Biochem. Soc. Symp. 73, 203–216
`(Printed in Great Britain)
` 2006 The Biochemical Society
`
`19
`
`The RNA polymerase I
`transcription machinery
`Jackie Russell and Joost C.B.M. Zomerdijk1
`
`Division of Gene Regulation and Expression, School of Life Sciences, University of
`Dundee, Wellcome Trust Biocentre, Dundee DD1 5EH, U.K.
`
`Abstract
`
`The rRNAs constitute the catalytic and structural components of the
`ribosome, the protein synthesis machinery of cells. The level of rRNA synthesis,
`mediated by Pol I (RNA polymerase I), therefore has a major impact on the life
`and destiny of a cell. In order to elucidate how cells achieve the stringent control
`of Pol I transcription, matching the supply of rRNA to demand under different
`cellular growth conditions, it is essential to understand the components and
`mechanics of the Pol I transcription machinery. In this review, we discuss: (i) the
`molecular composition and functions of the Pol I enzyme complex and the two
`main Pol I transcription factors, SL1 (selectivity factor 1) and UBF (upstream
`binding factor); (ii) the interplay between these factors during pre‑initiation
`complex formation at the rDNA promoter in mammalian cells; and (iii) the
`cellular control of the Pol I transcription machinery.
`
`Introduction
`
`The Pol I (RNA polymerase I) transcription machinery comprises three
`main components dedicated to the transcription of the rRNA genes: the Pol
`I enzyme, the TBP (TATA‑binding protein)–TAF (TBP‑associated factor)
`complex SL1 (selectivity factor 1)/TIF‑IB (transcription initiation factor‑IB)
`and the transactivator protein UBF (upstream binding factor) (reviewed
`in [1–6]). Confined to the nucleolar subcompartment of the nucleus, this
`machinery directs transcription from the rDNA promoter at some 200–400
`rRNA (ribosomal RNA) genes in the nucleolar organizer regions. The Pol I
`transcription machinery churns out 50% of the nascent RNA in the cell in the
`form of pre‑rRNA (47 S precursor rRNA), which is processed into three mature
`rRNAs (18 S, 5.8 S and 28 S), and these, together with the 5 S rRNA synthesized
`
`1To whom correspondence should be addressed (email j.zomerdijk@dundee.ac.uk).
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`J. Russell and J.C.B.M. Zomerdijk
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`by Pol III, comprise the enzymatic and structural scaffold of the ribosome [7].
`Transcription of the rRNA genes drives the biogenesis of the several millions
`of ribosomes needed in an actively growing cell, thereby dictating the overall
`level of cellular protein synthesis. Thus transcription by Pol I is essential for,
`and inextricably linked to, cell growth and, hence, to normal cell division
`(reviewed in [6,8]). Predictably, therefore, Pol I transcription is at the receiving
`end of many of the signalling pathways implicated in the control of cell growth,
`nutrient sensing and proliferation [1,6,9–17].
`
`The Pol I enzyme complexes
`
`DNA‑dependent RNA polymerases are highly conserved multisubunit
`enzyme complexes in eukaryotes. The composition of the Pol I enzyme complex
`has been derived primarily from yeast genetic and biochemical studies [18]. Our
`understanding of the Pol I enzyme complex in mammalian cells has advanced
`rapidly in recent years following identification of mammalian subunits both by
`comparison with yeast Pol I and by MS analysis of purified Pol I complexes.
`Pol I enzymes from yeast to humans show conservation with Pols II and III
`and with the α2ββ′ω subunit composition of prokaryotic core RNA polymerase
`(reviewed in [19]) (Table 1). The largest and second largest subunits of Pol I share
`substantial similarity with those of Pol II and Pol III and with prokaryotic β′ and
`β subunits respectively, possessing most of the enzymatic functions (substrate
`binding, active centre, template movement). The heterodimer AC40–AC19 of Pol
`I is shared with Pol III, and is homologous with the RPB3–RPB11 heterodimer
`of Pol II and functionally analogous to the prokaryotic α2 homodimer. The
`RPB6 subunit of Pol I, shared with Pols II and III, is a structural and functional
`homologue of the bacterial ω subunit. There are four additional subunits that
`are required to synthesize RNA from a non‑specific DNA template, shared
`between Pols I, II and III (RPB5, RPB8, RPB10 and RPB12). Subunit A12.2 is
`unique to Pol I (Table 1). In addition to these 10 subunits, collectively referred
`to as the ‘core’ complex, Pol I heterodimer A14–A43 shares genetic, biochemical
`and structural characteristics with heterodimers RPB4–RPB7 and C17–C25 of
`Pols II and III respectively, and there is sequence similarity between subunits
`RPB7, A43 and C25 [20–22]. There are two additional subunits, A49 and A34.5,
`that are specific to Pol I [18,23,24] and hence likely targets of the Pol I cognate
`transcription factors.
`The ∼600 kDa yeast Pol I complex is therefore composed of 14 subunits,
`and homologues of all of the yeast Pol I subunits are found in Metazoa, with the
`exception of the Pol I‑specific subunit A14, for which a mammalian homologue
`has yet to be identified. Mammalian homologues of the Pol I‑specific subunit
`A34.5 were discovered only recently: the mouse homologue is PAF49 (polymerase
`I‑associated factor of 49 kDa) [25]. Human Pol I proteomics led us to identify
`a 72 kDa subunit of Pol I with homology to the yeast Pol I subunit A34.5 (K.I.
`Panov, T.B. Panova and J.C.B.M. Zomerdijk, unpublished work), previously
`named ASE‑1 [antisense for ERCC‑1 (excision repair cross‑complementing‑1)]
`[26] or CAST (CD3ε‑associated signal transducer) [27] and, curiously, also
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`RNA polymerase I transcription machinery
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`UBF and SL1 [25,26]†
`
`hRRN3 [41]
`UBF [29]
`
`†K.I. Panov, T.B. Panova and J.C.B.M. Zomerdijk, unpublished work.
`*Structural and/or functional homologues with bacterial RNA polymerase (β′, β, α2, ω) and yeast Pol II (B).
`
`–
`–
`RPB9
`RPB4
`–
`α; RPB11(B12.5)
`ω
`–
`None
`α; RPB3 (B45)
`RPB7
`None
`β; RPB2 (B140)
`β′; RPB1 (B220)
`
`RPB12
`RPB10
`hRPA12.2
`?
`RPB8
`hRPA19
`hRPB6
`hRPB5
`CAST (PAF49)
`hRPA40
`hRPA43
`PAF53
`hRPA127
`hRPA190 (A194)
`
`I, II, III
`I, II, III
`I
`I
`I, II, III
`Ι, ΙΙΙ
`Ι, ΙΙ, ΙΙΙ
`I, II, III
`I
`Ι, ΙΙΙ
`I
`I
`Ι
`I
`
`RPB12 (ABC10α)
`RPB10 (ABC10β)
`RPA12c
`RPA14v
`RPB8 (ABC14.5)
`RPA19 (AC19)
`RPB6 (ABC23)
`RPB5 (ABC27)
`RPA34.5v
`RPA40 (AC40)
`RPA43
`RPA49c
`RPA135
`RPA190
`
`Interactions in mammalian PIC
`
`Ηomologues*
`
`Human Pol I subunits
`
`Unique or shared
`
`S. cerevisiae Pol I subunits
`
`primarily following yeast nomenclature
`[18]). ‘Unique or shared’ refers to subunits unique in Pol I, or shared with Pol III, or shared with Pols II and III. Human Pol I subunits are named
`cerevisiae (as determined by null mutant analysis); those subunits not essential are marked viable (v) or conditional mutants (c) (adapted from
`Table 1 Comparison of Pol I subunit composition in yeast and humans. Most Pol I subunits are essential for viability in Saccharomyces
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`implicated in T‑cell activation (Table 1). The A34.5 and A49 subunits are absent
`from yeast Pol I isolated under high‑salt conditions (referred to as A*), which
`displays a reduced specific activity in RNA synthesis from calf thymus DNA
`and a higher sensitivity to α‑amanitin, suggesting a role for these subunits in
`elongation or processivity [28]. PAF49 and PAF53, the mammalian homologues
`of yeast A34.5 and A49, also dissociate from Pol I under certain conditions,
`most notably upon serum starvation of cells [25,29].
`The structures of yeast Pol I and Pol II complexes have been studied
`extensively by electron microscopy and crystallography [30–33]. The
`cryo‑electron‑microscopic structure of yeast Pol I displays an overall similarity
`with the crystal structure of yeast Pol II (lacking RPB4 and RPB7), except
`at the positions of the Pol I‑specific subunits A34.5, A49, A43 and A14 [33].
`The A34.5 subunit of Pol I is positioned adjacent to the second largest subunit
`A135, at the entry to the cleft which binds the DNA template [31], where it
`is proposed to stabilize the interaction of the DNA template with the ‘core’
`enzyme. The A49 subunit is located on the head region of the clamp, formed
`by the largest subunit of Pol I [33,34] and constituting one side of the cleft. A49
`could potentially affect the conformation of the clamp [33], moving it inwards
`to hold downstream DNA more firmly, or interact directly with the DNA,
`thereby increasing the processivity of the enzyme [30,31]. Another structure
`specific to Pol I is the stalk, formed by subunits A43 and A14 and postulated to
`function as an interface for the assembly of initiation factors through its binding
`to the essential Pol I‑associated factor Rrn3p. Interestingly, the stalk protrudes
`from the Pol I structure at the same site as the C‑terminal domain of the largest
`subunit in Pol II, which has a role in initiation via its interaction with the basal
`transcription factors [33]. There are different conformational subpopulations
`of the yeast Pol I enzyme [34], consistent with conformational flexibility in
`other RNA polymerases, where the clamp maintains an open conformation in
`the initiation‑competent enzyme, being converted into a closed conformation
`during elongation [19,34].
`We have shown previously that the Pol I enzyme in human cells is found in
`at least two functionally distinct complexes (Pol Iα and Pol Iβ). These complexes
`are considerably larger than the yeast Pol I complex, at >1 MDa, and contain
`Pol I‑associated factors in addition to the core subunits [35] (K.I. Panov and
`J.C.B.M. Zomerdijk, unpublished work). Pol Iβ, comprising approx. 10% of
`the total Pol I extractable from the cell nucleus, is the initiation‑competent form
`of the enzyme, able to direct accurate initiation of transcription from the rDNA
`promoter. By contrast, Pol Iα, representing the bulk of the Pol I, is unable to
`direct rDNA promoter‑specific transcription, although it does possess Pol
`activity, catalysing the random synthesis of RNA. Given these properties and its
`relative abundance compared with Pol Iβ, the Pol Iα complex might represent
`the elongating Pol I complex, or polymerase with the capacity to be converted
`readily into initiation‑competent Pol Iβ, or spent polymerase complexes
`following transcription. The Pol Iβ enzyme complex is distinguishable by
`its associated factors, likely to be involved specifically in initiation. hRRN3
`(human RRN3; equivalent to mouse TIF‑IA/yeast Rrn3p) [35–39] is one such
`Pol Iβ‑specific factor. RRN3 is tethered to the A43 subunit of Pol I [40,41] and
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`links Pol I to the promoter‑bound essential transcription factor SL1. The role
`of RRN3 in bridging the polymerase and promoter recognition components of
`the Pol I transcription machinery (SL1 or core factor) is conserved from yeast to
`humans, and is required for PIC (pre‑initiation complex) assembly [35,37,38].
`The association of hRRN3/TIF‑IA with Pol I, to generate Pol Iβ [35,36],
`is an important point of control in pre‑rRNA synthesis. The hRRN3/TIF‑IA–
`Pol I interaction is impaired and cellular rRNA synthesis is down‑regulated
`in stationary‑phase, nutrient‑limited or cycloheximide‑treated mammalian
`cells [41,42]. There is evidence that rDNA transcription is regulated by the
`phosphorylation state of RRN3, which determines the steady‑state concentration
`of the RRN3–Pol I complex Pol Iβ [41]. Perhaps as a result of a reversal of
`phosphorylation events, Rrn3 is inactivated during the process of rDNA
`transcription [43]. The phosphorylation of TIF‑IA by JNK2 (c‑Jun N‑terminal
`kinase 2), which can occur upon cellular stress, leads to inactivation of TIF‑IA
`and down‑regulation of rRNA synthesis [44]. RRN3 activity and the RRN3–
`Pol I interaction are also regulated by the ERK (extracellular‑signal‑regulated
`kinase)/MAPK (mitogen‑activated protein kinase) and mTOR (mammalian
`target of rapamycin) signalling pathways, which are involved in growth factor
`signalling and nutrient sensing. Increased RRN3/TIF‑IA activity, rRNA gene
`transcription and cell proliferation correlate with phosphorylation of RRN3/
`TIF‑IA by ERK and RSK (p90 ribosomal S6 kinase) in growth factor‑stimulated
`mouse NIH 3T3 cells [10]. In separate studies, RRN3 activity has been reported
`to be down‑regulated [12] or unaffected [9] following inactivation of mTOR
`by rapamycin treatment of NIH 3T3 cells. Perhaps, therefore, the specific
`physiology of a cell determines the involvement of mTOR in the regulation of
`RRN3 activity. The mTOR pathway has been implicated in PIC formation, as
`the down‑regulation of RRN3 by rapamycin was correlated with changes in the
`phosphorylation status of RRN3, an inability of RRN3 to interact with Pol I and
`SL1, and translocation of TIF‑IA/RRN3 to the cytoplasm [12]. Furthermore,
`inactivation of yeast TOR by rapamycin is accompanied by release of Pol I
`from the nucleolus and inhibition of rDNA transcription [45], and the TOR
`signalling pathway has been shown to regulate the Rrn3p–Pol I interaction and
`the Rrn3‑dependent recruitment of Pol I to the promoter [14]. Curiously, in
`yeast, phosphorylation of Pol I, not Rrn3p, is necessary for the formation of a
`stable Pol I–Rrn3p complex for efficient transcription initiation in vitro, and a
`change in the phosphorylation state of Pol I is correlated with the association of
`Pol I with Rrn3p in vivo [46].
`
`TBP–TAF complex SL1
`
`RNA polymerase enzymes themselves have no intrinsic ability to recognize
`and bind specifically to promoter DNA sequences. Therefore PIC formation
`in transcription calls for the recruitment of the polymerases to the promoter
`via transcription factors. Sigma factors fulfil this role in bacteria, and basal or
`general transcription factors co‑operate in this function in eukaryotes, where
`the three classes of highly related enzymes, Pol I, Pol II and Pol III, catalyse the
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`J. Russell and J.C.B.M. Zomerdijk
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`transcription of specific sets of genes. A complex of TBP and TAFs is required for
`the accurate initiation of transcription by all three eukaryotic RNA polymerases
`[47], with few exceptions. The complement of TAFs in each complex, although
`variable in Pol II transcription, is specific to each class of genes. Binding to TBP
`of TAF subunits specific to one class of polymerases can preclude the binding
`of TAFs from a different class [48]. The TBP–TAF complexes specific to Pols I,
`II and III perform distinct roles in mediating a specific interaction between the
`polymerases and their respective promoters.
`In transcription by mammalian Pol II, the TBP–TAF complex TFIID
`(transcription factor IID) can bind the promoter through interaction of the
`TBP protein with TATA boxes and of the TAF proteins with other promoter
`sequence elements. Pol II and other factors are recruited subsequently to form
`the PIC [49]. In transcription by Pol III, the initial phases converge on the
`recruitment of TBP–TAF complex TFIIIB and the Pol III enzyme in formation
`of the PIC [23,50], although the type of Pol III promoter determines whether
`loading of promoter‑type specific TFIIIB at the promoter DNA is facilitated by
`the specific DNA‑binding abilities of TFIIIA and TFIIIC, TFIIIC alone, or the
`multisubunit complex PBP–PTF–SNAPC.
`In transcription by mammalian Pol I (reviewed in [6]), the TBP–TAF
`complex SL1 (murine TIF‑IB) is essential [48,51–53]. This ∼300 kDa complex
`is composed of TBP and at least three TAFs [48,51,52], TAFI48, TAFI63 and
`TAFI110 (mouse TAFI48, TAFI68 and TAFI95 respectively). Reconstitution
`of transcription in vitro using recombinant SL1 comprising only these four
`components is inefficient, suggesting that more components and/or specific
`modifications might be required to reproduce the full activity of SL1 [54]. We
`have recent evidence for the existence of another TAFI in SL1 (J.J. Gorski, S.
`Pathak and J.C.B.M. Zomerdijk, unpublished work). SL1 functions through
`the essential core element of the rDNA promoter, overlapping the start site
`of transcription [55]. SL1 alone does not produce a DNase I footprint, but a
`combination of SL1 and UBF extends the footprint of UBF alone, demonstrating
`co‑operativity between these proteins at the promoter [56,57].
`SL1 directs promoter‑ and species‑specific Pol I transcription. The SL1
`complexes of humans and mice are not interchangeable between these systems,
`in contrast with Pol I and the activator UBF. These findings imply a specific
`interaction between SL1 and its cognate rDNA promoter (reviewed in [58]).
`Indeed, we have shown recently that human SL1 can bind the human rDNA
`promoter independently and direct the initiation of transcription specifically
`from the human rDNA promoter in the absence of UBF [59], consistent with
`the findings that TIF‑IB and rat SL1 can bind and direct transcription from their
`cognate rDNA promoters independently of UBF [60,61].
`In addition to promoter recognition and binding, SL1 has an essential role
`in the recruitment of Pol I to the start site of transcription [35,59]. SL1 subunits
`TAFI63 and TAFI110 interact with the Pol I‑associated RRN3 [35–37], which
`in turn is attached to Pol I through subunit A43 in the ‘stalk’ structure of the
`enzyme complex and, perhaps, PAF67 [41,42].
`Another function for SL1 in PIC formation has been reported recently: we
`found that SL1 can stabilize UBF binding at the rDNA promoter [59]. In the
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`UBF
`
`SL1
`
`5
`
`1
`
`2
`
`3
`
`4
`
`rDNA
`
`Pol lβ
`
`hRRN3
`
`Pol lε
`
`6
`
`TTF-I
`PTRF
`
`rRNA
`
`Figure 1 The Pol I transcription cycle. The TBP–TAF complex SL1 interacts
`stably with the rDNA promoter, near the start site of transcription (black arrow).
`Dimers of UBF interact dynamically with rDNA promoter elements (grey arrows),
`such that UBF–rDNA complexes might be relatively short‑lived. UBF and SL1 interact
`co‑operatively at the promoter elements, and SL1 reduces the dissociation of UBF
`from the rDNA, thereby increasing the residence time of UBF at the promoter (1).
`rDNA promoter topology is altered dramatically by UBF binding. SL1 in the UBF–SL1–
`rDNA complex recruits the initiation‑competent Pol I–hRRN3 complex Pol Iβ through
`the interactions of TAFI subunits with hRRN3. This culminates in formation of the
`complete PIC, poised for initiation of transcription (2). UBF stimulates the promoter
`escape/clearance of Pol I. In the transition from initiation to elongation, hRRN3
`dissociates from Pol I, and the Pol I enzyme is converted into a transcript‑elongating
`enzyme complex, Pol Iε (3). SL1 and UBF remain promoter‑bound following promoter
`clearance by Pol I (4) and, thus, can function as a re‑initiation scaffold (5), to allow
`multiple rounds of transcription from the same rDNA promoter. Pol Iε transcription
`terminates and rRNA is released at termination sites (black rectangle on rDNA); this
`process is directed by TTF‑I (transcription termination factor‑I) and PTRF (polymerase
`I and transcript release factor). Following termination of transcription, it is possible that
`the spent Pol I complex is converted back into initiation‑competent Pol Iβ and thence
`recruited to the rDNA promoter by SL1 to initiate a new round of transcription (6).
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`absence of SL1, UBF associates with and dissociates from the rDNA rapidly,
`whereas the presence of SL1 at the rDNA promoter alters significantly the
`binding equilibrium of UBF, thereby increasing the residence time of UBF at the
`promoter. It is possible that the increased lifetime of the rDNA–UBF complex
`is essential to facilitate the UBF‑mediated activation of Pol I transcription.
`Collectively, these results support a unified model for SL1 function in the
`mammalian system, where SL1 drives or nucleates PIC formation specifically
`at the rDNA promoter, leading ultimately to the productive initiation of
`transcription by Pol I (Figure 1). SL1 remains stably bound to the rDNA
`promoter following initiation of transcription and escape by Pol I, and the SL1
`complex, together with UBF at the promoter, might serve as a re‑initiation
`scaffold to support multiple rounds of transcription [62].
`PIC assembly could perhaps be regulated through SL1, influencing the level
`of transcription by Pol I. Indeed, an increase in SL1 occupancy of the rDNA
`promoter correlates with increased Pol I transcription in IGF‑1 (insulin‑like
`growth factor‑1)‑stimulated HEK293 cells [13]. Although phosphorylation of
`SL1 in response to growth stimulatory factors has not yet been reported, some of
`the SL1 subunits are known to be phosphoproteins. SL1 activity can be controlled
`by acetylation, as acetylation of the mouse SL1 (TIF‑IB) subunit TAFI68 by
`P/CAF [p300/CBP (CREB‑binding protein)‑associated factor] stimulates
`binding of SL1 to the rDNA promoter and Pol I‑mediated transcription [63],
`and it is possible that this acetylation is a regulated, rather than constitutive,
`event in cells. Furthermore, SL1 could be a limiting component of rRNA gene
`expression in cells, and therefore an increase in its abundance would stimulate
`Pol I transcription. Although tissue‑specific or differential expression of the
`TAFs of SL1 has not been reported as yet, regulated expression of these Pol
`I‑specific TAFI subunits could impact on transcription by Pol I. Up‑regulation
`of TBP expression, following activation of the MAPK signalling pathway, can
`lead to stimulation of transcription from Pol I and Pol III promoters in certain
`cell types, implying that overexpression of this single subunit can drive the
`biogenesis of the separate TBP–TAF complexes required in the co‑ordinated
`transcription of rRNA and tRNA genes [64,65].
`
`Transcription activator UBF
`
`Reconstituted transcription assays have revealed the components minimally
`required for transcription initiation. ‘Basal’ levels of transcription from the
`rDNA promoter can be supported by SL1 and initiation‑competent Pol Iβ
`alone [60,61]. UBF efficiently activates rDNA transcription above this basal
`level. Stimulated transcription by UBF in the reconstituted system constitutes
`‘true’ or ‘net’ activation, as distinct from stimulation of transcription through
`the potential anti‑repression function of UBF in the context of chromatin
`transcription in vivo [66–68].
`UBF is proposed to function as an architectural protein at the rDNA
`promoter [69,70], where it binds as a dimer to the UCE (upstream control
`element), which has a modulatory role in Pol I transcription, and to the essential
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`core element. UBF (97 kDa) is composed of several distinct domains [69,70].
`The N‑terminal dimerization domain of UBF is essential for its transactivation
`function [69,71]. The large central portion contains six HMG (high‑mobility
`group) boxes [72,73] which exhibit a relaxed sequence specificity of DNA
`binding and display the ability to bend DNA [69,74–79]. In UBF, these HMG
`boxes do not contribute equally to DNA binding; rather, it is thought that HMG
`boxes 1–3 interact with the DNA minor groove in the interaction of UBF with
`the rDNA promoter [74,79]. Binding of a UBF dimer induces bending of the
`DNA, to create a loop of some 140 bp of DNA in a single 360o turn, generating
`a structure termed the enhancesome [78]. It has been proposed that tandem
`enhancesomes at the rDNA promoter would juxtapose the UCE and core
`promoter elements [78,79]. There is co‑operativity of binding of UBF and SL1
`to the UCE and the core elements of the rDNA promoter [56,57], although the
`precise arrangement and stoichiometry of UBF and SL1 at the rDNA promoter
`remain unknown. Interaction between UBF and SL1 [69,80,81] requires the
`highly acidic C‑terminal domain of UBF [69,82,83] and may involve subunits
`TAFI48 and TBP of SL1 [84,85]. UBF also interacts with Pol I [29,86] through
`Pol I subunits ASE1/CAST and PAF53 [29,87] (K.I. Panov, T.B. Panova and
`J.C.B.M. Zomerdijk, unpublished work) (Table 1).
`These interactions and the co‑operativity demonstrated between UBF and
`SL1 at the rDNA promoter led to the proposal that UBF recruits SL1 and Pol
`I to the rDNA promoter, activating transcription by facilitating PIC assembly.
`This is the principal mechanism used by activators of Pol II transcription
`[88]. However, in contrast with the sequence‑specific Pol II transcriptional
`activators, binding of UBF occurs rather indiscriminately [75,76] and extensively
`throughout the rDNA repeats in vivo [89,90], inconsistent with a role for UBF
`in nucleating PIC assembly specifically at the rDNA promoter. Moreover,
`as discussed, SL1 alone can nucleate PIC formation [59–61], stabilizing UBF
`binding at the rDNA promoter [59] and recruiting initiation‑competent Pol I to
`the core promoter [35].
`Recently, using a highly purified and reconstituted transcription system,
`combined with an immobilized ribosomal promoter DNA template, we have
`defined a specific and novel function for UBF in activating the rate of RNA
`synthesis at promoter escape or clearance by Pol I (K.I. Panov, J.K. Friedrich,
`J. Russell and J.C.B.M. Zomerdijk, unpublished work), a step we had identified
`previously as the rate‑limiting step for Pol I transcription in vitro [62]. This
`mechanism enables UBF to activate transcription both from previously inactive
`promoters following PIC assembly and from SL1‑engaged promoters at each
`successive round of transcription following re‑initiation (Figure 1). Stimulation
`of promoter escape/clearance by activators might also make an important
`contribution to efficient transcription by Pols II and III.
`Given its role in activating transcription, any variation in UBF expression
`levels or activity would be anticipated to have a dramatic effect on the level of
`transcription by Pol I. Indeed, UBF, limiting in some cells [91], is expressed at
`relatively high levels in cardiac hypertrophy and liver cancer cells, which support
`high levels of rRNA synthesis [92,93]. In mitogen‑stimulated lymphocytes, UBF
`overexpression is apparent prior to increased rRNA synthesis, and a decrease in
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`UBF expression occurs upon induced differentiation [94]. UBF expression can
`be up‑regulated by serum induction [9], operating through the mTOR pathway,
`and also by the myc proto‑oncogene [95].
`How is the activity of the UBF protein regulated? Phosphorylation of
`UBF can affect its interaction with other PIC components or DNA. C‑terminal
`phosphorylation of UBF increases its ability to interact with SL1 [69,82,83].
`The protein kinase CK2 phosphorylates UBF at serine residues within the
`C‑terminal acidic domain, and this contributes to transcriptional activation
`[96]. Phosphoinositide 3‑kinase phosphorylates UBF predominantly in the
`C‑terminus upon IGF‑1 (insulin‑like growth factor 1) treatment of serum‑starved
`mouse cells, where IRS‑1 (insulin receptor substrate‑1) targets phosphoinositide
`3‑kinase to UBF following translocation of IRS‑1 to the nucleolus [15], and this
`correlates with increased rRNA synthesis. C‑terminal phosphorylation of UBF
`can also be mediated by the mTOR signalling pathway and requires S6 kinase 1.
`This occurs upon serum induction of rDNA transcription in NIH 3T3 cells and
`promotes the interaction between UBF and SL1 [9]. The ERK/MAPK pathway
`targets UBF upon stimulation of Pol I transcription by epidermal growth factor
`in human neuroepithelioma cells. ERK1/2‑mediated phosphorylation in HMG
`boxes 1 and 2 alters the interaction of UBF with DNA [11]. UBF activity is also
`regulated by competitive acetylation/deacetylation. In this case, the ability of
`UBF to bind DNA is not affected [68]. Rather, binding of CBP acetyltransferase
`to UBF might up‑regulate UBF by preventing the binding of the tumour
`suppressor Rb (retinoblastoma) (and associated histone deacetylases) to UBF,
`precluding the negative effect of Rb on the binding of UBF to SL1 [97].
`
`Concluding remarks
`
`From the evidence obtained so far, we conclude that the Pol I TBP–TAF
`complex SL1 performs three important functions that, together, drive PIC
`formation at the rDNA promoter. SL1 recognizes and binds the core promoter
`elements through its TAFI proteins. The SL1 complex, positioned accurately
`at the rDNA promoter, recruits the initiation‑competent form of Pol I, the
`Pol I–RRN3 complex Pol Iβ, to the start site of transcription, through the
`interactions of TAFI110 and TAFI63 with RRN3. SL1 also stabilizes UBF at the
`rDNA promoter, and the consequential increase in residence time of UBF at the
`promoter is predicted to enhance the efficacy of UBF in its role as an activator
`of Pol I transcription. The dramatic alterations in DNA architecture created
`by UBF at the rDNA promoter are likely to be important for its activation
`function, although the significance of these has yet to be realized. We consider
`that the interaction of UBF with subunits of Pol I is crucial for the ability of
`UBF to stimulate the escape of Pol I from the promoter in the transition state
`between initiation and elongation of transcription.
`Pol I transcription can be regulated at the levels of PIC formation and
`UBF activity. These are influenced by the downstream effectors of growth
`factor‑responsive signalling pathways (including the MAPK, mTOR, and ERK
`pathways), the availability of nutrients (mTOR) and/or the cellular stress response
`
`© 2006 The Biochemical Society
`
`10
`
`

`

`RNA polymerase I transcription machinery
`
`213
`
`pathways (JNK2). The level of rRNA transcription activity has a direct effect
`on the overall level of cellular protein synthesis, thereby influencing the growth
`of the cell and cell division (reviewed in [6]). There is a correlation between
`the level of cellular rRNA transcription and the growth status of cells, in that
`slow‑growing or differentiated cells display low levels of rRNA transcription,
`whereas rapidly growing and cancerous cells maintain high levels of rRNA
`synthesis. Down‑regulation of Pol I transcription can lead to the activation of
`the p53 cellular response pathway, and therefore directly influence the cell’s
`decision to arrest or exit from the cell cycle in response to DNA damage, stress
`or malfunction [98]. Up‑regulation of rRNA transcription, with the growth
`and proliferative advantage it bestows on cells, could represent an early event
`in oncogenesis; significantly, the oncoprotein Myc has been shown recently to
`control cellular rRNA synthesis [99–101].
`
`We thank present and past members of our laboratory for their research and interest.
`We apologize to our colleagues whose work could not be cited due to space limitations.
`The Wellcome Trust funds research in the laboratory of J.C.B.M.Z., and J.C.B.M.Z. is a
`Wellcome Trust Senior Research Fellow in the Basic Biomedical Sciences.
`
`10.
`11.
`
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