Reconstitution Of RNA Polymerase Research Articles

Reconstitution of RNA polymerase I Upstream Activating Factor and the roles of histones H3 and H4 in complex assembly

RNA polymerase I (Pol I) transcription in Saccharomyces cerevisiae requires four separate factors that recruit Pol I to the promoter to form a pre‐initiation complex (PIC). Upstream Activating Factor (UAF) is one of two multi‐subunit complexes that regulate PIC formation by binding to the ribosomal DNA promoter and by stimulating recruitment of downstream Pol I factors. UAF is composed of Rrn9, Rrn5, Rrn10, Uaf30, and histones H3 and H4. We developed a recombinant E. coli based system to coexpress and purify transcriptionally active UAF complex and to investigate the importance of each subunit in complex formation. 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, and show that there are at least two copies of histone H3 and one copy of H4 present in the complex. Together, our results provide a new model suggesting that UAF contains a hybrid H3‐ H4 tetramer‐like subcomplex.Support or Funding InformationThe work was supported by B.A.K. grants from the US National Institutes of Health (NIH) NCI (5K22CA184235), a Sinsheimer Scholar award from the Alexandrine and Alexander L. Sinsheimer Fund, Central New York Community Foundation, Joseph C. George Fund, and Virginia Simons & Dr. C. Adele Brown Fund. Native MS measurements were financed by the NIH NIGMS (P41 GM103422).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reconstitution of RNA Polymerase I Upstream Activating Factor and the Roles of Histones H3 and H4 in Complex Assembly

RNA polymerase I (Pol I) transcription in Saccharomyces cerevisiae requires four separate factors that recruit Pol I to the promoter to form a pre-initiation complex. Upstream Activating Factor (UAF) is one of two multi-subunit complexes that regulate pre-initiation complex formation by binding to the ribosomal DNA promoter and by stimulating recruitment of downstream Pol I factors. UAF is composed of Rrn9, Rrn5, Rrn10, Uaf30, and histones H3 and H4. We developed a recombinant Escherichia coli-based system to coexpress and purify transcriptionally active UAF complex and to investigate the importance of each subunit in complex formation. 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, and show that there are at least two copies of histone H3 and one copy of H4 present in the complex. Together, our results provide a new model suggesting that UAF contains a hybrid H3–H4 tetramer-like subcomplex.

Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation.

A three-step procedure comprising (1) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (2) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (3) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a cross-linking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP.

9] Reconstitution of RNA polymerase

Publisher Summary The DNA-dependent RNA polymerase of Escherichia coli is composed of core enzyme and one of at least six molecular species of the σ subunit. Despite the complexity of its subunit composition and its extraordinarily large molecular size, RNA polymerase is one of the few large molecular assemblies that can be successfully reconstituted from isolated individual subunits. Core enzyme can be reconstituted by mixing α, β, and β' subunits under denaturing conditions, and then removing the denaturant by dialysis against the appropriate reconsitution buffer. The core enzyme reconstituted at low temperature is known to exist as an inactive premature from. It can be activated by heating at 30 ° for 30–60 min. Although the activated core enzyme can be used directly for many purposes, it contains considerable amounts of free subunits and α 2 β subassembly. For quantitative experiments, therefore, further purification of the reconstituted enzyme is essential.