General Transcription Factors for RNA Polymerase II1

General Transcription Factors for RNA Polymerase II’ RONALDC. CONAWAY AND

JOAN WELIKY CONAWAY

Program in Moleailar and Cell Biology O k t a h m Medical Research Foundation Oklahoma City, Oklahoma 73104 I. TFIID and Formation of the First Stable Intermediate in Assembly of the Preinitiation Complex ...................................... 11. TFIIB and Selective Binding of RNA Polymerase I1 to the TFIID-Core Promoter Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. TFIIF and Assembly ofthe Active Preinitiation Complex . . . . . n! Roles of TFIIE and TFIIH in Formation and Activation of the sembled Preinitiation Complex .......................... V. Overview of RNA Polymerase I1 General Elongation Factors . . . . . . . . . VI. SII and Nascent Transcript Cleavage ............. VII. The Elongation Activity of TFIIF ................................ VIII. The Elongin (SIII) Complex and von Hippel-Lindau Disease . . . . . . . . X. ELL and Acute Myeloid Leukemia .............................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........

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Messenger RNA synthesis is a major site for the regulation of gene expression. Eukaryotic mRNA synthesis is an elaborate biochemical process catalyzed by multisubunit RNA polymerase I1 and controlled by the concerted action of a diverse collection of transcription factors that fall into at least three functional classes: (1)DNA binding transuctiwators,which activate expression of specific genes or gene families by increasing the rate of initiation ( I ) or, as shown recently, the efficiency of elongation (2-4) by RNA polymerase 11; (2)coactiwators, such as the SRB-containingMediator complex (5, 6), Creb binding protein (CBP) (7-9), and PC4 (10, I]), which are required for transcriptional activation and appear to function by promoting essential communication between DNA binding transactivators and the RNA polymerase I1 initiation complex; and (3)the general transcriptionfactors, which are characterized by their ability to function in intimate association with RNA polymerase I1 as components of the preinitiation, initiation, and elongation complexes, by their apparent roles in transcription of most, if not all, eukaryotic protein-coding genes, and by their strilung structural and functional conservation in eukaryotes from yeast to man (12-14). Abbreviations: RAP, RNA Polymerase associated Protein; TFG, h-anscriptionfactor g. Progress in Nucleic Acid Research and Molecular Biology, Vol. 56

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The past decade was a golden age for biochemical studies of the general transcription factors. During this time, many general transcription factors were identifed and purified to homogeneity, and working models for their roles in transcription were established. Among the most striking features of the general transcription factors are their sheer number and rich functional diversity. To date, more than 10 general transcription factors have been purified to homogeneity and classified according to the transcriptional stage they regulate. Biochemically defined general transcription factors include the general initiation factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, which function in the preinitiation and initiation stages of transcription to expedite selective binding of RNA polymerase I1 to promoters and to promote synthesis of the first few phosphodiester bonds of nascent transcripts (12), and the general elongation factors SII, P-TEFb, TFIIF, elongin (SIII), and ELL, which function by a variety of mechanisms to promote efficient elongation of transcripts by RNA polymerase I1 (13-15 ). Here we review the general transcription factors with particular emphasis on their roles in the preinitiation, initiation, and elongation stages of transcription by RNA polymerase 11.

1. TFllD and Formation of the First Stable Intermediate in Assembly of the Preinitiation Complex

Transcription initiation by RNA polymerase I1 is preceded by assembly of polymerase and the general initiation factors into an active preinitiation complex at the core region of a class I1promoter. Core promoters transcribed by RNA polymerase I1 are structurally diverse (16).Whereas a small subset of core promoters contains both a consensus TATA box (consensus sequence TATAAAA) located -30 base pairs upstream of the transcriptional start site and a strong initiator (Inr) element [consensus sequence (Y),-,CANT(Y),-,] surrounding the transcriptional start site, a large number of core promoters lack a consensus TATA box, a strong Inr element, or both. In some TATA-less promoters, the TATA box is replaced by an (A + T)-rich sequence that differs from the consensus TATA box, but is nevertheless a functional promoter element. In other TATA-less promoters, a functional promoter element at -30 appears to be lacking entirely. RNA polymerase I1 preinitiation complexes assemble at many TATA and TATA-less core promoters by a common pathway (17)(Fig. 1). Assembly of the preinitiation complex is nucleated by sequence-specific binding of TFIID to the core promoter to form the nucleoprotein recognition site for RNA polymerase I1 and the other general initiation factors on the DNA. RNA polymerase 11, assisted by TFIIB and TFIIF, can then bind selectively to the

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-

TATA+ Inr TATA+ In* (AdML)

TATA- In+

p

4v

rpoL +rUNSTABLE COMPLEX

STABLE COMMITTED COMPLEX

' I 1 'IB

llB

COMMIITED COMPLEX

+Lr

[IF

COMPLETE COMPLEX

STABLE COMMITTED COMPLEX IIE, IIH COMPLETE COMPLEX

FIG.1. Similar pathways for assembly of the RNA polymerase I1 preinitiation complex at many TATA and TATA-lesspromoters. TATA, Consensus TATA element; Inr, strong initiator element; A W L , adenovirus 2 major late promoter; POL 11, RNA polymerase 11; IIB, TFIIB; IIF, TFIIF; IIE, TFIIE; IIH, TFIIH.

TFIID-promoter complex. Assembly of the complete preinitiation complex is accomplished by binding of TFIIE and TFIIH. TFIID is the only general initiation factor that possesses measurable sequence-specific DNA binding activity and is therefore believed to be largely responsible for sequence-specific recognition of the core promoter by the preinitiation complex. TFIID is a multisubunit complex composed of the TATA box binding protein (TBP) and as many as 10 additional polypeptides, TBP-associated factors (TAFs) (18-22). At least two TFIID subunits possess sequence-specific DNA binding activities. TBP, which binds to a variety of consensus and nonconsensus TATA boxes (23,24),and TAF150, which binds the initiator (Inr) element (25-27). The combined DNA binding activities of these two TFIID subunits may account for the sequence-specific DNA binding properties of TFIID. TFIID is believed to duect assembly of the RNA polymerase I1 preinitiation complex at all class I1 core promoters, even though it binds most avidly to promoters that have consensus TATA boxes and strong Inr elements, such as the AdML promoter. At the AdML promoter, TFIID alone is sufficient for formation of the first stable intermediate on the pathway to assembly of the preinitiation complex. At this promoter, sequence-specific interactions between the TATA box and TBP and between the Inr and TAF150 or

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other subunits of TFIID apparently provide sufficient binding energy for formation of a stable, committed intermediate. At many other promoters, which lack consensus TATA boxes or strong Inr elements, TFIID binds sequence specifically but weakly to form unstable preinitiation intermediates. At these promoters, formation of the first stable preinitiation intermediate depends strongly on TFIID and both TFIIB and RNA polymerase 11;these intermediates are further stabilized by binding of TFIIF (17).

II. TFllB and Selective Binding of RNA Polymerase II to the T F I I M o r e Promoter Complex Selective binding of RNA polymerase I1 to the TFIID-core promoter complex requires TFIIB (28).TFIIB from higher eukaryotes is a protein of -35 kDa; it was initially purified to homogeneity from rat liver (29) and subsequently from human cells (30) and Drosophila melanogaster (31).TFIIB from yeast is a protein of -41 kDa (32,33). TFTIB functions as a ‘loridging factor” that promotes binding of RNA polymerase I1 to core promoters by direct and stable interactions with polymerase and TFIID or its TBP subunit (28).Distinct regions of TFIIB appear to mediate its interactions with RNA polymerase I1 and TBP. A proteaseresistant C-terminal core, which contains two 84-amino acid direct repeats, is sufficient for interaction with the TBP-promoter complex. A proteasesusceptibleN-terminal region, which contains a putative zinc-finger structure (CysX,CysX,,CysX,Cys), is required for selective binding of polymerase to the TBP-promoter complex (34-40). Substantial evidence indicates that TFIIB plays an important role in establishing the proper spacing between the TATA box and transcriptional start site. Some mutations in Saccharomyces cermisiae TFIIB lead to dramatic alterations in the position of the transcriptional start site (33).In addition, in an S . cereuisiae in vitro transcription system, replacement of TFIIB and RNA polymerase I1 from S. cerevisiae with their Schizosaccharomyces pombe counterparts is sufficient to change the transcriptional start site to the position characteristic of S . pombe (41).

111. TFllF and Assembly of the Active Preinitiation Complex

Although TFIIF is not essential for entry of RNA polymerase I1 into the preinitiation complex (17, 42, 43), it strongly stabilizes the binding of polymerase and TFIIB to the TFIID-core promoter complex (17, 44-46). The

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role of TFIIF in transcription initiation is most likely not limited to stabilizing binding of RNA polymerase I1 to the core promoter, however, because initiation depends strongly on TFIIF even under conditions where the factor makes only a relatively small contribution to the strength of polymerase binding (17).In addtion, although transcription initiation from nearly all promoters tested shows a very strong dependence on TFIIF, RNA polymerase I1 is capable of initiating transcription in vitro from a human IgH promoter in the absence of TFIIF,in a reaction requiring a negatively supercoiled DNA template and depending only on TFIID and TFIIB (42). In higher eukaryotes,TFIIF is a heterodimer composed of -30- (RAP30) and -70-kDa (RAP74) subunits, which were first identified among a small group of proteins capable of binding to immobilized RNA polymerase I1 ( 4 7 ) . TFIIF was initially identified as one of the general initiation factors (29, 48) and purified to homogeneity from rat liver (49),Drosophih melanogaster Kc cells (50),and subsequently human cells (51, 52). In S. cerevisiae, TFIIF is a heterotrimer composed of -1O5- F G l ) , -54- (TFG2),and -30-kDa (TGF3) subunits (53, 54). TFIIF shares structural and functional properties with bacterial u factors (12, 55).First, like bacterial u factors, TFIIF binds stably to its cognate RNA polymerase. As mentioned above, human RAP30 and RAP74 were initially identified among a small group of proteins that bind to immobilized RNA polymerase I1 (47, 56, 57); in addition, human (52, 58, 59), D. melunogaster (50), rat (60),and S. cerevisiae (53)TFIIF have each been shown to associate stably with RNA polymerase I1 in solution. On isolating a cDNA encoding human RAP30, Greenblatt and co-workers identified a RAP30 region sharing sequence similarity with the RNA polymerase bindmg domains of Escherichia coli u70 and Bacillirs subtilis u43 (61). Ln subsequent experiments, it was observed that a serine residue located at the C terminus of this RAP30 region is protected from phosphorylation by CAMP-dependent protein kinase when TFIIF and RNA polymerase I1 are mixed, suggesting that TFIIF binds to RNA polymerase TI at least in part through this region (62). In the same study, it was also observed that TFIIF is capable of binding to E . coli RNA polymerase and can be dsplaced by u70. Taken together, these results suggest that RAP30 and u70 have related RNA polymerase-binding domains. Second, a variety of evidence suggests that, llke bacterial u factors, TFIIF promotes stable binding of its cognate polymerase to promoters through a direct interaction with DNA. Results from resbiction-site protection (44)and phenanthrolene-copper-footprinting (45) experiments indicate that TFIIF promotes formation of stable protein-DNA contacts between the TATA box and transcriptional start site, either by promoting binding of RNA polymerase 11, TFIIB, TFIID, or some combination of these proteins to this

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core promoter region or by binding directly to DNA. More recent results suggest that the RAP30 and RAP74 subunits of TFIIF contain C-terminal regions capable of interacting directly with DNA (63, 64). The RAP30 C-terminus exhibits statistically sigdicant similarity to the C terminus of B. sub& ~ ~ ( 6 This 5 ) . region of uKincludes the highly conserved u homology region 4. Within this region is a helix-turn-helix DNA binding motif that is a cryptic DNA binding domain that interacts with the -35 element of bacterial promoters (66, 67). Using the approach of Gross and co-workers (66, 67), we obtained evidence that the RAP30 C terminus is also a cryptic DNA binding domain (63).Consistent with the hypothesis that the DNA binding activity of RAP30 is relevant to its function in transcription initiation, (1) RAP30 can be cross-linked in the preinitiation complex to core promoter sequences between the TATA box and transcriptional start site (68), (2)the RAP30 C terminus, including the cryptic DNA binding domain, is critical for TFIIF activity in transcription initiation (63, 65, 69, 70), and (3)there is a strong correlation between the effects of RAP30 C-terminal deletion mutations on RAP30 DNA binding and transcription activities (63).Despite the ability of both of its subunits to interact with DNA, TFIIF most likely does not make a major contribution to sequence-specific recognition of promoter sequences by RNA polymerase 11, because substantial evidence argues that TFIID is the primary determinant of sequence specificity in assembly of the preinitiation complex. Third, TFIIF has been shown to prevent nonselective binding of RNA polymerase I1 to nonpromoter sites on DNA, much as E. coli u70 prevents nonselective binding of E. coli RNA polymerase to nonpromoter sites on DNA (71, 72). Pwified RNA polymerase I1 binds DNA nonselectively but quite stably to form a binary complex that dissociateswith a half-life of more than 1 hr. Both formation and stability of these binary complexes are substantially reduced in the presence of TFIIF. Thus, another contribution of TFIIF to selective binding of RNA polymerase I1 to core promoters may be to suppress formation of nonproductive binary complexes of polymerase and DNA.

IV. Roles of TFllE and TFllH in Formation and Activation of the Fully Assembled Preinitiation Complex

Entry of the final two general initiation factors, TFIIE and TFIIH, into the preinitiation complex results in formation of stable protein-DNA contacts near the transcriptional start site (44, 73). Whether TFIIE and TFIIH bind DNA directly or whether they induce RNA polymerase I1 or the other

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general initiation factors to bind h s region of the core promoter is unclear. Substantial evidence argues, however, that the major contribution of TFIIE and TFIIH to formation of the productive initiation complex is to promote AT€-dependent formation of an open promoter complex by unwindmg a short stretch of promoter DNA near the transcriptional start site prior to initiation (74, 75). TFIIE from higher eukaryotes is a heterodimer composed of -34- and -58-kDa polypeptides and has been purified to homogeneity independently from human cells (76) and rat liver (77). TFIIE from s. cerevisiae is a heterodimer of -43- and -66-kDa subunits (78, 79). TFIIH from higher eukaryotes and S. cerevisiae is a nine-subunit protein that has now been implicated in cellular processes as diverse as transcriptional regulation, DNA repair, and cell cycle control. TFIIH was initially identified and purified from rat liver (73,80) and subsequently from S. cerevisiue (8482) and human cells (83, 84). TFIIH is the only general transcription factor known to possess associated enzymatic activities. Purified rat TFIIH possesses a weak DNA-dependent ATPase activity specific for adenine nucleoside triphosphates (80).Although this ATPase was stimulated by a variety of single- and double-stranded DNAs, it was most strongly stimulated by DNA fragments containing the TATA regions of the Adh4L and mouse interleukin-3 core promoters, which are both strong promoters in uitro. Saccharomyces cermisiae (85) and human TFIIH (86,87) were subsequently shown to possess closely associated DNA-dependent ATPase activity. Human (88),rat (89),and S. cerevisiae (90) TFIIH also possess An-dependent DNA helicase activities. The largest subunit of human TFIIH is a DNA helicase encoded by the product of the nucleotide excision repair (NER) gene XPB/ERCC3 (88), which is mutated in individuals suffering from the human genetic disorders xeroderma pigmentosum (group B) and Cockayne’s syndrome. In other studies, TFIIH was shown to function &rectly in NER in cells (91, 92) and to be composed of additional subunits encoded by known NER genes, including TFBl (93-95), Ssll (95, 96), and XPD/ERCC2 (yeast B a d ) (90, 97, 98), which encodes a second TFIIH-associated DNA helicase. Substantial evidence suggests that the TFIIH DNA helicase mediates AT€-dependent formation of an open promoter complex prior to synthesis of the first phosphodiester bond of nascent transcripts. It is well established that, under most conditions, promoter-specific transcription requires a hydrolyzable ATP cofactor. It was frst observed that the nonhydrolyzable ATP analog, AMP-PNP, does not replace ATP in synthesis of accurately initiated transcripts from the AdML promoter, even though AMP-PNP is a substrate for elongation by RNA polymerase I1 (99). Subsequently it was demonstrat-

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ed that ATP is required at some stage during synthesis of the first eight phosphodiester bonds of transcripts initiated at the AdML promoter (100).Furthermore, ATP is required for synthesis of dinucleotide-primed, abortive trinucleotide transcripts initiated at the AdML promoter, providing evidence that ATP is essential for synthesis of the first phosphodiester bond of nascent transcripts (101).Using a partially fractionated rat liver transcription system, we identified ATPyS as a potent inhibitor of the ATP-requiring step in transcription initiation and used this inhibitor to demonstrate that ATP activates the preinitiation complex in a reversible step preceding synthesis of the first phosphodiester bond of nascent transcripts (102).The requirement for ATP in activation of the preinitiation complex, although somewhat controversial (103),has recently been confirmed in studies carried out with crude extracts (104)and fully reconstituted, purified transcription systems (105)These studies have also provided strong evidence that TFIIH is inhspensible for ATPdependent transcription initiation by RNA polymerase 11. Evidence consistent with the idea that ATP-dependent activation of the preinitiation complex involves formation of an open promoter complex by the TFIIH DNA helicase has come from several laboratories. First, using KMnO, as a probe for DNA melting, Gralla and co-workers (74)obtained evidence that ATP hydrolysis provides energy to drive unwinding of a short stretch of DNA surrounding the transcriptional start site prior to transcription initiation in crude HeLa cell extracts (74).Like ATP-dependent activation of the preinitiation complex, formation of the open promoter complex requires ATP or dATP (100,102);neither AMP-PNP, CTP, UTP, nor GTP support DNA melting. Second, Timmers and co-workers confirmed and extended these findmgs by demonstrating that TFIIH is essential for ATPdependent open complex formation in a fully reconstituted, purified transcription system (75).Finally, additional evidence that the TFIIH DNA helicase plays a role in Am-dependent activation of the preinitiation complex has come from biochemical studies indicating that an ATP cofactor is not required for transcription initiation under a limited set of conditions where TFIIH is dispensible for initiation. These conditions include initiation by RNA polymerase I1 from promoters on negatively supercoiled DNA templates (42, 43, 106) or “preopened promoters containing a short stretch of mismatched base pairs surrounding the transcriptional start site (105, 107, 108). In addition to DNA-dependent ATPase and DNA helicase activities, S. cereuisiae TFIIH (85)and, later, rat (109)and human (110)TFIIH were found to possess a protein kinase activity capable of phosphorylating the heptapeptide repeats in the C-terminal domain (CTD) of the largest subunit of RNA polymerase 11. The mammalian TFIIH kinase is composed of cdk 7 (M015) (111-113), cyclin H (111-113), and MAT1 (84).The S. cerevisiae

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TFIIH kinase is a three-subunit complex composed of the cdk-like kinase KIN28, the cyclin-like protein CCLI, and TFB3, a homolog of MAT1 (82, 114, 115; R. Kornberg, personal communication). Based on findings indicating (1) that the CTDs of RNA polymerase I1 molecules actively engaged in transcription are highly phosphorylated (116-118) and (2) that polymerases containing hypophosphorylated CTDs preferentially enter the preinitiation complex (119-121),where they are subsequently phosphorylated (119),it was proposed that CTD phosphorylation could play a role in transcription initiation or in the transition of polymerase from initiation to elongation. Several lines of evidence argue, however, that CTD phosphorylation is not an essential step in promoter-specific transcription by RNA polymerase 11. First, the TFIIH kinase utilizes both ATP and GTP as phosphate donors, whereas transcription initiation by RNA polymerase I1 exhibits a strict requirement for adenine nucleoside triphosphates (85, 89, 109).Second, the isoquinoline sulfonamide derivatives H-7 and H-8, which potently inhibit the TFIIH kinase, have no effect on promoter-specific transcription by RNA polymerase I1 in vitro in highly purified basal transcription systems (122, 123).Finally, TFIIH containing a mutant cdk7 subunit that lacks CTD kmase activity is fully functional in both basal and activated transcription in vitro (124).

V. Overview of RNA Polymerase II General Elongation Factors

A requirement for a class of elongation factors that promotes eukaryotic messenger RNA synthesis was predicted by early biochemical studies of the catalybc properties of RNA polymerase 11. These studies revealed that purified RNA polymerase I1 lacks the capacity to catalyze RNA chain elongation in vitro at rates sufficient to account for the observed rates of messenger RNA synthesis in vivo; whereas eukaryotic messenger RNA synthesis is estimated to proceed in vivo at rates of 1200-2000 nucleotides per minute (125-127), RNA polymerase I1 synthesizes RNA in vitro at 100-300 nucleotides per minute under optimal conditions (128).Furthermore, RNA chain elongation by purified RNA polymerase I1 is an inherently discontinuous process interrupted by frequent pausing and, in some cases, premature arrest at a variety of sequences found within eukaryotic protein-coding genes (13, 129). Consequently, elongation factors that increase the overall rate of messenger RNA synthesis by suppressing transient pausing or preventing premature arrest by transcribing RNA polymerase I1 might be expected to play important roles in eukaryotic gene expression by expediting passage of polymerase through the long stretches of chromosomal DNA comprising most eukaryoticproteincoding genes. Indeed, eukaryotes have evolved such a family of elongation

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factors. Biochemically defined members of this family include P-TEF'b, SII, TFIIF, elongm (SIII), and ELL. P-TEF'b functions in partially fractionated transcription systems to convert early, promoter-specific, termination-prone transcription complexes into productive elongation complexes (15,130).SII, TFIIF, elongin (SIII),and ELL all regulate the activity of the purified RNA polymerase I1 ternary elongation complex. These four factors are the subject of the following sections.

VI. SII and Nascent Transcript Cleavage SII is an -38-kDa protein originally identified and purified to homogeneity from Ehrlich ascites tumor cells (131).SII does not appear to increase the overall catalytic rate of nucleotide addition to growing RNA chains by RNA polymerase 11. Instead, SII expedites passage of polymerase through transcriptional impediments, including various nucleoprotein complexes as well as a collection of related DNA sequences that act as intrinsic arrest sites (132).On encountering transcriptional impediments, a fraction of transcribing polymerase arrests, but can be reactivated by SII. SII-sensitive intrinsic arrest sites are found in many genes, including the human histone H3.3, adenovirus 2 major late, and adenosine deaminase genes (14, 129). Typical intrinsic arrest sites include two or more closely spaced stretches of T residues in the nontemplate strand. Why particular DNA sequences function as arrest sites is not clear. However, the DNA at some arrest sites can adopt a bent conformation, and it has been proposed that these DNA bends are directly responsible for inducing arrest (133). Although it is not known whether SII-sensitive arrest sites play a role in regulating the expression of particular eukaryotic genes, SII-sensitive arrest sites are distributed throughout many eukaryotic protein-coding genes. Yeast cells lacking the SII gene (PPR2) are sensitive to the uracil analog, 6-azauracil, which lowers intracellular UTP and GTP pools and thus might be expected to affect transcription elongation. Notably, the phenotypic effects of some yeast RNA polymerase I1 large subunit mutations that render messenger RNA synthesis sensitive to 6-azauracil are overcome by increasing the dosage of the wild-type PPR2 gene (134). Efforts to understand how SII promotes passage of RNA polymerase I1 through intrinsic arrest sites led to the discovery that SII-dependent readthrough is accompanied by reiterative endonucleolytic cleavage and reextension of nascent transcripts in the ternary elongation complex (135-137 ). Many observations argue that SII-activatedtranscript cleavage is an essential step in SII-dependent transcription. The SII-dependent readthrough of all transcriptional impediments examined is accompanied by nascent transcript

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cleavage (138-141). Also, the appearance of SII-dependent cleavage products precedes the appearance of readthrough products (138, 139, 144, and all SII-dependent transcription through intrinsic arrest sites in the human histone H3.3 gene or past DNA-bound Lac repressor is preceded by cleavage (138, 141).Finally, SII deletion or point mutants that fail to activate nascent transcript cleavage also fail to promote readthrough (138, 142-144). It is noteworthy that, although this evidence argues that cleavage is necessary for readthrough, it may not be sufficient in light of the recent identification of an SII mutant that promotes transcript cleavage but not readthrough (143,144 ). Attempts to iden* the catalyhc site responsible for SII-activated transcript cleavage show that RNA polymerase I1 participates directly in the cleavage reaction. The SII-activated transcript cleavage requires a physical interaction between polymerase and the cleaved transcript and is inhibited by low concentrations of the drug, a-amanitin, which inhibits elongation by RNA polymerase I1 (135-137). Whereas purified SII has no detectable transcript cleavage activity in the absence of polymerase, highly purified RNA polymerase I1 elongation complexes exhibit low but detectable levels of transcript cleavage activity (135-137). Finally, evidence that the RNA polymerase I1 catalyhc site is responsible for transcript cleavage has come from the observation that SII-activated transcript cleavage and pyrophosphorolysis result in cleavage of the same internal phosphodiester bonds of nascent transcripts (145).

VII. The Elongation Activity of TFllF TFIIF is unique among RNA polymerase I1 general transcription factors by virtue of its abihty to function in both the initiation and the elongation stages of transcription. Greenleaf and co-workers first demonstrated that TFIIF is capable of stimulating elongation by RNA polymerase (50).Their work and subsequent studies from several laboratories demonstrated that TFIIF stimulates elongation by a mechanism that involves suppression of the frequency or duration of transient pausing by RNA polymerase I1 at many sites on DNA templates (58, 128, 146-149). Unlike SII, TFIIF does not promote nascent transcript cleavage by the RNA polymerase I1 elongation complex, and it is not capable of releasing polymerase from arrest at intrinsic arrest sites. Nevertheless, interaction of TFIIF with transcribing RNA polymerase I1 decreases the likelihood that polymerase will suffer arrest at these sites (150 and references therein). Thus, TFIIF and SII appear to regulate the activity of transcribing RNA polymerase I1 in different yet complementary ways: whereas TFIIF suppresses transient pausing by polymerase and protects the elongation complex from becoming arrested, SII reactivates the elongation complex once it has arrested.

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Although the mechanism by which TFIIF stimulates elongation by RNA polymerase I1 has not been established unequivocally, evidence suggests that TFIIF elongation activity is executed through a direct but transient interaction with transcribing polymerase. TFIIF is capable of stimulating the elongation rate of purified RNA polymerase I1 on oligo(dC)-tailedDNA templates in the absence of other transcription factors (50).Although TFIIF does not remain stably bound to transcribing polymerase during elongation in vitro (50),TFIIF is capable of binding stably to RNA polymerase 11 in solution (47, 50, 53, 58, 60, 151). Phosphorylation of RAP74 both stabilizes binding of TFIIF to RNA polymerase I1 and increases TFIIF elongation activity (152). Analysis of a series of mutant TFIIFs containing RAP30 deletion mutations revealed that TFIIF elongation activity is strongly dependent on the RAP30 region proposed (62) to bind RNA polymerase 11, but not on RAP30 C-termind sequences required for initiation (69).

VIII. The Elongin (SIII)Complex and von Hippel-Lindau Disease Like TFIIF, elongin (SIII)increases the overall rate of elongation by RNA polymerase I1 by decreasing the frequency or duration of transient pausing by polymerase at many sites on DNA templates (148).Elongin (SIII) was initially identified and purified to homogeneity from rat liver nuclei (153)as a heterotrimeric complex of A, B, and C subunits with apparent molecular masses of -110, -18, and -15 kDa. Subsequent biochemical studies have shown (1) that elongin A is the transcriptionally active subunit of elongin (SIII) (154) and stimulates elongation by a novel, inducible transcriptional activation domain that exhibits an overall architecture similar to the ligandinducible activation domains of members of the nuclear receptor superfamily (T. Aso, J. W. Conaway and R.C. Conaway, unpublished results) and (2) that elongin B and C are positive regulatory subunits (155, 156). Elongin B and C regulate elongm A by different mechanisms (154) (Fig. 2). Elongin C functions as a direct activator of elongin A, because it is capable of interacting directly with elongin A in the absence of elongin B to form an AC complex with increased specific activity relative to that of elongin A. In contrast, elongin B, a member of the ubiquitin homology (UbH) gene family, neither activates elongin A nor is capable of interacting with elongin A in the absence of elongin C; instead, elongin B binds directly to elongin C and promotes assembly and stability of the elongin (SIII) complex. The elongin BC complex has recently been shown to be a potential target for regulation by the product of the von Hippel-Lindau (VHL)tumor suppressor gene (157, 158). The VHL gene is mutated in families with VHL

.J

low specific activity

-

r n R N A .

high specific activity (unstable)

FAST

rnRNAJ

FAsEl?

high specific activity (stable) FIG.2. Assembly and activities of elongin (SIII) and elongin subassemblies. Pol 11, RNA polymerase 11; A, elongin A; B, elongin B; C, elongin C; mRNA, messenger RNA.

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disease, a rare genetic disorder (incidence -1 in 36,000) that predisposes affected individuals to a variety of cancers, including retinal hemangiomas, central nervous system hemangioblastomas, multiple endocrine neoplasias, and clear-cell renal carcinoma (159-161). Of more general clinical importance, somatic mutations of the VHL gene are found in most sporadic clearcell renal carcinomas (161-164). The VHL protein binds tightly and specifically to the elongin BC complex both in vitro and in cells (157,158).A subset of naturally occurring VHL mutants from VHL tumors and clear-cell renal carcinomas exhibits substantially reduced binding to the elongin BC complex, arguing that the VHL-elongin BC interaction is likely to be important for the tumor suppressor activity of the VHL protein. Binding of the VHL protein and elongin A to the elongin BC complex in vitro is mutually exclusive, and binding of the VHL protein to the elongin BC complex inhibits its ability to activate elongin A transcriptional activity (157).Taken together, these results suggest that the normal tumor suppressor function of the VHL protein could involve regulation of elongin (SIII) transcriptional activity.

IX. ELL and Acute Myeloid Leukemia We isolated a novel -80-kDa elongation factor from rat liver nuclei and found that the purified protein exhibits sigdicant sequence similarity to the product of the human ELL gene (165).Subsequent experiments led to the discovery that the human ELL protein is also an RNA polymerase I1 elongation factor (165).Like TFIIF and elongin (SIII), ELL stimulates the rate of elongation by RNA polymerase I1 by suppressing transient pausing by polymerase at many sites on DNA templates (165). The human ELL (eleven-nineteen lysine-rich leukemia) gene on chromosome 19~13.1was initially isolated as a gene that undergoes frequent translocations with the MLL (mixed-lineageleukemia) gene on chromosome l l q 2 3 in acute myeloidleukemia (166,167).ELL encodes a 621-amino acid protein that is highly conserved and ubiquitously expressed in higher eukaryotes, but that contains no obvious structural motifs characteristic of transcription factors (166,167).The N-terminalhalfof the 3968-amino acid MLL ) methyltransgene product contains (A-7")-hook DNA binding ( 1 6 7 ~and ferase-like domains, whereas the C-terminal half of the MLL-encoded protein contains several regions that resemble the Drosophilu trithorux gene product, including multiple contiguous zinc-finger motifs and a highly conserved 215-amino acid region at the C terminus of the protein (168, 169). Like its potential Drosophita counterpart, the MLL gene product regulates expression of homeotic genes (170).

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ELL

-

(19~13.1)

A-T -. . hooks

MLL

-

TRITHORAX like

MT

Zn-finaers

I )

MLL FIG.3. The chimeric MLL-ELL gene in acute myeloid leukemia. MT, Methylbansferaselike domain; Zn, zinc.

Chromosomal translocations involving MLL and ELL result in formation of a chimeric gene encoding all but the first 45 amino acids of ELL fused to the N-terminal-1400 amino acids of the MLL protein, including its (A-T)hook and methyltransferase domains, but lacking the C-terminal trithoraxlike regions (Fig. 3). Whether leukemogenesis results from expression of the MLL-ELL chimera or from loss of one allele of ELL, MLL, or both remains unclear. Nevertheless, the identification of two RNA polymerase I1 elongation factors, elongin (SIII) and ELL, which are implicated in oncogenesis, supports the idea that there may be a close connection between the regulation of transcription elongation and cell growth. A c KNOWLED GM E NTS Work in our laboratory is supported by National Institutes of Health Grant GM41628 and by funds provided to the Oklahoma Medical Research Foundation by the H. A. and Mary K. Chapman Charitable Trust.

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