Species specificity of transcription by RNA polymerase I

Species specificity of transcription by RNA polymerase

I

Jutta Heix and lngrid Grummt German An unusual specificity. RNA

Cancer property

This

Research

of ribosomal

results

polymerase

characterization

from distinct

I transcription of TIF-IB/SLl,

Center,

Heidelberg,

gene transcription

Germany

is its marked species

promoter-recognition

apparatus.

The

properties

purification

a promoter-recognition

of the

and functional

factor containing

protein,

three subunits

(TAFls) of the respective human and mouse factor, will facillitate

the molecular transcription

Current

as well as the recent cloning of cDNAs

the

TATA-binding

analysis of the mechanisms

underlying

and reveal how the basal transcriptional

Opinion

in Genetics

Introduction Initiation of ribosomal gene transcription is mediated by a specific multiprotein complex comprising RNA polymerase I (Pol I) and at least four transcription initiation factors. The first of these factors to be cloned was upstream-binding factor (UBF), an 85-97 kDa nucleolar protein that contains multiple high-mobility group (HMG) domains (i.e. 80-90 amino acid motifs with homology to the DNA-binding domain of HMG proteins 1 and 2) flanked by an amino-terminal dimerization motif and an acidic carboxy-terminal tail (for recent reviews, see [1,2]). The structure and DNA-binding properties of UBF are conserved among different vertebrate species [3-6]. When UBF binds to the promoter, it forms a nucleoprotein structure in which DNA is wrapped around the contiguous HMG domains, thus placing start site proximal and distal promoter elements in close proximity [7*].

The other Pol I specific factors are known by a variety of nomenclatures. For the sake of brevity and clarity, we will use the term TIF-I (for transcription initiation factor) with letters appendend to indicate different factors (i.e. TIF-IA, TIF-IB, and TIF-IC) that have been purified from mouse cells [S]. Promoter selectivity is mediated by a multisubunit factor called TIF-IB in mouse and SLl in humans. TIF-IB/SLl, as for the functionally analogous Pol II and Pol III specific factors TFIID and TFIIIB, contains the TATA-binding protein (TBP) and a set of specific TBP-associated factors (TAFs) [9,10]. Promoter-bound TIF-IB/SLl recruits Pol I, together with TIF-IA and TIF-IC, to the rDNA and nucleates the assembly of a transcription initiation complex [8]. Both TIF-IA and TIF-IC are basal Pol I transcription factors that are associated with Pol I and serve an essential role in initiation.

& Development

encoding the

species-specific machinery

1995,

rDNA

has evolved.

5:652-656

The amount or activity of TIF-IA correlates with cell proliferation, so this factor is likely to play a key role in growth-dependent regulation of rRNA synthesis [l 11. TIF-IC is functionally homologous to the Pol II specific factor TFIIF. In addition to its function in transcription initiation, TIF-IC plays a role in the elongation of nascent RNA chains [12]. Early studies demonstrated that ribosomal templates are transcribed by extracts from only corresponding or closely related species [13]. The progress that has been made in the purification and functional characterization of individual transcription factors together with the availability of specific antibodies and recombinant transcription factors has facilitated the molecular analysis of the mechanism underlying this species-specific transcription. In this review, we will highlight the most current advances in the understanding of rDNA promoter selectivity and focus on the transcription factors that govern mouse and human Pol I promoter recognition.

rDNA

promoter

elements

Although the rRNA-coding regions are among the most highly conserved of gene sequences, the signal sequences that mediate transcription initiation have diverged significantly [2]. One theory for the divergence of the control sequences is based on the fact that they are members of a single multigene family and therefore undergo concerted evolution [14]. In short, mutations in the controlling region of one gene that enhance its interaction with an essential transcription factor may be selected for and fixed in all other copies of the genes because of the genetic exchanges (gene conversion,

Abbreviations H&K-high-mobility TIF-transcription

652

group;

Pal I-RNA

initiation

factor:

0

polymerase

I; TAF-TBP-associated

UBF-upstream-binding

Current

Biology

Ltd ISSN

factor;

factor;

TBP-TATA-binding

UCE-upstream

0959-437X

control

element.

protein;

Species specificity of transcription by RNA polymerase I Heix and Grummt unequal crossing over, and excision-integration) that occur among them. Thus, the repetitive nature of the ribosomal genes, along with the use of a dedicated transcription machinery, permits rapid evolutionary changes, inevitably leading to species specificities. In agreement with this interpretation, the promoter sequences show little, if any, sequence homology between distantly related organisms.

Nevertheless, despite the lack of obvious sequence similarities, the overall structural organization of rDNA promoters from eukaryotes is comparable. In general, the promoter can be divided into a start site proximal ‘core’ domain and upstream elements, including the upstream control element (UCE), enhancers, spacer promoters and upstream terminators [2]. Whereas the core is sufficient for accurate initiation, the upstream elements stimulate promoter activity without affecting transcriptional specificity. Interestingly, the UCE shares a region of significant sequence homology with the core domain of the rDNA promoter (Fig. 1). This sequence homology, together with the finding that the correct spacing and helical alignment between the UCE and the core domain is crucial for promoter function [15,16], suggests that two factor molecules (presumably UBF and/or TIF-IB/SLl) bind to both the UCE and the core. Experiments with chimeric mouse/human rDNA promoters have shown that the core element mediates species-specific transcription. Mouse sequences upstream of -32 or downstream of -14 can be replaced with the corresponding human sequence without affecting promoter specificity. Human rDNA promoter activity, on the other hand, has been shown to require nucleotides -43 to +17 [17], a finding that indicates a functional asymmetry of the rDNA promoter. Paradoxically, if half of a helical turn (5 bp) is inserted in, or removed from, the middle of the Xermpus promoter, it becomes non-functional in the homologous system, but functions efficiently and correctly in the mouse extract [15]. This observation indicates that the spacing between individual promoter domains can play an important role in promoter selectivity of PO1 I.

-110 -81 GTCCGTG'?CGcgcgtcgccTGGECGXGZ

TIF-IB/SLl

confers promoter

polymerase

selectivity

to RNA

I

The development of species-specific transcription appears to involve the co-evolution of the rDNA promoter sequences with proteins that interact at the promoter. Early studies suggested an important role for UBF in promoter selectivity because the requirement of UBF for basal transcription in humans and rodents is different. Binding of SLl to human rDNA requires UBE whereas TIF-IB forms a committed complex in the absence of UBE However, UBF is a highly conserved protein which shows only a few amino acid changes between primates and rodents. Consistent with the conservation of structure, human and mouse UBF have been demonstrated to be functionally interchangeable [4]. Despite the almost complete lack of sequence homology between the mammalian and Xenoprrs rDNA promoters, UBF 6om mammals or Xenopur specifically recognizes both homologous and heterologous promoter sequences. Nevertheless, Xenopus UBF does not functionally substitute for mammalian UBF in reconstituted transcription assays [3]. However, hybrid proteins containing the amino-terminal half of Xcnopus UBF fused to the carboxyl terminus of human UBF were able to restore transcriptional activity in such assays [6]. Thus, the failure of the Xenopus factor to promote transcription from the mammalian template appears to derive from its inability to form a productive complex with the heterologous TIF-IB/SLl. These studies point to the importance of species-specific protein-protein interactions in mediating transcription complex assembly. In addition to its interaction with TIF-IB/SLl, UBF contacts the central player of the transcription complex, Pol I [ 181. This interaction between UBF and Pol I is mediated by one defined subunit of Pol I in organisms as divergent as mouse and yeast. The subunits that interact with Pol I (34.5 kDa in yeast and 62 kDa in mouse) may be functionally homologous, because both in yeast and mammals, two forms of Pol I can be separated (Pol A and Pol I,) that

-33 CTCCGAGTCGgcatttTGGGCCGCC~

Humar -12

Mouse

8 1995 Current Opinion in Genetics & Oevefopment

Fig. 1. Schematic representation of the spatial array of rDNA promoter elements. The core and the upstream control element WCE) are shown as boxes. The nucleotide sequence of both regions from human and mouse are shown above. The nucleotide positions are numbered with respect to the transcription start site. Capital letters mark homologous bases between the UCE and the core of both species. The consensus sequences are shown in the box below.

653

654

Differentiation and gene regulation

lack this subunit and differ in their template specificity. Thus, at least on a functional level, the interaction between UBF and Pol I is highly conserved across a wide phylogenetic range. To find out which of the factors is responsible for the template stringency, individual factors purified from mouse and human cells were assayed in a reconstituted system on either the homologous or heterologous rDNA templates. These experiments demonstrated that both UBF and Pol I (together with its associated factors TIF-IA and TIF-IC), either individually or in combination, can be replaced by the heterologous factor without changing template specificity. TIF-IB/SLl, on the other hand, must derive horn the same species to support rDNA transcription. That is, transcription of mouse rDNA requires addition of TIF-IB and transcription of human rDNA requires SLl [4,19*]. This result is in accord with previous studies showing that the human factor SLl can reprogram a mouse extract to transcribe human rDNA [9,20]. Surprisingly, the reverse experiment, reprogramming of a human nuclear extract with TIF-IB, did not work [21]. In unfractionated HeLa cell extracts, apparently, TIF-IB activity is counteracted by an (as yet) unknown component.

Differences

in TAFls determine

the specificity

of

TIF-IB/SLl To determine the molecular basis for the inability of TIF-IB/SLl to direct transcription of heterologous rDNA, the properties and the subunit compositions of the murine and human factors were compared. SLl and TIF-IB are multiprotein complexes comprising TBP and three TAFs [9,10]. Two of the human and mouse Pol I specific TAFs (TAFt48 and TAFt68/63) are similar in size, but the largest TAFt and TBP from these two species both differ in size from their counterpart. The largest TAFt has an apparent molecular mass of 95 kDa in mouse compared with 110 kDa in human. Mouse and human TBP differ in their variable amino-terminal domain [22]. A chimeric complex containing human TBP and murine TAFls exhibits specificity for the mouse promoter, indicating that differences in the amino terminus of human and mouse TBP do not contribute to the promoter selectivity of TIF-IB/SLl [19*]. Moreover, the core domain of TBP has been shown to be sufficient for the assembly of transcriptionally active TBP/TAFl complexes [23-l, and a chimeric complex comprising yeast TBP and human TAFls faithfully promotes human rDNA transcription [24*]. Thus, the interactions between TBP and TAFls have been evolutionarily conserved. Given the interchangeability of TBP in TIF-IB/SLl, on one hand, and the differences in individual TAFts, on the other hand, the molecular basis for speciesspecific promoter recognition. should reside in differ-

ences between the human and mouse TAFts. This implies that at least one of the TAFls should interact with DNA. Indeed, UV-crosslinking experiments have demonstrated that TAF148 and TAFt68/63 contact DNA [19*]. Unexpectedly, these two TAFls bind to both the homologous and heterologous promoter. This result, together with the observation that TIF-IB/SLl promotes transcription from only the homologous template, suggests that binding of TIF-IB/SLl to the heterologous promoter precludes formation of productive initiation complexes. We propose that binding to the respective species-specific promoter elements results in a defined conformation of TIF-IB/SLl which in turn is a prerequisite for functional interactions with other polypeptides present in the initiation complex (Fig. 2). Recently, the cDNAs encoding each of the human TAFts have been isolated [25**]. The TAFts are novel proteins with no significant similarities to any proteins in the database, except for the fact that TAF168/63 contains two putative zinc fingers that may play a role in DNA binding. Analyses of subunit interactions indicate that the three TAFts can bind individually and specifically to TBP. In addition, human TAFts interact with each other to form a stable TBP/TAF complex [25”] which is as active in supporting transcription from the human ribosomal gene promoter as endogenous SLl, whereas partial complexes do not efficiently direct transcription in oitro [26*-l. Thus, the three TAFts together with TBP are necessary and sufficient to reconstitute a transcriptionally active SLl complex. The cDNAs for the murine TAF,s, mTAF148, mTAFt68, and mTAF195, have also been cloned recently (J Heix, J Zomerdijk, R Tjian, I Grummt, unpublished data). The three mouse TAFts show 89-76X homology to their human counterparts and it seems, therefore, that subtle differences between individual TAFls may affect the overall conformation of the TBP/TAF complex. Work is in progress to assemble chimeric TIF-IB/SLl complexes from recombinant human and mouse subunits to find out whether one or all of the TAFts dictates species-specific transcription. We expect comparison of Pol I transcription machineries from different species to help our understanding of how changes in interactions between components of the transcription apparatus can lead to new promoter specificities.

Conclusions Molecular evolution of the rDNA promoter has resulted in a high degree of incompatibility between the transcription machineries of different organisms. This rapid evolution of the rRNA promoters is likely to provide a major driving force for compensatory changes in the transcription machinery resulting in species-specific transcription. Despite the rDNA promoter sequences of different species being so disparate, the universal structural organization of both the &-acting elements

Species specificity of transcription by RNA polymerase I Heix and Grummt

(a)

POL I --\

Homologous

SSE

6)

POL I

an important role in assembly of productive initiation complexes. Although we cannot exclude the possibility that other, yet to be identified, factors present in the polymerase fraction contribute to species-specific transcription, the major player in transcription complex assembly is the TBP-containing factor TIF-IB/SLl. This factor recognizes the rDNA promoter, forms a cooperative complex with UBF and recruits Pol I to the transcription start site. This variety of functions possibly involves interactions between the TAFs and many of the components participating in the formation of transcription initiation complexes. Therefore, subtle differences in the interaction of TIF-IB/SLl with species-specific promoter elements and components of the transcriptional machinery are probably the major determinants of species-specific rDNA transcription. The availability of recombinant transcription factors, specific antibodies, and reconstituted transcription systems comprising highly purified protein factors will allow rapid advances in our understanding of the multiple molecular interactions occurring at the rDNA promoter in the near future. The final explanation for species-specific transcription, however, may come only when the structure of the transcription initiation complex is resolved at the atomic level.

Acknowledgements We thank

Stephen Mason for critical reading of the manuscript colleagues in both our laboratory and Robert Tjian’s group for making possible our contribution to this area. Work performed in the authors’ laboratory was supported by the Deutsche Forschunggemeinschaft (Leibniz-Programm and SFU 319) and the Fond7 der Chemischen Industrie. and our

Fig. 2. Model depicting TIF-IB/SLl interactions at (a) the homologous and (b) the heterologous species-specific element (SSE) of the core promoter. The model is not meant to indicate specific interactions between the proteins shown, as such interactions have yet to be demonstrated. We hypothesize that TIF-IB/SLl binds to both the homologous and heterologous promoter. Binding to the homologous promoter induces a conformational change of TIFIB/SLl which in turn is a prerequisite for productive interaction with Pol I and/or the associated factors TIF-IA and TIF-IC leading to transcription. Whether or not upstream-binding factor (UBF) remains associated with the initiation complex is not known.

and tram-acting factors of species as phylogenetically distant as protozoa, fungi, insects, and mammals suggests that the general mechanisms of rDNA transcriptional regulation have been evolutionarily conserved. Thus, the functional differences between yeast and mammals are not as extensive as the disparity in promoter sequences may suggest. Significantly, protein-DNA interactions alone are not sufficient to confer promoter selectivity but the correct interaction between transcription factors appears to play

References and recommended Papers of particular interest, published review, have been highlighted as: . of special interest _ .. of outstanding interest

reading within

the annual period of

1.

Paule MR: Transcription of ribosomal RNA by eucaryotic RNA polymerase I. In Transcription: Mechanisms and Reguhfion. Edited by Conaway RC, Conaway JW. New York: Raven Press, Ltd; 1994:83-l 06.

2.

Moss T, Stefanovsky VY: Promotion and regulation of ribosomal transcription in eucaryotes by RNA polymerase I. Prog Nucleic Acids Res MO/ Bio/ 1995, 50:25-66.

3.

Bell SP, Pikaard CS, Reeder RH, Tjian R: Molecular mechanism governing species-specific transcription of ribosomal RNA. Cell 1989, 59:489-497.

4.

Bell SP, Jantzen HM, Tjian R: Assembly of multiprotein complexes directs rRNA promoter Genes Dev 1990, 4:943-954.

5.

Pikaard CS, Pape LK, Henderson SL, Ryan K, Paalman MH, Lopata MA, Reeder RH, Sollner-Webb 8: Enhancers for RNA polymerase I in mouse ribosomal DNA. MO/ Cell Biol 1990, 10:4816-4825.

alternative selectivity.

655

656

Differentiation and gene regulation 6.

Jantzen HM, Chow AM, King DS, Tjian R: Multiple domains of the RNA polymerase I activator UBF interact with the TATAbinding protein complex hSL1 to mediate transcription. Genes Dev 1992, 6:1950-l 963.

7. .

Bazett-Jones DP, Leblanc B, Herfott M, Moss T: Short range DNA looping by the Xenopus HMC-box transcription factor, xUBF. Science 1994, 264: 113&l 137. The authors analyzed the interactions of Xenopus UEF with the ribosomal gene promoter and demonstrated that binding of a UBF dimer loops 180 bp of enhancer DNA into an approximately 360’ turn. The structure of this DNA-protein complex was analyzed by electron spectroscopic imaging. 8.

Schnapp A, Crummt I: Transcription complex formation at the mouse rDNA promoter involves the stepwise association of four transcription factors and RNA polymerase I. / Viol Chem 1991, 266:24588-24595.

9.

Comai L, Tanese N, Tjian R: The TATA-binding protein and associated factors are integral components of the RNA polymerase I transcription factor, Sll. Cell 1992, 68:965-976.

10.

Eberhard D, Tora L, Egly JM, Crummt I: A TBP-containing multiprotein complex (TIF-IB) mediates transcription specificity of murine RNA polymerase I. Nucleic Acids Res 1993, 21:4180-4186.

11.

Schnapp A, Schnapp G, Erny B, Grummt I: Function of the growth-regulated transcription initiation factor TIF-IA in initiation complex formation at the murine ribosomal gene promoter. MO/ Ce// Biol 1993, 13:6723-6732.

12.

Schnapp C, Schnapp A, Rosenbauer H, Grummt I: TIF-IC, a factor involved in both transcrition initiation and elengation of RNA polymerase I. EM60 / 1994, 13:4028-4035.

13.

Crummt I, Roth E, Paule MR: Ribosomal RNA transcription vitro is species specific. Nature 1982, 2%:173-l 74.

14.

Arnheim N: Concerted evolution of multigene families. In Evolution of Genes and Proteins. Edited by Koehn R, Nei M. Sunderland, MA: Sinauer; 1983:38-61.

15.

Pape LK, Windle changes convert upstream domain side. Genes Dev

16.

Xie WQ, Rothblum LI: Domains of the rat rDNA promoter must be aligned stereospecifically. MO/ Cell Biol 1992, 12:1266-l 275.

1 7.

T, lshikawa Y, Kato Safrany G, Tanaka N, Kishimoto H, Kominami R, Muramatsu M: Structural determinant of the species-specific transcription of the mouse rRNA gene promoter. MO/ Ce// Biol 1989 9:349-353.

18.

in

JJ, Sollner-Webb B: Half helical turn spacing a frog into a mouse promoter: a distant determines the helix face of the initiation 1990, 4:52-62.

Schnapp G, Santori F, Carles C, Riva M, Grummt I: The HMC box-containing nucleolar transcription factor UBF interacts with a specific subunit of RNA polymerase I. EMBO / 1994, 13:190-l 99.

Rudloff U, Eberhard D, Tora L, Stunnenberg H, Grummt I: TBP-associated factors interact with DNA and govern species specificity of RNA polymerase I transcription. EM80 / 1994, 13:2611-2616. To investigate whether the differences in the amino termini of mouse and human TBP contribute to the species specificity of TIF-IB/SLl, a mouse cell line stably expressing epitope-tagged human TBP (ehTBP) was used. The data demonstrate that ehTBP associates with mouse TAF,s to form an active hybrid complex that exhibits specificity for 19. .

the mouse promoter. This result demonstrates that differences between the amino-terminal domains of mouse and human TBP do not play a significant role in rDNA promoter selectivity, suggesting that structural differences between mouse and human TAFls determine species-specific promoter recognition. 20.

Learned RM, Cordes S, Tjian R: Purification and characterization of a transcription factor that confers promoter specificity to human RNA polymerase I. MO/ Cell Biol 1985, 5:1358-l 369.

21.

Schnapp A, Rosenbauer H, Grummt I: Transacting factors involved in species-specificity and control of mouse ribosomal gene transcription. MO/ Ce//.Viol 1991, 104:137-l 47.

22.

Tamura T, Sumita K, Fujino I, Aoyama A, Horikoshi M, Hoffmann A, Roeder RG, Muramatsu M, Mikoshiba K: Striking homology of the “variable” N-terminal as well as the “conserved core” domains of the mouse and human TATA-factors (TFIID). Nucleic Acids Res 1991, 19:3861-3865.

23. .

Rudloff U, Stunnenberg HC, Keaveney M, Grummt I: Yeast TBP can replace its human homologue in the RNA polymerase l-specific multisubunit factor SLl. / MO/ Biol 1994, 243:84&845. The authors show that yeast TBP assembles with the human TAF,s in viva and that the resulting hybrid complex faithfully promotes rDNA transcription in vitro, indicating that the interactions between TBP and the Pol I specific TAFs have been evolutionarily conserved. 24. .

Rudloff U, Eberhard D, Grummt I: The conserved core domain of the TATA binding protein is sufficient to assemble the multisubunit RNA polymerase l-specific transcription factor 91. Proc Nat/ Acad Sci USA 1994, 91:8229-8233. This report demonstrates that the conserved core domain of TBP alone is sufficient for the correct assembly of an active Pol I specific TBP/TAF complex. 25. ..

Comai L, Zomerdijk JCBM, Beckmann H, Zhou S, Admon A, Tjian R: Reconstitution of transcription factor Sll: exclusive binding of TBP by Sll or TFIID subunits. Science 1994, 266:1966-l 972. Describes the isolation of the cDNAs encoding the full set of human Pol I specific TAFs, hTAF,48, hTAF163, and hTAFIll0. Analyses of subunit interactions using recombinant proteins revealed that each hTAFI specifically contacts TBP and that in addition all TAFls interact with each other to form a stable TBPDAF complex. Furthermore, protein-protein interaction studies using Pol I and Pol II specific TAFs indicate that a mutually exclusive binding specificity for TBP is intrinsic to TAFls and TAFlls. This specificity explains the formation of the different promoter-specific and RNA polymerase specific TBP/TAF complexes. 26. ..

Zomerdijk JCBM, Beckmann H, Comai L, Tjian R: Assembly of transcriptionally active RNA polymerase I initiation factor Sll from recombinant subunits. Science 1994, 266:2015-2018. This report shows the in viva and in vitro assembly of transcriptionally active SLl from recombinant TBP and TAFls. Partial complexes were not functional for Pol I specific transcription demonstrating that human TBP and hTAF,48, hTAF,63, and hTAF,l10 are necessary and sufficient to reconstitute SLl activity.

J Heix and I Grummt, II, German

lM59130 I Grummt

Cancer

Heidelberg, E-mail:

I)ivision

of Molecular

Center, Germany.

Icesearch

Uioloby

of the Cell

Im Neuenheimer

Feld 280,

I.Grurnmt@l)KFZ-Heidelberg.de