Abstract
RNA polymerase II (Pol II) is the central enzyme of eukaryotic gene expression machinery. Complete definition of the three-dimensional structure of Pol II is essential for understanding the mechanisms that regulate transcription via protein-protein interactions within the Pol II apparatus. To date a series of Pol II-related crystal structures have been reported. However, certain peptide regions, including several that are implicated to interact with regulatory factors, remain obscure. Here we describe conformations for two such regions that are close to the Pol II surface and assume seemingly flexible loop structures. One is located in the TFIIF-interacting Protrusion domain, whereas the other is in the TFIIE-interacting Clamp domain. This structural definition was aided by the application of an advanced crystallographic refinement approach that utilizes the single anomalous diffraction (SAD) from zinc ions bound intrinsically in Pol II. The SAD-based strategy allowed the 12-subunit Pol II model to be fully refined up to 3.8 A with excellent stereochemical properties, demonstrating the effectiveness of the SAD approach for the refinement of large structures at low-to-moderate resolutions. Our results also define additional components of the free Pol II, including the functionally critical Fork Loop-1 and Fork Loop-2 elements. As such, this refined Pol II model provides the most complete structural reference for future analyses of complex structures formed between Pol II and its regulatory factors.
Highlights
RNA polymerase II (Pol II)2 and its associated factors form an elaborate protein machinery that transcribes DNA sequences into pre-mRNAs to carry out the first step of gene expression in eukaryotic cells [1]
We describe a full refinement of the 12-subunit Pol II structure at 3.8 Å using a newly developed algorithm that is capable of parameter refinement for crystals that diffract to moderate resolutions (e.g. 3– 4 Å)
Observations, improving the ratio of observations to parameters. When this technique was applied to make use of single anomalous diffraction (SAD) from the zinc ions bound intrinsically in Pol II, a Pol II structure was obtained with significant improvements as judged by comparison to available models of the 12-subunit Pol II with regard to stereochemical properties
Summary
Purification of Pol II from yeast (Saccharomyces cerevisiae) and its crystallization, x-ray diffraction data collection, and experimental phasing have been described previously [15]. The SAD phase information was included into the refinement target by invoking REFMAC5D with inputs of the anomalous-scatterer model (8 zinc sites), scattering factors (fЈ ϭ Ϫ6.4306, fЉ ϭ 3.8873), and column label for SAD amplitudes in the data file. The positional parameters of the anomalous scatterers were refined in concert with the polymerase model during both stages, allowing for simultaneous updating of the SAD phase information. Two approaches were tested to define initial TLS groups; the first was to use previously known rigid-body domains of Pol II [6, 15], and the second was to derive TLS groups for each subunit by conducting several cycles of atomic B-factor refinement without positional refinement and submitting output to the TLSMD [31] server, which produced suggested TLS groups for each subunit.
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