Dissertation

ATP-mediated activation of RNA polymerase II transcription complexes

Kopytek, Stephan Jack

1997

Overview

Transcription initiation by RNA polymerase II (pol II) is a complex, multistep process that minimally involves transcription complex assembly, open complex formation, and promoter clearance. Hydrolysis of the $\\beta$-$\\gamma$ phosphoanhydride bond of ATP has previously been shown to be required for open complex formation, as well as for the phosphorylation of the carboxy-terminal domain of the largest subunit of pol II. The observation that ATP-dependent activation of pol II transcription complexes can be blocked by ATP analogs that contain non-hydrolyzable $\\beta$-$\\gamma$ phosphoanhydride bonds (such as $\\rm ATP\\gamma S)$ was exploited to develop a functional kinetic assay for ATP-mediated activation of transcription complexes. Activation is dependent on treatment of assembled preinitiation complexes with ATP (or dATP) prior to addition of $\\rm ATP\\gamma S.$ The mouse mammary tumor virus (MMTV) and the adenovirus major late (AdML) wild type promoters were assayed along with mutant versions that did not contain functional binding sites for proteins germane to transcription from these promoters. Elements of the MMTV promoter that were mutated include a site recognized by initiation site binding protein, a binding site for nuclear factor 1, and two adjacent binding sites for Oct-1. AdML promoter mutants included mutations in the binding site for upstream stimulatory factor (USF) and in the AdML initiator element. The time course of formation of activated complexes in the presence of dATP is characterized by two kinetic phases, a rapid formation followed by a relatively slow decay, and activated complexes are estimated to form with a half time of less than 1 min. Similar results were obtained for all promoters tested. A fraction of the total number of preinitiation complexes present become activated within 1 to 1.5 min in the presence of (d)ATP, and activation appears to be rapidly reversible. At least 30% of the total number of preinitiation complexes assembled on all promoters, with the exception of the AdML promoter containing a mutation in the binding site for USF, in which only 20% of the complexes become activated. USF was found to stabilize activated transcription complexes on the AdML promoter.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biochemistry / Microbiology / Molecular biology
• ISBN: 9780591669350, 0591669358