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Although MDM2 plays a major role in regulating the stability of the p53 tumor suppressor protein, other poorly understood MDM2-independent pathways also exist. Human adenoviruses have evolved strategies to regulate p53 function and stability to permit efficient viral replication. One mechanism involves adenovirus E1B55K and E4orf6 proteins, which collaborate to target p53 for degradation. To determine the mechanism of this process, a multiprotein E4orf6-associated complex was purified and shown to contain a novel Cullin-containing E3 ubiquitin ligase that is (1) composed of Cullin family member Cul5, Elongins B and C, and the RING-H2 finger protein Rbx1(ROC1); (2) remarkably similar to the von Hippel-Lindau tumor suppressor and SCF (Skp1-Cul1/Cdc53-F-box) E3 ubiquitin ligase complexes; and (3) capable of stimulating ubiquitination of p53 in vitro in the presence of E1/E2 ubiquitin-activating and -conjugating enzymes. Cullins are activated by NEDD8 modification; therefore, to determine whether Cullin complexes are required for adenovirus-induced p53 degradation, studies were conducted in ts41 Chinese hamster ovary cells that are temperature sensitive for the NEDD8 pathway. E4orf6/E1B55K failed to induce the degradation of p53 at the nonpermissive temperature. Thus, our results identify a novel role for the Cullin-based machinery in regulation of p53.

The heterodimeric Elongin BC complex has been shown to interact in vitro and in mammalian cells with a conserved BC-box motif found in a growing number of proteins including RNA polymerase II elongation factor Elongin A, SOCS-box proteins, and the von Hippel-Lindau (VHL) tumor suppressor protein. Recently, the VHL-Elongin BC complex was found to interact with a module composed of Cullin family member Cul2 and RING-H2 finger protein Rbx1 to reconstitute a novel E3 ubiquitin ligase that activates ubiquitylation by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. In the context of the VHL ubiquitin ligase, Elongin BC functions as an adaptor that links the VHL protein to the Cul2/Rbx1 module, raising the possibility that the Elongin BC complex could function as an integral component of a larger family of E3 ubiquitin ligases by linking alternative BC-box proteins to Cullin/Rbx1 modules. In this report, we describe identification and purification from rat liver of a novel leucine-rich repeat-containing BC-box protein, MUF1, which we demonstrate is capable of assembling with a Cullin/Rbx1 module containing the Cullin family member Cul5 to reconstitute ubiquitin ligase activity. In addition, we show that the additional BC-box proteins Elongin A, SOCS1, and WSB1 are also capable of assembling with the Cul5/Rbx1 module to reconstitute potential ubiquitin ligases. Taken together, our findings identify MUF1 as a new member of the BC-box family of proteins, and they predict the existence of a larger family of Elongin BC-based E3 ubiquitin ligases.

TFIIF, ELL, and Elongin belong to a class of RNA polymerase II transcription factors that function similarly to activate the rate of elongation by suppressing transient pausing by polymerase at many sites along DNA templates. SII is a functionally distinct RNA polymerase II elongation factor that promotes elongation by reactivating arrested polymerase. Studies of the mechanism of SII action have shown (i) that arrest of RNA polymerase II results from irreversible displacement of the 3'-end of the nascent transcript from the polymerase catalytic site and (ii) that SII reactivates arrested polymerase by inducing endonucleolytic cleavage of the nascent transcript by the polymerase catalytic site thereby creating a new transcript 3'-end that is properly aligned with the catalytic site and can be extended. SII also induces nascent transcript cleavage by paused but non-arrested RNA polymerase II elongation intermediates, leading to the proposal that pausing may result from reversible displacement of the 3'-end of nascent transcripts from the polymerase catalytic site. On the basis of evidence consistent with the model that TFIIF, ELL, and Elongin suppress pausing by preventing displacement of the 3'-end of the nascent transcript from the polymerase catalytic site, we investigated the possibility of cross-talk between SII and transcription factors TFIIF, ELL, and Elongin. These studies led to the discovery that TFIIF, ELL, and Elongin are all capable of inhibiting SII-induced nascent transcript cleavage by non-arrested RNA polymerase II elongation intermediates. Here we present these findings, which bring to light a novel activity associated with TFIIF, ELL, and Elongin and suggest that these transcription factors may expedite elongation not only by increasing the forward rate of nucleotide addition by RNA polymerase II, but also by inhibiting SII-induced nascent transcript cleavage by non-arrested RNA polymerase II elongation intermediates.

RNA polymerase II elongation factor ELL was recently purified from rat liver as a component of a multiprotein complex containing ELL and three ELL-associated proteins (EAPs) of approximately 45 (EAP45), approximately 30 (EAP30), and approximately 20 (EAP20) kDa (Shilatifard, A. (1998) J. Biol. Chem. 273, 11212-11217). Cloning of cDNA encoding the EAP30 protein revealed that it shares significant sequence similarity with the product of the Saccharomyces cerevisiae SNF8 gene (Schmidt, A. E., Miller, T., Schmidt, S. L., Shiekhattar, R., and Shilatifard, A. (1999) J. Biol. Chem. 274, 21981-21985), which is required for efficient derepression of glucose-repressed genes. Here we report the cloning of cDNAs encoding the EAP45 and EAP20 proteins. In addition, we identify the S. cerevisiae VPS36 and YJR102c genes as potential orthologs of EAP45 and EAP20 and show that they are previously uncharacterized SNF genes with properties very similar to SNF8.

TFIIH is a multifunctional RNA polymerase II general initiation factor that includes two DNA helicases encoded by the Xeroderma pigmentosum complementation group B (XPB) and D (XPD) genes and a cyclin-dependent protein kinase encoded by the CDK7 gene. Previous studies have shown that the TFIIH XPB DNA helicase plays critical roles not only in transcription initiation, where it catalyzes ATP-dependent formation of the open complex, but also in efficient promoter escape, where it suppresses arrest of very early RNA polymerase II elongation intermediates. In this report, we present evidence that ATP-dependent TFIIH action in transcription initiation and promoter escape requires distinct regions of the DNA template; these regions are well separated from the promoter region unwound by the XPB DNA helicase and extend, respectively, approximately 23-39 and approximately 39-50 bp downstream from the transcriptional start site. Taken together, our findings bring to light a role for promoter DNA in TFIIH action and are consistent with the model that TFIIH translocates along promoter DNA ahead of the RNA polymerase II elongation complex until polymerase has escaped the promoter.

Recently, key advances in biochemical and structural studies of RNA polymerase II (pol II) and the basal transcriptional machinery have shed considerable light on the basic mechanisms underlying the initiation stage of eukaryotic mRNA synthesis. The development of methods for obtaining crystal structures of pol II and its complexes has revolutionized transcriptional studies and holds promise that aspects of initiation will soon be understood at atomic resolution; crosslinking studies have revealed intriguing features of the topology of the pol II initiation complex and provided working models for dynamic steps of initiation; and mechanistic studies have identified promoter escape as a critical step during initiation and brought to light novel roles for the general initiation factors TFIIE, TFIIF, and TFIIH in this process.

Inherited mutations of the TFIIH helicase subunits xeroderma pigmentosum (XP) B or XPD yield overlapping DNA repair and transcription syndromes. The high risk of cancer in these patients is not fully explained by the repair defect. The transcription defect is subtle and has proven more difficult to evaluate. Here, XPB and XPD mutations are shown to block transcription activation by the FUSE Binding Protein (FBP), a regulator of c-myc expression, and repression by the FBP Interacting Repressor (FIR). Through TFIIH, FBP facilitates transcription until promoter escape, whereas after initiation, FIR uses TFIIH to delay promoter escape. Mutations in TFIIH that impair regulation by FBP and FIR affect proper regulation of c-myc expression and have implications in the development of malignancy.

Mutations in the VHL tumor suppressor gene result in constitutive expression of many hypoxia-inducible genes, at least in part because of increases in the cellular level of hypoxia-inducible transcription factor HIF1alpha, which in normal cells is rapidly ubiquitinated and degraded by the proteasome under normoxic conditions. The recent observation that the VHL protein is a subunit of an Skp1-Cul1/Cdc53-F-box (SCF)-like E3 ubiquitin ligase raised the possibility that VHL may be directly responsible for regulating cellular levels of HIF1alpha by targeting it for ubiquitination and proteolysis. In this report, we test this hypothesis directly. We report development of methods for production of the purified recombinant VHL complex and present direct biochemical evidence that it can function with an E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzyme to activate HIF1alpha ubiquitination in vitro. Our findings provide new insight into the function of the VHL tumor suppressor protein, and they provide a foundation for future investigations of the mechanisms underlying VHL regulation of oxygen-dependent gene expression.

The elongation stage of eukaryotic mRNA synthesis can be regulated by transcription factors that interact directly with the RNA polymerase II (pol II) elongation complex and by activities that modulate the structure of its chromatin template. Recent studies have revealed new elongation factors and have implicated the general initiation factors TFIIE, TFIIF and TFIIH, as well as the C-terminal domain (CTD) of the largest subunit of pol II, in elongation. The recently reported high-resolution crystal structure of RNA polymerase II, which provides insight into the architecture of the elongation complex, marks a new era of investigation into transcription elongation.

Transcription factor IIIA (TFIIIA) activates 5S ribosomal RNA gene transcription in eukaryotes. The protein from vertebrates has nine contiguous Cys(2)His(2)zinc fingers which function in nucleic acid binding, and a C-terminal region involved in transcription activation. In order to identify protein partners for TFIIIA, yeast two-hybrid screens were performed using the C-terminal region of Xenopus TFIIIA as an attractor and a rat cDNA library as a source of potential partners. A cDNA clone was identified which produced a protein in yeast that interacted with Xenopus TFIIIA but not with yeast TFIIIA. This rat clone was sequenced and the primary structure of the human homolog (termed TFIIIA-intP for TFIIIA-interacting protein) was determined from expressed sequence tags. In vitro interaction of recombinant human TFIIIA-intP with recombinant Xenopus TFIIIA was demonstrated by immuno-precipitation of the complex using anti-TFIIIA-intP antibody. Interaction of rat TFIIIA with rat TFIIIA-intP was indicated by co-chromatography of the two proteins on DEAE-5PW following fractionation of a rat liver extract on cation, anion and gel filtration resins. In a HeLa cell nuclear extract, recombinant TFIIIA-intP was able to stimulate TFIIIA-dependent transcription of the Xenopus 5S ribosomal RNA gene but not TFIIIA-independent transcription of the human adenovirus VA RNA gene.

Elongin is a transcription elongation factor that was first identified in mammalian systems and is composed of the three subunits, elongin A, B, and C. Sequence homologues of elongin A and elongin C, but not elongin B, were identified in the yeast genome. Neither yeast elongin A nor C sequence homologues was required for cell viability. The two gene products could be purified from yeast as a complex. A recombinant form of the complex, which could only be produced in bacteria if the gene products were co-expressed, was purified over several chromatographic steps. The complex did not stimulate transcription elongation by yeast RNA polymerase II. Using limited proteolysis, the N-terminal 144 residues of yeast elongin A were shown to be sufficient for interaction with yeast elongin C. The purified complex of yeast elongin C/elongin A(1-143) was analyzed using circular dichroism and nuclear magnetic spectroscopy. These studies revealed that yeast elongin A is unfolded but undergoes a dramatic modification of its structure in the presence of elongin C, and that elongin C forms a stable dimer in the absence of elongin A.

Transcription factor (TF) IIF is a multifunctional RNA polymerase II transcription factor that has well established roles in both transcription initiation, where it functions as a component of the preinitiation complex and is required for formation of the open complex and synthesis of the first phosphodiester bond of nascent transcripts, and in transcription elongation, where it is capable of interacting directly with the ternary elongation complex and stimulating the rate of transcription. In this report, we present evidence that TFIIF is also required for efficient promoter escape by RNA polymerase II. Our findings argue that TFIIF performs dual roles in this process. We observe (i) that TFIIF suppresses the frequency of abortive transcription by very early RNA polymerase II elongation intermediates by increasing their processivity and (ii) that TFIIF cooperates with TFIIH to prevent premature arrest of early elongation intermediates. In addition, our findings argue that two TFIIF functional domains mediate TFIIF action in promoter escape. First, we observe that a TFIIF mutant selectively lacking elongation activity supports TFIIH action in promoter escape, but is defective in suppressing the frequency of abortive transcription by very early RNA polymerase II elongation intermediates. Second, a TFIIF mutant selectively lacking initiation activity is more active than wild type TFIIF in increasing the processivity of very early elongation intermediates, but is defective in supporting TFIIH action in promoter escape. Taken together, our findings bring to light a function for TFIIF in promoter escape and support a role for TFIIF elongation activity in this process.

The von Hippel-Lindau tumor suppressor protein (pVHL) negatively regulates hypoxia-inducible mRNAs such as the mRNA encoding vascular endothelial growth factor (VEGF). This activity has been linked to its ability to form multimeric complexes that contain elongin C, elongin B, and Cul2. To understand this process in greater detail, we performed a series of in vitro binding assays using pVHL, elongin B, and elongin C variants as well as synthetic peptide competitors derived from pVHL or elongin C. A subdomain of elongin C (residues 17-50) was necessary and sufficient for detectable binding to elongin B. In contrast, elongin B residues required for binding to elongin C were not confined to a discrete colinear domain. We found that the pVHL (residues 157-171) is necessary and sufficient for binding to elongin C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that directly bind to elongin C and/or alter the conformation of pVHL such that binding to elongin C is at least partially diminished. These results are consistent with the view that diminished binding of pVHL to the elongins plays a causal role in VHL disease.

The RING-H2 finger protein Rbx1 is a subunit of the related SCF (Skp1-Cdc53/Cul1-F-box protein) and von Hippel-Lindau (VHL) tumor suppressor (elongin BC-Cul2-VHL) E3 ubiquitin ligase complexes, where it functions as a component of Cdc53/Rbx1 and Cul2/Rbx1 modules that activate ubiquitination of target proteins by the E2 ubiquitin-conjugating enzymes Cdc34 and Ubc5. Here we demonstrate that the Cdc53/Rbx1 and Cul2/Rbx1 modules also activate conjugation of the ubiquitin-like protein Rub1 to Cdc53 and Cul2 by the dedicated E2 Rub1 conjugating enzyme Ubc12. Our findings identify Rbx1 as a common component of enzyme systems responsible for ubiquitin and Rub1 modification of target proteins.

Mutations of von Hippel-Lindau disease (VHL) tumor-suppressor gene product (pVHL) are found in patients with dominant inherited VHL syndrome and in the vast majority of sporadic clear cell renal carcinomas. The function of the pVHL protein has not been clarified. pVHL has been shown to form a complex with elongin B and elongin C (VBC) and with cullin (CUL)-2. In light of the structural analogy of VBC-CUL-2 to SKP1-CUL-1-F-box ubiquitin ligases, the ubiquitin ligase activity of VBC-CUL-2 was examined in this study. We show that VBC-CUL-2 exhibits ubiquitin ligase activity, and we identified UbcH5a, b, and c, but not CDC34, as the ubiquitin-conjugating enzymes of the VBC-CUL-2 ubiquitin ligase. The protein Rbx1/ROC1 enhances ligase activity of VBC-CUL-2 as it does in the SKP1-CUL-1-F-box protein ligase complex. We also found that pVHL associates with two proteins, p100 and p220, which migrate at a similar molecular weight as two major bands in the ubiquitination assay. Furthermore, naturally occurring pVHL missense mutations, including mutants capable of forming a complex with elongin B-elongin C-CUL-2, fail to associate with p100 and p220 and cannot exhibit the E3 ligase activity. These results suggest that pVHL might be the substrate recognition subunit of the VBC-CUL-2 E3 ligase. This is also, to our knowledge, the first example of a human tumor-suppressor protein being directly involved in the ubiquitin conjugation system which leads to the targeted degradation of substrate proteins.

Rat pheochromocytoma (PC12) cells were stably transfected with either wild type or mutated human von Hippel-Lindau tumor suppressor protein (hpVHL). These proteins have opposing effects on regulating expression of the gene encoding tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Whereas wild type hpVHL represses levels of TH mRNA and protein 5-fold, a truncated pVHL mutant, pVHL(1-115), induces accumulation of TH mRNA and protein 3-fold. hpVHL-induced inhibition of TH gene expression does not involve either a decrease in TH mRNA stability or repression of TH promoter activity. However, repression results from inhibition of RNA elongation at a downstream region of the TH gene. This elongation pause is accompanied by hpVHL sequestration in the nuclear extracts of elongins B and C, regulatory components of the transcription elongation heterotrimer SIII (elongin A/B/C). Hypoxia, a physiological stimulus for TH gene expression, alleviates the elongation block. A truncated pVHL mutant, pVHL(1-115), stimulates TH gene expression by increasing the efficiency of TH transcript elongation. This is the first report showing pVHL-dependent regulation of specific transcript elongation in vivo, as well as dominant negative activity of pVHL mutants in pheochromocytoma cells.

TFIIH is an RNA polymerase II transcription factor that performs ATP-dependent functions in both transcription initiation, where it catalyzes formation of the open complex, and in promoter escape, where it suppresses arrest of the early elongation complex at promoter-proximal sites. TFIIH possesses three known ATP-dependent activities: a 3' --> 5' DNA helicase catalyzed by its XPB subunit, a 5' --> 3' DNA helicase catalyzed by its XPD subunit, and a carboxyl-terminal domain (CTD) kinase activity catalyzed by its CDK7 subunit. In this report, we exploit TFIIH mutants to investigate the contributions of TFIIH DNA helicase and CTD kinase activities to efficient promoter escape by RNA polymerase II in a minimal transcription system reconstituted with purified polymerase and general initiation factors. Our findings argue that the TFIIH XPB DNA helicase is primarily responsible for preventing premature arrest of early elongation intermediates during exit of polymerase from the promoter.

Elongin is a heterotrimeric transcription elongation factor composed of subunits A, B, and C in mammals. Elongin A and C are F-box-containing and SKP1 homologue proteins, respectively, and are therefore of interest for their potential roles in cell cycle-dependent proteolysis. Mammalian elongin C interacts with both elongin A and elongin B, as well as with the von Hippel-Lindau tumor suppressor protein VHL. To investigate the corresponding interactions in yeast, we have utilized NMR spectroscopy combined with ultracentrifugal sedimentation experiments to examine complexes of yeast elongin C (Elc1) with yeast elongin A (Ela1) and two peptides from homologous regions of Ela1 and human VHL. Elc1 alone is a homotetramer composed of subunits with a structured N-terminal region and a dynamically unstable C-terminal region. Binding of a peptide fragment of the Elc1-interaction domain of Ela1 or with a homologous peptide from VHL promotes folding of the C-terminal region of Elc1 into two regular helical structures and dissociates Elc1 into homodimers. Moreover, analysis of the complex of Elc1 with the full Elc1-interaction domain of Ela1 reveals that the Elc1 homodimer is dissociated to preferentially form an Ela1/Elc1 heterodimer. Thus, elongin C is found to oligomerize in solution and to undergo significant structural rearrangements upon binding of two different partner proteins. These results suggest a structural basis for the interaction of an F-box-containing protein with a SKP1 homologue and the modulation of this interaction by the tumor suppressor VHL.

Over the past few years, biochemical and genetic studies have shed considerable light on the structure and function of the RNA polymerase II (pol II) elongation complex and the transcription factors that control it. Novel elongation factors have been identified and their mechanisms of action characterized in increasing detail; new insights into the biological roles of elongation factors have been gained from genetic studies of the regulation of mRNA synthesis in yeast; and intriguing links between the pol II elongation machinery and the pathways of DNA repair and recombination have emerged.

Mammalian Elongin B is a 118-amino acid protein composed of an 84-amino acid amino-terminal ubiquitin-like domain and a 34-amino acid carboxyl-terminal tail. Elongin B is found in cells as a subunit of the heterodimeric Elongin BC complex, which was originally identified as a positive regulator of RNA polymerase II elongation factor Elongin A and subsequently as a component of the multiprotein von Hippel-Lindau tumor suppressor and suppressor of cytokine signaling complexes. As part of our effort to understand how the Elongin BC complex regulates the activity of Elongin A, we are characterizing Elongin B functional domains. In this report, we show that the Elongin B ubiquitin-like domain is necessary and sufficient for interaction with Elongin C and for positive regulation of Elongin A transcriptional activity. In addition, by site-directed mutagenesis of the Elongin B ubiquitin-like domain, we identify a short Elongin B region that is important for its interaction with Elongin C. Finally, we observe that both the ubiquitin-like domain and carboxyl-terminal tail are conserved in Drosophila melanogaster and Caenorhabditis elegans Elongin B homologs that efficiently substitute for mammalian Elongin B in reconstitution of the transcriptionally active Elongin ABC complex, suggesting that the carboxyl-terminal tail performs an additional function not detected in our assays.

The von Hippel-Lindau (VHL) tumor suppressor gene is mutated in most human kidney cancers. The VHL protein is part of a complex that includes Elongin B, Elongin C, and Cullin-2, proteins associated with transcriptional elongation and ubiquitination. Here it is shown that the endogenous VHL complex in rat liver also includes Rbx1, an evolutionarily conserved protein that contains a RING-H2 fingerlike motif and that interacts with Cullins. The yeast homolog of Rbx1 is a subunit and potent activator of the Cdc53-containing SCFCdc4 ubiquitin ligase required for ubiquitination of the cyclin-dependent kinase inhibitor Sic1 and for the G1 to S cell cycle transition. These findings provide a further link between VHL and the cellular ubiquitination machinery.

Control of cyclin levels is critical for proper cell cycle regulation. In yeast, the stability of the G1 cyclin Cln1 is controlled by phosphorylation-dependent ubiquitination. Here it is shown that this reaction can be reconstituted in vitro with an SCF E3 ubiquitin ligase complex. Phosphorylated Cln1 was ubiquitinated by SCF (Skp1-Cdc53-F-box protein) complexes containing the F-box protein Grr1, Rbx1, and the E2 Cdc34. Rbx1 promotes association of Cdc34 with Cdc53 and stimulates Cdc34 auto-ubiquitination in the context of Cdc53 or SCF complexes. Rbx1, which is also a component of the von Hippel-Lindau tumor suppressor complex, may define a previously unrecognized class of E3-associated proteins.

Eukaryotic mRNA synthesis is catalyzed by multisubunit RNA polymerase II and proceeds through multiple stages referred to as preinitiation, initiation, elongation, and termination. Over the past 20 years, biochemical studies of eukaryotic mRNA synthesis have largely focused on the preinitiation and initiation stages of transcription. These studies led to the discovery of the class of general initiation factors (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH), which function in intimate association with RNA polymerase II and are required for selective binding of polymerase to its promoters, formation of the open complex, and synthesis of the first few phosphodiester bonds of nascent transcripts. Recently, biochemical studies of the elongation stage of eukaryotic mRNA synthesis have led to the discovery of several cellular proteins that have properties expected of general elongation factors and that have been found to play unanticipated roles in human disease. Among these candidate general elongation factors are the positive transcription elongation factor b (P-TEFb), eleven-nineteen lysine-rich in leukemia (ELL), Cockayne syndrome complementation group B (CSB), and elongin proteins, which all function in vitro to expedite elongation by RNA polymerase II by suppressing transient pausing or premature arrest by polymerase through direct interactions with the elongation complex. Despite their similar activities in elongation, the P-TEFb, ELL, CSB, and elongin proteins appear to play roles in a diverse collection of human diseases, including human immunodeficiency virus-1 infection, acute myeloid leukemia, Cockayne syndrome, and the familial cancer predisposition syndrome von Hippel-Lindau disease. here we review our current understanding of the P-TEFb, ELL, CSB, and elongin proteins, their mechanisms of action, and their roles in human disease.

The Elongin BC complex was identified initially as a positive regulator of RNA polymerase II (Pol II) elongation factor Elongin A and subsequently as a component of the multiprotein von Hippel-Lindau (VHL) tumor suppressor complex, in which it participates in both tumor suppression and negative regulation of hypoxia-inducible genes. Elongin B is a ubiquitin-like protein, and Elongin C is a Skp1-like protein that binds to a BC-box motif that is present in both Elongin A and VHL and is distinct from the conserved F-box motif recognized by Skp1. In this report, we demonstrate that the Elongin BC complex also binds to a functional BC box present in the SOCS box, a sequence motif identified recently in the suppressor of cytokine signaling-1 (SOCS-1) protein, as well as in a collection of additional proteins belonging to the SOCS, ras, WD-40 repeat, SPRY domain, and ankyrin repeat families. In addition, we present evidence (1) that the Elongin BC complex is a component of a multiprotein SOCS-1 complex that attenuates Jak/STAT signaling by binding to Jak2 and inhibiting Jak2 kinase, and (2) that by interacting with the SOCS box, the Elongin BC complex can increase expression of the SOCS-1 protein by inhibiting its degradation. These results suggest that Elongin BC is a multifunctional regulatory complex capable of controlling multiple pathways in the cell through interaction with a short degenerate sequence motif found in many different proteins.

We previously identified and purified Elongin by its ability to stimulate the rate of elongation by RNA polymerase II in vitro (Bradsher, J. N., Jackson, K. W., Conaway, R. C., and Conaway, J. W. (1993) J. Biol. Chem. 268, 25587-25593). In this report, we present evidence that stimulation of elongation by Elongin requires that the early RNA polymerase II elongation complex undergoes conversion to an Elongin-activable form. We observe (i) that Elongin does not detectably stimulate the rate of promoter-specific transcription initiation by the fully assembled preinitiation complex and (ii) that early RNA polymerase II elongation intermediates first become susceptible to stimulation by Elongin after synthesizing 8-9-nucleotide-long transcripts. Furthermore, we show that the relative inability of Elongin to stimulate elongation by early elongation intermediates correlates not with the lengths of their associated transcripts but, instead, with the presence of transcription factor IIF (TFIIF) in transcription reactions. By exploiting adenovirus 2 major late promoter derivatives that contain premelted transcriptional start sites and do not require TFIIF, TFIIE, or TFIIH for transcription initiation, we observe (i) that Elongin is capable of strongly stimulating the rate of synthesis of trinucleotide transcripts by a subcomplex of RNA polymerase II, TBP, and TFIIB and (ii) that the ability of Elongin to stimulate synthesis of these short transcripts is substantially reduced by addition of TFIIF to transcription reactions. Here we present these findings, which are consistent with the model that maximal stimulation of elongation by Elongin requires that early elongation intermediates undergo a structural transition that includes loss of TFIIF.

A multiprotein complex isolated from murine cells is identified as a counterpart of the yeast Mediator of transcriptional regulation on the basis of the following: homologs of two subunits of yeast Mediator, Srb7 and Med7, copurify with the complex; peptide sequencing reveals, in addition, homologs of the yeast Mediator subunits Rgr1 and Med6; as with yeast Mediator, the mouse complex binds to the RNA polymerase II C-terminal domain (CTD) and stimulates phosphorylation of the CTD by TFIIH. Peptide sequencing also identifies a component of mouse Mediator as a relative of Ring-3 protein, a mitogen-activated nuclear protein kinase, raising the possibility of Mediator as an end point of signal transduction pathways.

The C-terminal part of the largest subunit of eukaryotic RNA polymerase II is composed solely of the highly repeated consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. This domain, called the C-terminal domain (CTD), is phosphorylated mostly at serine residues during transcription initiation, but the precise role of this phosphorylation remains controversial. Several protein kinases are able to phosphorylate this sequence in vitro. The aim of this work was to define the positions of the amino acids phosphorylated by four of these CTD kinases (transcription factor (TF) IIH-kinase, DNA-dependent protein kinase, and the mitogen-activated protein kinases ERK1 and ERK2) and to compare the specificity of these different protein kinases. We show that TFIIH kinase and the mitogen-activated protein kinases phosphorylate only serine 5 of the CTD sequence, whereas DNA-dependent protein kinase phosphorylates serines 2 and 7. Among the different CTD kinases, only TFIIH kinase is appreciably more active on two repeats of the consensus sequence than on one motif. These in vitro results can provide some clues to the nature of the protein kinases responsible for the in vivo phosphorylation of the RNA polymerase CTD. In particular, the ratio of phosphorylated serine to threonine observed in vivo cannot be explained if TFIIH kinase is the only protein kinase involved in the phosphorylation of the CTD.

The von Hippel-Lindau tumor suppressor protein (pVHL) binds to elongins B and C and posttranscriptionally regulates the accumulation of hypoxia-inducible mRNAs under normoxic (21% O2) conditions. Here we report that pVHL binds, via elongin C, to the human homolog of the Caenorhabditis elegans Cul2 protein. Coimmunoprecipitation and chromatographic copurification data suggest that pVHL-Cul2 complexes exist in native cells. pVHL mutants that were unable to bind to complexes containing elongin C and Cul2 were likewise unable to inhibit the accumulation of hypoxia-inducible mRNAs. A model for the regulation of hypoxia-inducible mRNAs by pVHL is presented based on the apparent similarity of elongin C and Cul2 to Skp1 and Cdc53, respectively. These latter proteins form complexes that target specific proteins for ubiquitin-dependent proteolysis.

Shortly after initiating promoter-specific transcription in vitro, mammalian RNA polymerase II becomes highly susceptible to arrest in a promoter-proximal region 9-13 base pairs downstream of the transcriptional start site (Dvir, A., Conaway, R. C., and Conaway, J. W. (1996) J. Biol. Chem. 271, 23352-23356). Arrest by polymerase in this region is suppressed by TFIIH in an ATP-dependent reaction (Dvir, A., Conaway, R. C., and Conaway, J. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9006-9010). In this report, we present evidence that, in addition to TFIIH and an ATP cofactor, efficient transcription by RNA polymerase II through this promoter-proximal region requires formation of an "escape-competent" transcriptional intermediate. Formation of this intermediate requires template DNA 40-50 base pairs downstream of the transcriptional start site. This requirement for downstream DNA is transient, since template DNA downstream of +40 is dispensable for assembly of the preinitiation complex, for initiation and synthesis of the first 10-12 phosphodiester bonds of nascent transcripts and for further extension of transcripts longer than approximately 14 nucleotides. Thus, promoter escape requires that the RNA polymerase II transcription complex undergoes a critical structural transition, likely driven by interaction of one or more components of the transcriptional machinery with template DNA 40-50 base pairs downstream of the transcriptional start site.

Elongin C is a 112-amino acid protein that is found in mammalian cells as a positive regulatory subunit of heterotrimeric RNA polymerase II elongation factor Elongin (SIII) and as a component of a multiprotein complex containing the von Hippel-Lindau (VHL) tumor suppressor protein. As a subunit of the Elongin complex, Elongin C interacts directly with the transcriptionally active Elongin A subunit and potently induces its elongation activity; in addition, Elongin C interacts with the ubiquitin-like Elongin B subunit, which regulates the interaction of Elongin C with Elongin A. As a component of the VHL complex, Elongin C interacts directly with both Elongin B and the VHL protein. Binding of the VHL protein to Elongin C was found to prevent Elongin C from interacting with and activating Elongin A in vitro, leading to the proposal that one function of the VHL protein may be to regulate RNA polymerase II elongation by negatively regulating the Elongin complex. In this report, we identify Elongin C sequences required for its interaction with the VHL protein. We previously demonstrated that the ability of Elongin C to bind and activate Elongin A is sensitive to mutations in the C-terminal half of Elongin C, as well as to mutations in an N-terminal Elongin C region needed for formation of the Elongin BC complex. Here we show that interaction of Elongin C with the VHL tumor suppressor protein depends strongly on sequences in the C terminus of Elongin C but is independent of the N-terminal Elongin C region required for binding to Elongin B and for binding and activation of Elongin A. Taken together, our results are consistent with the proposal that the VHL protein negatively regulates Elongin C activation of the Elongin complex by sterically blocking the interaction of C-terminal Elongin C sequences with Elongin A. In addition, our finding that only a subset of Elongin C sequences required for its interaction with Elongin A are critical for binding to VHL may offer the opportunity to develop reagents that selectively interfere with Elongin and VHL function.

The human ELL gene on chromosome 19p13.1 undergoes frequent translocations with the trithorax-like MLL gene on chromosome 11q23 in acute myeloid leukemia. Recently, the human ELL gene was shown to encode an RNA polymerase II elongation factor that activates elongation by suppressing transient pausing by polymerase at many sites along the DNA. In this report, we identify and characterize two overlapping ELL functional domains that govern its interaction with RNA polymerase II and the ternary elongation complex. Our findings reveal that, in addition to its elongation activation domain, ELL contains a novel type of RNA polymerase II interaction domain that is capable of negatively regulating polymerase activity in promoter-specific transcription initiation in vitro. Notably, the MLL-ELL translocation results in deletion of a portion of this functional domain, and ELL mutants lacking sequences deleted by the translocation bind RNA polymerase II and are fully active in elongation, but fail to inhibit initiation. Taken together, these results raise the possibility that the MLL-ELL translocation could alter ELL-RNA polymerase II interactions that are not involved in regulation of elongation.

TFIIH is a multifunctional RNA polymerase II transcription factor that possesses DNA-dependent ATPase, DNA helicase, and protein kinase activities. Previous studies have established that TFIIH enters the preinitiation complex and fulfills a critical role in initiation by catalyzing ATP-dependent formation of the open complex prior to synthesis of the first phosphodiester bond of nascent transcripts. In this report, we present direct evidence that TFIIH also controls RNA polymerase II activity at a postinitiation stage of transcription, by preventing premature arrest by very early elongation complexes just prior to their transition to stably elongating complexes. Unexpectedly, we observe that TFIIH is capable of entering the transcription cycle not only during assembly of the preinitiation complex but also after initiation and synthesis of as many as four to six phosphodiester bonds. These findings shed new light on the role of TFIIH in initiation and promoter escape and reveal an unanticipated flexibility in the ability of TFIIH to interact with RNA polymerase II transcription intermediates prior to, during, and immediately after initiation.

With the availability of the general initiation factors (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH), it is now possible to investigate aspects of the mechanism of eukaryotic messenger RNA synthesis in purified, reconstituted RNA polymerase II transcription systems. Rapid progress in these investigations has been spurred by use of a growing number of assays that are proving valuable not only for dissecting the molecular mechanisms of transcription initiation and elongation by RNA polymerase II, but also for identifying and purifying novel transcription factors that regulate polymerase activity. Here we describe a variety of these assays and discuss their utility in the analysis of transcription by RNA polymerase II.

We recently isolated an RNA polymerase II elongation factor from rat liver nuclei and found it to be homologous to the product of the human ELL gene, a frequent target for translocations in acute myeloid leukemia. To further our understanding of the possible role(s) of ELL in transcriptional regulation and human disease, we initiated a search for ELL-related proteins. In this report we describe molecular cloning, expression, and characterization of human ELL2, a novel RNA polymerase II elongation factor 49% identical and 66% similar to ELL. Mechanistic studies indicate that ELL2 and ELL possess similar transcriptional activities. Structure-function studies localize the ELL2 elongation activation domain to an ELL2 N-terminal region that is highly homologous to ELL. Finally, Northern blot analysis reveals that the ELL2 and ELL genes are transcribed in many of the same tissues, but that the ratio of their transcripts exhibits tissue-to-tissue variation, raising the possibility that ELL2 and ELL may not perform completely general functions, but, instead, may perform gene- or tissue-specific functions.

Over the past year, key advances in several areas of research on the structure and function of the RNA polymerase (pol II) elongation complex have shed considerable light on the mechanisms governing the elongation stage of eukaryotic mRNA synthesis. Novel features of the regulation of elongation by DNA and RNA binding transcriptional activators have been brought to light; the mechanisms of action of elongation factors that suppress pausing and premature arrest by transcribing pol II have been defined in greater detail; and novel elongation factors implicated in human disease have been identified and characterized.

The elongin (SIII) complex strongly stimulates the rate of elongation by RNA polymerase II by suppressing transient pausing by polymerase at many sites along the DNA. Elongin (SIII) is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, which bind stably to each other to form a binary complex that interacts with elongin A and strongly induces its transcriptional activity. The elongin (SIII) complex is a potential target for negative regulation by the von Hippel-Lindau (VHL) tumor suppressor protein, which is capable of binding stably to the elongin BC complex and preventing it from activating elongin A. Here, we identify an elongin A domain sufficient for activation of elongation and demonstrate that it is a novel type of inducible activator that targets the RNA polymerase II elongation complex and is evolutionarily conserved in species as distantly related as Caenorhabditis elegans and man. In addition, we demonstrate that both the elongin A elongation activation domain and the VHL tumor suppressor protein interact with the elongin BC complex through a conserved elongin BC binding site motif that is essential for induction of elongin A activity by elongin BC and for tumor suppression by the VHL protein.

The Elongin (SIII) complex stimulates the rate of elongation by RNA polymerase II by suppressing transient pausing by polymerase at many sites along DNA templates. The Elongin (SIII) complex is composed of a transcriptionally active A subunit, a chaperone-like B subunit, which promotes assembly and enhances stability of the Elongin (SIII) complex, and a regulatory C subunit, which (i) functions as a potent activator of Elongin A transcriptional activity, (ii) interacts specifically with Elongin B to form an isolable Elongin BC complex, and (iii) is bound and negatively regulated in vitro by the product of the von Hippel-Lindau tumor suppressor gene. As part of our effort to understand how Elongin C regulates the activity of the Elongin (SIII) complex, we are characterizing Elongin C functional domains. In this report, we identify Elongin C mutants that fall into multiple functional classes based on their abilities to bind Elongin B and to bind and activate Elongin A under our assay conditions. Characterization of these mutants suggests that Elongin C is composed of multiple overlapping regions that mediate functional interactions with Elongin A and B.

Human hepatitis B virus genome encodes a protein, termed HBx, that is widely recognized as a transcriptional transactivator. While HBx does not directly bind cis-acting transcriptional control elements, it has been shown to associate with cellular proteins that bind DNA. Because HBx transactivated a large number of viral/cellular transcriptional control elements, we looked for its targets within the components of the basal transcriptional machinery. This search led to the identification of its interactions with TFIIH. Here, we show that HBx interacts with yeast and mammalian TFIIH complexes both in vitro and in vivo. These interactions between HBx and the components of TFIIH are supported by several lines of evidence including results from immunoprocedures and direct methods of measuring interactions. We have identified ERCC3 and ERCC2 DNA helicase subunits of holoenzyme TFIIH as targets of HBx interactions. Furthermore, the DNA helicase activity of purified TFIIH from rat liver and, individually, the ERCC2 component of TFIIH is stimulated in the presence of HBx. These observations suggest a role for HBx in transcription and DNA repair.

It is well established that TFIIH-dependent transcription by RNA polymerase II requires a hydrolyzable ATP cofactor for synthesis of the first phosphodiester bond of nascent transcripts. Whether an ATP cofactor is also required after initiation for escape of RNA polymerase II from the promoter has, however, been controversial. We have now addressed this question directly by investigating the ability of RNA polymerase II transcription complexes containing short, approximately 5-8-nucleotide transcripts synthesized in the presence of limiting nucleotides to escape the promoter in the absence of an ATP cofactor in a basal transcription system reconstituted with purified RNA polymerase II and general initiation factors. Depletion of ATP had a profound effect on the ability of initiated complexes to progress into the elongation phase: whereas in the presence of ATP, the majority of transcription complexes could be chased away from the promoter-proximal region, most complexes deprived of ATP catalyzed synthesis of only a few phosphodiester bonds and then ceased elongation after synthesizing transcripts less than 10-14 nucleotides in length. A significant fraction of these transcripts could be extended following addition of ATP, indicating that they were contained in arrested, but potentially active elongation complexes. Like the ATP-requiring step in initiation, ATP-dependent suppression of arrest by RNA polymerase II at promoter-proximal sites is inhibited by adenosine 5'-O-(thio)triphosphate. Transcription complexes containing transcripts longer than 9-10 nucleotides are insensitive to inhibition by ATPgammaS, indicating that susceptibility to ATP-sensitive arrest is a property of very early elongation complexes. Taken together, our findings reveal a novel role for an ATP cofactor in transcription by RNA polymerase II.

Synthesis of eukaryotic messenger RNA by RNA polymerase II is governed by the concerted action of a set of general transcription factors that control the activity of polymerase during both the initiation and elongation stages of transcription. To date, five general elongation factors [P-TEFb, SII, TFIIF, Elongin (SIII) and ELL] have been defined biochemically. Here, we discuss these transcription factors and their roles in controlling the activity of the RNA polymerase II elongation complex.

The human ELL gene on chromosome 19 undergoes frequent translocations with the trithorax-like MLL gene on chromosome 11 in acute myeloid leukemias. Here, ELL was shown to encode a previously uncharacterized elongation factor that can increase the catalytic rate of RNA polymerase II transcription by suppressing transient pausing by polymerase at multiple sites along the DNA. Functionally, ELL resembles Elongin (SIII), a transcription elongation factor regulated by the product of the von Hippel-Lindau (VHL) tumor suppressor gene. The discovery of a second elongation factor implicated in oncogenesis provides further support for a close connection between the regulation of transcription elongation and cell growth.

A requirement for an ATP cofactor in synthesis of the first 8-10 bonds of promoter-specific transcripts by RNA polymerase II is well established. Whether ATP is required for synthesis of the first phosphodiester bond or at a slightly later stage in synthesis of nascent transcripts, however, remains controversial. Goodrich and Tjian (Goodrich, J.A., and Tjian, R. (1994) Cell 77, 145-156) recently proposed that synthesis of the first phosphodiester bond of promoter-specific transcripts by RNA polymerase II is independent of ATP and general transcription factors TFIIE and TFIIH. Here we investigate this model. Taken together, our findings indicate that ATP, TFIIE, and TFIIH can have a profound effect on the efficiency of transcription initiation. First, we observe that synthesis of the first phosphodiester bond of transcripts initiated at the adenovirus 2 major late promoter depends strongly on ATP, TFIIE, and TFIIH in a transcription system reconstituted with RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE, and TFIIF. Second, we demonstrate that, in this enzyme system, ATP-dependent activation of transcription initiation can occur immediately prior to synthesis of the first phosphodiester bond of nascent transcripts. Finally, we demonstrate that the activated initiation complex is unstable and decays rapidly to an inactive state in the presence of the inhibitor ATP-gammaS (adenosine 5'-O-(thio)triphosphate), even during reiterative synthesis of abortive transcripts.

A full-length cDNA encoding a human homolog of the approx. 110-kDa subunit (elongin A; El A) of the RNA polymerase II transcription factor, elongin, was isolated and sequenced. Comparison of the open reading frames of the human el A cDNA and the previously characterized rat El A cDNA [Aso et al., Science 269 (1995) 1439-1443] indicates that they are 84% conserved in nucleotide sequence and encode 84% identical proteins.