Structure of the 12-subunit RNA polymerase II refined with the aid of anomalous diffraction data.
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.
Meyer
Peter A
PA
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Ye
Ping
P
Suh
Man-Hee
MH
Zhang
Mincheng
M
Fu
Jianhua
J
D001665:Binding Sites; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D011487:Protein Conformation; D021122:Protein Subunits; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D019473:Transcription Factors, TFII / Q000737:chemistry / Q000378:metabolism
D021122:Protein Subunits; D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE; D012319:RNA Polymerase II; C056604:transcription factor TFIIF