A dual interface determines the recognition of RNA polymerase II by RNA capping enzyme.

RNA capping enzyme (CE) is recruited specifically to RNA polymerase II (Pol II) transcription sites to facilitate cotranscriptional 5'-capping of pre-mRNA and other Pol II transcripts. The current model to explain this specific recruitment of CE to Pol II as opposed to Pol I and Pol III rests on the interaction between CE and the phosphorylated C-terminal domain (CTD) of Pol II largest subunit Rpb1 and more specifically between the CE nucleotidyltransferase domain and the phosphorylated CTD. Through biochemical and diffraction analyses, we demonstrate the existence of a distinctive stoichiometric complex between CE and the phosphorylated Pol II (Pol IIO). Analysis of the complex revealed an additional and unexpected polymerase-CE interface (PCI) located on the multihelical Foot domain of Rpb1. We name this interface PCI1 and the previously known nucleotidyltransferase/phosphorylated CTD interface PCI2. Although PCI1 and PCI2 individually contribute to only weak interactions with CE, a dramatically stabilized and stoichiometric complex is formed when PCI1 and PCI2 are combined in cis as they occur in an intact phosphorylated Pol II molecule. Disrupting either PCI1 or PCI2 by alanine substitution or deletion diminishes CE association with Pol II and causes severe growth defects in vivo. Evidence from manipulating PCI1 indicates that the Foot domain contributes to the specificity in CE interaction with Pol II as opposed to Pol I and Pol III. Our results indicate that the dual interface based on combining PCI1 and PCI2 is required for directing CE to Pol II elongation complexes.

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.

Phasing RNA polymerase II using intrinsically bound Zn atoms: an updated structural model.

Macromolecular assemblies as large as RNA polymerase II (Pol II) can be phased by a few intrinsically bound Zn atoms, by using MAD experiments as described here. A phasing effectiveness of 570 aa/Zn is attained for Pol II. The resulting experimental, unbiased electron density map is of such quality that it confirms the existing crystallographic model and further reveals structural regions not shown by model phases, thus updating the Pol II model at three sites. The mechanistically important fork loop-1 element is observed to be ordered in the absence of nucleic acids, suggesting additional insights into the mechanisms that maintain the stability of the transcription ternary complex and allow its release. Furthermore, a computational experiment with simulated MAD data sets demonstrates that 1 Zn site is able to provide adequate experimental phase information for as many as 1100 amino acids of polypeptide, under the conditions of the current synchrotron and detector technologies.

Fcp1 directly recognizes the C-terminal domain (CTD) and interacts with a site on RNA polymerase II distinct from the CTD.

Fcp1 is an essential protein phosphatase that hydrolyzes phosphoserines within the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). Fcp1 plays a major role in the regulation of CTD phosphorylation and, hence, critically influences the function of Pol II throughout the transcription cycle. The basic understanding of Fcp1-CTD interaction has remained ambiguous because two different modes have been proposed: the "dockingsite" model versus the "distributive" mechanism. Here we demonstrate biochemically that Fcp1 recognizes and dephosphorylates the CTD directly, independent of the globular non-CTD part of the Pol II structure. We point out that the recognition of CTD by the phosphatase is based on random access and is not driven by Pol II conformation. Results from three different types of experiments reveal that the overall interaction between Fcp1 and Pol II is not stable but dynamic. In addition, we show that Fcp1 also interacts with a region on the polymerase distinct from the CTD. We emphasize that this non-CTD site is functionally distinct from the docking site invoked previously as essential for the CTD phosphatase activity of Fcp1. We speculate that Fcp1 interaction with the non-CTD site may mediate its stimulatory effect on transcription elongation reported previously.

An agarose-acrylamide composite native gel system suitable for separating ultra-large protein complexes.

An agarose-acrylamide composite native gel (CNG) system has been developed for separating protein complexes of ultra-large molecular sizes (over 500kDa) and for analyzing protein-protein interactions in their native states. Various native gel conditions were explored and techniques were improved to facilitate the formation and performance of the CNG system. We demonstrate here that the CNG technique is capable of resolving a complex of RNA polymerase II and an associated factor from the free components, which had not been previously achieved with other methods. Furthermore, this CNG electrophoresis can be conveniently coupled to second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identification of protein components within discrete complexes separated during the CNG run. The CNG technique is particularly suitable for capturing dynamic protein-protein interactions as exemplified here by the formation and demonstration of RNA polymerase II-Fcp1 complex.

Functional expression of a fusion-dimeric MoFe protein of nitrogenase in Azotobacter vinelandii.

The MoFe protein component of the complex metalloenzyme nitrogenase is an alpha2beta2 tetramer encoded by the nifD and the nifK genes. In nitrogen fixing organisms, the alpha and beta subunits are translated as separate polypeptides and then assembled into tetrameric MoFe protein complex that includes two types of metal centers, the P cluster and the FeMo cofactor. In Azotobacter vinelandii, the NifEN complex, the site for biosynthesis of the FeMo cofactor, is an alpha2beta2 tetramer that is structurally similar to the MoFe protein and encoded as two separate polypeptides by the nifE and the nifN genes. In Anabaena variabilis it was shown that a NifE-N fusion protein encoded by translationally fused nifE and nifN genes can support biological nitrogen fixation. The structural similarity between the MoFe protein and the NifEN complex prompted us to test whether the MoFe protein could also be functional when synthesized as a single protein encoded by nifD-K translational fusion. Here we report that the NifD-K fusion protein encoded by nifD-K translational fusion in A. vinelandii is a large protein (as determined by Western blot analysis) and is capable of supporting biological nitrogen fixation. These results imply that the MoFe protein is flexible in that it can accommodate major structural changes and remain functional.

Functional expression of the FeMo-cofactor-specific biosynthetic genes nifEN as a NifE-N fusion protein synthesizing unit in Azotobacter vinelandii.

The nifEN encodes an E2N2 tetrameric metalloprotein complex that serves as scaffold for assembly of the FeMo cofactor of nitrogenase. In most diazotrophs, the NifE and NifN are translated as separate polypeptides and then assembled into tetrameric E2N2 complex. However, in Anabaena variabilis which has two nif clusters that encode two different NifEN complexes, the NifEN2 is encoded by a single nifE-N like gene, which has high homology to the NifE at amino-terminus and to the NifN at the carboxy-terminus. These observations implied that a metalloprotein like NifEN can accommodate large variations in their amino acid composition and also in the way they are synthesized (as two separate proteins or as a single protein) and yet remain functional. In Azotobacter vinelandii NifE and NifN are synthesized separately. To test whether NifEN could retain its functionality when encoded by a single gene, we generated a translational fusion of the nifE and nifN genes of A. vinelandii that could encode a large NifE-N fusion protein. When expressed in the nifEN-minus strain of A. vinelandii, the nifE-N gene fusion could complement the NifEN function. Western blot analysis by using polyclonal NifEN antibodies revealed that the complementing nifEN product is a large NifE-N fusion protein unit. The fact that the gene fusion of nifE-N specifies a functional NifE-N fusion protein reflects that these metalloproteins can accommodate a wide range of flexibility in their gene organization, structure, and assembly.