PIC composition and the transcription bubble size dynamics.
Projektbezogene Publikationen (Auswahl)
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Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on a consensus promoter. Nucleic Acids Research, 50(13), 7511-7528.
Bera, Subhas C.; America, Pim P. B.; Maatsola, Santeri; Seifert, Mona; Ostrofet, Eugeniu; Cnossen, Jelmer; Spermann, Monika; Papini, Flávia S.; Depken, Martin; Malinen, Anssi M. & 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
