Browsing Upstate Medical University by Subject "Reconstitution"
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Reconstitution and Characterization of RNA Polymerase I Upstream Activating Factor.RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and one of the most critical steps in ribosome biosynthesis. Pol I transcription initiation is coordinated by four Pol I factors that include the Upstream Activating Factor (UAF), TATA-binding protein (TBP), Core Factor (CF), and Rrn3. These factors work together to recruit Pol I to the rDNApromoter and to initiate transcription.UAF is a six-subunit complex composed of Rrn9, Rrn5, Uaf30, Rrn10, and histones H3 and H4.To investigate the importance of each UAF subunit in UAF complex formation and complex integrity, we developed a recombinant Escherichia coli-based system to coexpress and purify transcriptionally active UAF complex. Here, we found that no single subunit is required for UAF assembly, including histones H3 and H4. We also demonstrate that histone H3 is able to interact with each UAF-specific subunit. Last, wedetermined the stoichiometry of the subunits of the UAF complex, revealing there are two copies of histoneH3 and one copy of the remaining UAF subunits, including histone H4. Together, our results provide a new model suggesting that UAF contains a hybrid H3–H4 tetramer-like subcomplex.The results from this thesiswill help to reveal key mechanisms in Pol Itranscription activation.