The transcription factors interferon regulatory factor 3 (IRF3) and NF-kappaB are required for the expression of many genes involved in the innate immune response. Viral infection, or the binding of double-stranded RNA to Toll-like receptor 3, results in the coordinate activation of IRF3 and NF-kappaB. Activation of IRF3 requires signal-dependent phosphorylation, but little is known about the signaling pathway or kinases involved. Here we report that the noncanonical IkappaB kinase homologs, IkappaB kinase-epsilon (IKKepsilon) and TANK-binding kinase-1 (TBK1), which were previously implicated in NF-kappaB activation, are also essential components of the IRF3 signaling pathway. Thus, IKKepsilon and TBK1 have a pivotal role in coordinating the activation of IRF3 and NF-kappaB in the innate immune response.

The RNA polymerase II holoenzyme consists of RNA polymerase II, a subset of general transcription factors, and regulatory proteins known as SRB proteins. The genes encoding SRB proteins were isolated as suppressors of mutations in the RNA polymerase II carboxy-terminal domain (CTD). The CTD and SRB proteins have been implicated in the response to transcriptional regulators. We report here the isolation of two new SRB genes, SRB10 and SRB11, which encode kinase- and cyclin-like proteins, respectively. Genetic and biochemical evidence indicates that the SRB10 and SRB11 proteins form a kinase-cyclin pair in the holoenzyme. The SRB10/11 kinase is essential for a normal transcriptional response to galactose induction in vivo. Holoenzymes lacking SRB10/11 kinase function are strikingly deficient in CTD phosphorylation. Although defects in the kinase substantially affect transcription in vivo, purified holoenzymes lacking SRB10/11 kinase function do not show defects in defined in vitro transcription systems, suggesting that the factors necessary to elicit the regulatory role of the SRB10/11 kinase are missing in these systems. These results indicate that the SRB10/11 kinase is involved in CTD phosphorylation and suggest that this modification has a role in the response to transcriptional regulators in vivo.

Here we report the identification of a novel PMA-inducible IkappaB kinase complex, distinct from the well-characterized high-molecular weight IkappaB kinase complex containing IKKalpha, IKKbeta, and IKKgamma. We have characterized one kinase from this complex, which we designate IKKepsilon. Although recombinant IKKepsilon directly phosphorylates only serine 36 of IKBalpha, the PMA-activated endogenous IKKepsilon complex phosphorylates both critical serine residues. Remarkably, this activity is due to the presence of a distinct kinase in this complex. A dominant-negative mutant of IKKepsilon blocks induction of NF-kappaB by both PMA and activation of the T cell receptor but has no effect on the activation of NF-KB by TNFalpha or IL-1. These observations indicate that the activation of NF-kappaB requires multiple distinct IkappaB kinase complexes, which respond to both overlapping and discrete signaling pathways.

Two cyclin-dependent kinases have been identified in yeast and mammalian RNA polymerase II transcription initiation complexes. We find that the two yeast kinases are indistinguishable in their ability to phosphorylate the RNA polymerase II CTD, and yet in living cells one kinase is a positive regulator and the other a negative regulator. This paradox is resolved by the observation that the negative regulator, Srb10, is uniquely capable of phosphorylating the CTD prior to formation of the initiation complex on promoter DNA, with consequent inhibition of transcription. In contrast, the TFIIH kinase phosphorylates the CTD only after the transcription apparatus is associated with promoter DNA. These results reveal that the timing of CTD phosphorylation can account for the positive and negative functions of the two kinases and provide a model for Srb10-dependent repression of genes involved in cell type specificity, meiosis, and sugar utilization.

RNA polymerase II holoenzymes have been described that consist of RNA polymerase II, a subset of general transcription factors, and four SRB proteins. The SRB proteins, which were identified through a selection for genes involved in transcription initiation by RNA polymerase II in vivo, are a hallmark of the holoenzyme. We report here the isolation and characterization of additional SRB genes. We show that the products of all nine SRB genes identified thus far are components of the RNA polymerase II holoenzyme and are associated with a holoenzyme subcomplex termed the mediator of activation. The holoenzyme is capable of responding to a transcriptional activator, suggesting a model in which activators function, in part, through direct interactions with the holoenzyme. Immunoprecipitation experiments with anti-SRB5 antibodies demonstrate that the acidic activating domain of VPI6 specifically binds to the holoenzyme. Furthermore, the holoenzyme and the mediator subcomplex bind to a VP16 affinity column. These results provide a more complete description of the RNA polymerase II holoenzyme and suggest that this form of the transcription apparatus can be recruited to promoters via direct interactions with activators.[Key Words: RNA polymerase II; holoenzyme; carboxy-terminal domain; genetic suppressors; transcription initiation; SRBs; transcription activation] Received December 13, 1994; revised version accepted March 14, 1995.Large muhisubunit complexes containing RNA polymerase II, a subset of the general transcription factors, and additional factors implicated in regulation of transcription initiation in vivo, can assemble independently of promoter DNA (Kim et al. 1994;Koleske and Young 1994). These complexes, termed RNA polymerase II holoenzymes, have been purified from Saccharomyces cerevisiae. The larger form of holoenzyme contains RNA polymerase II, TFIIB, TFIIF, TFIIH, and SRB (suppressor of RNA polymerase B) proteins (Koleske and Young 1994). Another form of holoenzyme has been described that contains RNA polymerase II, TFIIF, and SRB proteins but lacks TFIIB and TFIIH (Kim et al. 1994). The two holoenzyme forms may exist simultaneously in vivo, or the isolation of the smaller complex may be a consequence of the instability of the RNA polymerase II holoenzyme during purification.Selective transcription initiation in vitro by the 12-subunit core RNA polymerase II was shown previously to require the action of at least five general initiation factors: TATA-binding protein (TBPJ, TFIIB, TFIIE, TFIIF, and TFIIH (for review, see Conaway and Conaway 1993;Zawel and Reinberg 1993). Consistent with these data, selective transcription initiation in vitro with the larger form of RNA polymerase II holoenzyme required TBP and TFIIE (Koleske and Young 1994), and initiation with the smaller form required TBP, TFIIB, TFIIE, and TFIIH (Kim et al. 1994).The holoenzymes were discovered by virtue of their association with SRB proteins. SRB genes were obtained through a genetic selection designed to identify genes involved in RNA ...

Dysregulation of the alternative complement pathway (AP) predisposes individuals to a number of diseases including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Moreover, glomerular Ig deposits can lead to complement-driven nephropathies. Here we describe the discovery of a highly potent, reversible, and selective small-molecule inhibitor of factor B, a serine protease that drives the central amplification loop of the AP. Oral administration of the inhibitor prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats. In addition, inhibition of factor B prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes. These data demonstrate the potential therapeutic value of using a factor B inhibitor for systemic treatment of complement-mediated diseases and provide a basis for its clinical development.

Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.

RNA polymerase H subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed oa-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that inmunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional colunm chromatography. The Eucaryotic nuclear RNA polymerases I, II, and III are responsible for the synthesis of rRNA, mRNA, and small stable RNAs, respectively. These enzymes were originally defined by their ability to catalyze RNA chain initiation and elongation on nonspecific DNA templates and were distinguished by their chromatographic properties and their differential sensitivity to a-amanitin (26,36). It was subsequently shown that purified RNA polymerases I, II, and III can selectively initiate transcription in vitro in the presence of appropriate templates and associated factors (19,22,27,30,39).While the factors that provide specificity for the initiation of transcription are under intensive scrutiny, the RNA polymerases remain poorly understood. In contrast to their procaryotic counterparts, whose components have been well defined genetically and biochemically (13, 42), eucaryotic RNA polymerase subunits have been described largely as the polypeptides that copurify with nonspecific transcriptional activity. These eucaryotic RNA polymerases consist of 10 to 14 polypeptides that range from 220 to 10 kilodaltons (26,31,36). It is not clear whether these proteins are the complete set of RNA polymerase II subunits in vivo, whether they are present in equimolar amounts, and how they contribute to the function of the enzyme.The components of Saccharomyces cerevisiae RNA polymerases have been a focus of attention because RNA polymerases appear to be highly conserved among eucaryotes and because of the relative ease of genetic and biochemical experimentation in S. cerevisiae. Yeast RNA polymerases are perhaps the most thoroughly studied eucaryotic RNA polymerases, largely because of the work of Sentenac and his colleagues (for a review, see reference 36). The genes that encode the 10 putative RNA polymerase II subunits have been isolated and sequenced (2, 23, 38, 40a, 41, 41a; Woychik and Young, unpublished data). The two largest subunits of RNA polymerase II, RPB1 and RPB2, are * Corresponding author.homologs of the ,B' and ,B subunits of procaryotic RNA polymerase (2, 38). These two large subunits are essential for mRNA synthesis (28,35) and are able to bind nucleoside triphosphates and DNA (12,14,32). The smaller RNA polymerase II subunits (RPB3, RPB4, RPB5, RPB6, RPB7, RPB8, RPB9, and RPB10) have no obvious procaryotic counterparts. Nonetheless, at least some of these proteins have roles in transcription. Mutations in the genes for RPB3 and RPB4 can cause defects in mR...