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.In eukaryotic cells, RNA polymerase II (RNA Pol II) 5 and its associated factors carry out transcription of pre-mRNAs, snRNAs, telomerase RNA, and other noncoding RNAs. Pol II also couples transcription to nuclear processes including pre-mRNA modifications (1-4), mRNA export, and chromatin reconfiguration (5-7). Coupling RNA processing with synthesis is presumed to be critical in restricting the temporal window during which unmodified transcripts are vulnerable to degradation by endogenous ribonuclease activities (8 -10) and directing RNA processing factors to sites of Pol II transcription at specific steps during Pol II progression through a gene. The Pol II elongation complex coordinates these transactions to help orchestrate control over gene expression (for review, see Ref. 11). Such coordination is mediated by specific and reversible interactions among Pol II and the factors involved in elongation.The formation of RNA 5Ј cap structure, m 7 GpppN, is the first transcription-coordinated RNA modification event, and it occurs as soon as the transcript attains ϳ25 nucleotides (12-15). The RNA cap is formed in three enzymatic steps (16, 17); (i) removal of the 5Ј ␥-phosphate catalyzed by the RNA triphosphatase; (ii) attachment of a GMP to the 5Ј diphosphate by the guanylyltransferase; (iii) methylation of the 5Ј guanine by the cap methyltransferase. The first two of these steps are closely linked and coupled to Pol II transcription in most org...

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.C-terminal domain dephosphorylation ͉ C-terminal domain hyperphosphorylation R NA polymerase II (Pol II) is distinguished from RNA polymerases I and III by the unique C-terminal domain (CTD) of its largest subunit, Rpb1 (1-3). The CTD, through its interaction with the Mediator complex, plays a central role in transcription activation (4-6), and it also plays critical roles in coupling mRNA synthesis to mRNA processing events, such as 5Ј capping, splicing, and 3Ј cleavage and polyadenylation (7,8). Composed of a tandemly repeated heptapeptide of the consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 , the CTD undergoes reversible phosphorylation (9-11). Whereas the hypophosphorylated Pol II isoform (Pol IIA) assembles with general transcription factors into the transcription preinitiation complex (12-14), the hyperphosphorylated isoform (Pol IIO) is the dominant species found in transcription elongation complexes in vivo (15)(16)(17). The phosphorylation state of the CTD is controlled by the activities of CTD-specific kinases and phosphatases. Several transcription factor-associated CTD kinases have been identified, including CDK7͞Kin28, CDK9͞Bur1͞Ctk1 and CDK8͞Srb10 (11,(18)(19)(20). Other kinases (e.g., Cdc2 kinase and mitogen-activated protein kinase 2) can phosphorylate the CTD in vitro, but their contributions to CTD phosphorylation in vivo are uncertain (18,(21)(22)(23).Pol IIO is believed to be dephosphorylated before it can recycle for another round of transcription initiation (13,14,21,24). Fcp1 was the first CTD phosphatase discovered to fulfill such a function (25, 26). Fcp1 was reported to remove phosphates specifically from the Pol II CTD, but not from the other phosphoproteins tested (25). The human and...

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.

No abstract

RNA polymerase II (Pol II)

Several lines of experimental analyses on the ploidy status of Azotobacter vinelandii genome lead to the conclusion that it contains more than 40 copies of its chromosome and therefore it is a polyploid organism. The genetic evidence argues against the existence of polyploidy in these cells since the segregation pattern of genetic markers under lack of selection pressure mimic that of haploids. However, when A. vinelandii was made Nif- by inserting a kanamycin resistance marker gene in the nifDK sequence and the cells were selected for kanamycin resistance and Nif+ phenotype, we were able to score colonies that are both kanamycin resistant and Nif+. Therefore, when the cells were subjected to forced double selection of the same locus, they behaved as if they carried at least two chromosomes, one carrying the kanamycin resistance marker in the nifDK genes and the other carrying the intact nifDK genes. These analyses suggested that at least a diploidy status can be induced in these cells under selection pressure.

The MoFe protein component of the complex metalloenzyme nitrogenase is an ␣2␤2 tetramer encoded by the nifD and the nifK genes. In nitrogen fixing organisms, the ␣ and ␤ 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 ␣2␤2 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.The nitrogenase enzyme, which is responsible for conversion of atmospheric nitrogen to ammonia, is found in all diazotrophs. It is actually composed of two separately purified, oxygen-labile, metalloproteins designated the Fe protein and the MoFe protein (1-7). The Fe protein is a homodimer of two identical subunits encoded by the nifH. Both subunits are bridged by one 4Fe-4S metal center and contain two nucleotide (MgATP or MgADP)-binding sites (5, 8 -10). The Fe protein serves as the obligate electron donor to MoFe protein during catalysis in a MgATP-and reductant-dependent process (11,12). The MoFe protein exists as an ␣2␤2 tetramer of about 240 kDa in size, encoded by nifD and nifK genes, respectively (13,14). Each ␣␤ heterodimer subunit binds two unique metal clusters, the FeMo cofactor and the P cluster. The FeMo cofactor that serves as the site of dinitrogen binding and reduction by the enzyme is located in the ␣ subunit, and the P cluster, which is positioned at the interface of ␣ and ␤ subunits of the heterodimer, is believed to mediate electron transfer from the Fe protein to the FeMo cofactor (15-17). During catalysis, the Fe protein forms a complex with the MoFe protein and transfers one electron to the MoFe protein with concomitant hydrolysis of two ATPs per Fe-MoFe protein interaction. It is accepted that the electron flow is from the Fe protein cluster to the MoFe protein P cluster and then to the FeMo cofactor, which is the substrate-binding and reduction site (13, 18 -20).Molecular evolutionary history of the nifDK, based on comparative analysis of the amino acid sequence of NifD, NifK, NifE, and NifN peptides, suggested that the genes encoding the NifDK and NifEN constitute a novel paralogous gene fam...

Azotobacter vinelandii UW97 is defective in nitrogen fixation due to a replacement of serine at position 44 by phenylalanine in the Fe-protein [Pulakat, L., Hausman, B.S.,