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Biochimica et Biophysica Acta, 1050 (1990) 351-355 Elsevier BBAEXP 92144

Multifunctional DNA-binding proteins mediate concerted transcription activation of yeast ribosomal protein genes W i U e m H. M a g e r a n d R u d i J. P l a n t a Biochemisch Laboratorium, Vr(je Unioersiteit, Amsterdam (The Netherlands) (Received 16 May 1990)

Key words: Yeast; Ribosomal protein gene; Multifunctional protein

Transcription activation of ribosomal protein genes (rp genes) in yeast is mediated through two different abundant transacting proteins, RAP1 and ABF1. These factors are multifunctional proteins playing a part in diverse cellular processes, all related to cellular growth.

Introduction Ribosome biogenesis in yeast encompasses the equimolar production of some 80 different constituents, ribosomal proteins and rRNAs. Ribosomal proteins, synthesized in the cytoplasm, are transported to the nucleolus where they assemble with precursor rRNA in a defined and ordered fashion (for a review see Ref. 1). Ribosome formation in yeast is a highly balanced process. No pool of free ribosomal proteins exists. After increasing the copy number of a particular rp gene, excess ribosomal protein appeared to be degraded very rapidly, suggesting 'ribosome assembly-mediated control' as the ultimate level of coordination in the production of the ribosomal components (reviewed in Refs. 2,3). Coordination of rp synthesis occurs primarily at the transcriptional level [2,3]. Molecular evolution of the rp gene promoters, supposedly, has been proceeded in such a way that transcription of the different rp genes leads to nearly equal amounts of mRNA. In this respect, rp genes should be considered as house-keeping genes displaying a constitutive transcription during steady state growth conditions. To meet a changing need for protein biosynthetic capacity upon alterations in physiological circumstances, however, the rate of rp gene transcription can be accurately adjusted. Under such conditions the balance in cellular rp mRNA levels is maintained, indicating a coordinate regulation of transcription [2,3].

Abbreviations: rp, ribosomal protein; UAS, upstream activation site. Correspondence: R.J. Planta, Biochemisch Laboratorium, Vrije Universiteit, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.

Such a coordinate response can be observed, for instance, when yeast cells are shifted from an ethanolbased medium to a glucose-based medium (see Fig. 1). In general, yeast rp genes are scattered over the genome. In the few cases that two different rp genes are physically linked they are nevertheless transcribed independently. Therefore, we, and others, have searched for cis-acting dements and trans-acting factors that might mediate the concerted transcriptional regulation of yeast rp genes.

Transcriptional activators of yeast rp genes Comparison of the upstream sequences of many yeast rp genes initially revealed the presence of one conserved nucleotide element, the RPG box, with the consensus sequence ACACCCATACATTT [4]. RPG boxes occur at a distance of 250-450 bp from the ATG, mostly in a O' 5' 10' 15' 20'30' 60'120'

$10 L45

Fig. 1. Coordinate response of rp gene expression to a nutritional upshift. RNA was isolated from yeast cells first grown on 25[ ethanol as a carbon course and then transferred to a medium containing 25[ glucose. Samples were taken at several time points after the shift. The Northern blot was probed with gene-specific oligonucleotides: L45 as an example of an ABFl-controUed rp gene and SIO for an RAP1controlled rp gene.

016%4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

352

TABLE I Nomenclature of abundant DNA-binding factors in yeast

Name TUF SBF-E RAP1 GRF1 TBA SUF TAF SBF-B ABF1 GFI BAF1 CP1 GFII CBP1

Ref. 7/ 24 21 22 25 13} 14 24 22 31 16 27 } 31 28

To be designated RAP1

ABF1

CP1

tandem arrangement and in both possible orientations. Deletion analyses of the promoter regions of several rp genes (commonly fused to a reporter gene) demonstrated these RPG boxes to represent upstream transcription activation sites (UAS~,,; [2,3]). Removal of RPG boxes abolishes transcription completely, though in some cases a residual transcription activity remained, due to the presence of a T stretch downstream of the boxes [5]. T stretches often occur in rp gene promoters (as well as in the promoters of many other highly expressed genes in yeast) and attribute to the strength of the UAS. A synthetic oligonucleotide harbouring the consensus RPG box sequence appeared to be sufficient to activate transcription of a heterologous gene when placed at the position where normally its own UAS is located [6]. By bandshift assays and footprint analyses, RPG boxes have been identified as the DNA binding site for an abundant protein factor, initially designated TUF [7,8], hereafter referred to as RAP1 (see below; Table I). RAP1 has an apparent molecular mass of 120 kDa in SDS gels. The geae has been isolated and sequenced and appeared to encode a protein with a predicted molecular mass of 92.5 kDa, the discrepancy probably reflecting some electrophoretic anomaly [9]. RAP1 is an acidic protein showing an unusually high content of Asn residues, but otherwise not containing characteristic protein domains. No obvious DNA binding motifs (such as helix-turn-helix, leucine zipper or Zn -finger) could be recognized, though partial proteolysis generated a fragment of 50 kDa that is still able to bind its recognition sequence [10]. Notably, no distinct transcription activation domain could be demarcated so far. Methylation interference studies indicated that all G-residues in the RPG box consensus sequence are contact points with the protein factor [11]. On the other hand, analysis of single site mutations showed that

mainly the core sequence YCYR at position 4-7 is essential for transcription activity in vivo [11]. For this group of rp genes the cis/trans combination of RPG box/RAP1 is indispensable for efficient transcription activation. Therefore it is difficult to assess whether it is sufficient for regulation in response to, e.g., a nutritional upshift. Evidence is available that RPG boxes at least mediate the enhanced transcription activation upon the transfer of yeast cells from ethanol to glucose [12]. Not all rp genes harbour RPG boxes. The genes encoding $33 and L3, for instance, contain a binding site for a second abundant yeast factor, initially called SUF [13] or TAF [14], hereafter referred to as ABF1 (to be discussed below; Table I). In the pertinent rp gene promoter regions ABF1 binds at approx. 160-200 bp from the ATG start codon, the orientation of the ABFl-responsive site upstream of the L3 gene being opposite to that in front of the $33 gene. Deletion analysis showed the ABFl-site to be part of the UAS of these genes [13,14]. Analysis of the $33 gene, however, suggested a different role of ABF1 under different growth conditions [13]. Contrary to the RPG box, the ABFl-responsive site is not a strong enhancer. The use of synthetic oligonucleotides encompassing the ABF1binding site strongly suggests that the action of ABF1 as a transcriptional activator depends upon additional factors. ABF1 is a protein with an apparent molecular mass of 130-140 kDa in SDS gels. The corresponding gene has been isolated and sequenced [15-17]. It encodes a protein with a predicted molecular mass of 81.7 kDa. A putative transcription activating domain has not been elucidated so far, but the protein does exhibit a potential DNA-binding motif, the atypical metal-binding 'finger' structure CXTHX3HX4CX4C. Comparison of the primary structures of ABF1 and RAP1 revealed a striking homology [17]. Except for the putative DNAbinding domain of ABF1, the remaining p ~ t of the protein is 40% conserved relative to RAP1. This finding suggest an evolutionary relationship between both proteins, but may also reflect an extensive functional similarity of the two factors. Methylation interference studies of the ABFI-responsive site revealed a dual binding motif, RTCRYNsACG, suggesting that the factor binds at one side of the DNA helix [18]. Consistent with this result a single mutation ACG ~ AGG appeared to prevent ABFl-binding in vitro [18] and abolishes transcription activity in vivo (Doorenbosch M.M., Mager, W.H. and Planta, R.J., unpublished data). As a matter of fact, due to the split feature of this motif the putative identity of SUF, TAF and ABF1 initially remained uncovered. Competition retardation analyses making use of oligomers, however, proved these factors very likely to be identical [18].

353 -263

-279

-245

-193

V CPI

SUF

- 177

8DUF

I -284

-232

-277

RTCACGTG GTCACGTG

ulmtrouu doleUeum

-218

-194

RTCRYNNNNNACG GTAACTTTACACG

-z7g

-236

-245

-182

ttTCaTttTCttt ctTCtTttTCcaa

-193

-177

Y

Fig. 2. Deletion analysis of the extended promoter of the L45 gene. Above, a schematic map of the L45 gene promoter is depicted, including potential protein binding sites. The upper sequences represent the corresponding consensus binding sites. Below, the result of a deletion analysis is shown. Deletions were performed up to the positions indicated in the map. The L45 genes carrying the shortened promoter regions were cloned on a multicopy yeast vector. RNA was isolated from the respective transformed cells and hybridized with L45 and $10 gene-specific probes.

Upstream of the gene encoding the acidic ribosomal protein L45, a binding site occurs for a third abundant yeast factor, CP1 (for explanation of this designation see below), giving rise to a prominent complex in bandshift assays (Kraakman, L., Mager, W.H. and Planta, R.J., unpublished data). Removal or mutation of this binding site, however, did not affect the transcription activity of the L45 gene (see Fig. 2). A second, rather weak, complex turned out to reflect the in vitro binding of ABF1 to the L45 gene promoter fragment, notably, however, to a deviating nucleotide motif: RTARYNsACG. Also for the L45 gene the ABFl-responsive site is essential for efficient transcription activation (see Fig. 2). Recently, a similar conclusion was drawn for the yeast genes encoding ribosomal proteins L2A and L2B (Bozzoni, I., personal communication). In view of the abundancy in yeast cells of both RAP1 and ABF1, identified as transcriptional activators of rp genes, it is not surprising that these factors play a part in transcription activation of other yeast genes as well. It was highly remarkable, however, to find out that these proteins fulfil very different functions in the yeast cell. RAP1 and ABF1 are general, multifunctionai proteins

RAP1 and ABF1 are not exclusively involved in the transcription activation of yeast rp genes. Many other genes including those encoding glycolytic enzymes (PGK, EN01, PYK1, ADH1), harbour RPG boxes in

their upstream activation site (Refs. cited in Refs. 11,19). In addition, the 5'-flanking sequences of many yeast genes contain an ABFl-responsive site (Doorenbosch M.M., Mager, W.H. and Planta, R.I., unpublished data), though at presently its functional significance has been established experimentally for only a limited number of these genes. The extended promoter regions of several genes, e.g., PGK, even carry both a RAP1 and an ABF1 site [19,20]. In particular those genes that are under RAPl-control can be considered as house-keeping genes, the expression of which is supposed to be regulated in a cell growth rate dependent fashion. Therefore, we decided to estimate the cellular level of RAP1 and ABF1 proteins able to bind to their DNA sites in vitro, in cells grown in ethanol and glucose as carbon source. Using S100 extracts from glucose-grown cells, the amount of complex formation turned out to be much higher than with S100 extracts from ethanol-grown cells, indicating a considerable difference in the cellular level of the DNA-binding factors. On the other hand, Northern analyses revealed hardly any differences in the cellular levels of the corresponding mRNAs (Kraakman, L., Hoekstra, R., Mager, W.H. and Planta, R.J., unpublished results). These findings suggest that either these factors are post-translationally modified to modulate their DNA-binding efficiency or, less likely, that expression of the respective genes is subject to a translational control. Surprisingly, the factors shown to serve as transcriptional activators of rp genes and many other genes in yeast, can also fulfil quite different functions in the yeast cell. First of all, the DNA regions responsible for the maintenance of the transcriptional quiescence of the silent mating type loci HML and HMR (the 'silencers') contain binding sites for RAP1, ABF1 and an ARSbinding factor [21-23]. For the actual silencing function of these multiple protein-DNA complexes additional factors, products of the SIR genes, are required [24]. In addition, RAP1 binds to telomeric sequences [22,23,25]. Telomeres contain the repeated sequence (C1_3A),, thus occasionally representing RPG-box motifs. Finally, RAP1 is required for attaching chromosomal DNA to the nuclear scaffold [26]. At present it is unknown whether a correlation exists between RAP1 transcription activation sites and chromosomeattachment sites. ABF1, in addition to being a transcriptional activator or silencer, was identified as a protein that binds specific nucleotide elements associated with autonomously replicating sequences, ARS [22]. A third abundant yeast factor, CP1, was found to associate with the conserved centromere region I, but the actual binding site occurs at many places in the yeast genome [27,28]. No activating function for this protein could be elucidated so far (see above). We have

354 summarized the various designations for RAP1, ABF1 and CP1 in Table I. Since RAP1 and ABF1 are involved in such diverse cellular processes as transcriptional regulation, replication, telomere function etc., the question arises of how their actual functional role is determined. The complicated interplay of factors involved in transcriptional silencing but also, e.g., the complicated architecture of the UAS of the PGK gene suggest that the nucleotide context of the protein binding sites and/or the interaction of RAP1 and ABF1 with additional factors will define their mode of action. Additional protein factors are involved in rp gene transcription Since transcription activation of rp genes appears to be mediated by (at least) two distinct abundant DNA binding factors, again the question arises of how coordinate regulation upon changes in growth conditions through these two general factors may be achieved. Are these factors modified in a similar and perhaps rp gene specific fashion, for instance by phosphorylation? Or do common additional factors, interacting with unidentified cis-acting DNA elements or through protein-protein contacts, play a part? Several lines of evidence suggest that, indeed, additional activating factors are involved in RAP1- and ABFl-mediated transcription activation. By ultraviolet cross-linking, proteins with a similar size were found to be associated with both RAP1 and ABF1 [14]. In addition, GCR1 deficiency was found to have a similar effect on ADH1 gene transcription as deletion of the RPG box [20]. GCR1 has been identified as a positive trans-acting transcriptional regulator of glycolytic enzymes. In gcrl- strains also the cellular level of rp59 mRNA was significantly reduced [29]. GCR1 does not affect the in vitro DNA binding activity of RAP1, but the results support the hypothesis that RAP1 may activate the transcription of glycolytic genes, and perhaps also rp genes, through the interaction with the GCR1 gene product [29]. Additional proof for the interaction with additional factors is provided by the analysis of the PGK gene. In the extended promoter of the PGK gene, in addition to an ABFl-site and an RPG-box, several so-called CTTCC blocks occur [19,20]. For these different cis-acting elements, a synergistic interaction has been proposed, since efficient transcription of the PGK gene only took place having the ABFl-site and the CTTCC block in conjunction with the RPG box [20]. With respect to the ABF1mediated transcription activation of rp genes, evidence for additional factors came forward from our studies of the $33 and L45 genes. After deletion of the ABFl-site located upstream of the $33 gene, the resulting promoter fragment still gave rise to complex formation in bandshift assays and, in addition, showed residual tran-

scription activity [13]. Notably, differences were observed dependent on the carbon source used for growth of the yeast cells. A factor predominantly present in glucose-grown cells was designated GDUF and a factor only observed in S100 extracts from ethanol-grown cells was named EDUF [13]. The binding site for GDUF was determined by footprint analysis and a subsequent methylation interference study suggested that the following bases are involved in protein binding: GAAAATGAA (Doorenbosch, M.M., Mager, W.H. and Planta, R.J., unpublished data). Making use of an oligonucleotide encompassing the GDUF-responsive site in bandshift assays of yeast protein fractions more than one protein was able to bind to this site, probably in a mutually exclusive fashion. Also downstream of the ABF1 site in the L45 gene promoter a GDUF site was found. This nucleotide element also turned out to be a weak activating site in this promoter region. Experiments are in progress to determine the possible synergistic action of the ABF1 and GDUF sites in these promoters. Recently, the synergistic action of ABF1 and factors interacting with an adjacent T-rich sequence was proposed for the DED1 gene, an essential yeast gene with unknown function, [30]. Interestingly, in this T-rich sequence a potential GDUF site occurs. Computer searches so far have elucidated only a limited number of genes harbouring the combination of an ABF1 site and a GDUF site (result not shown). We hypothesize that an additional protein factor interacting with the GDUF site is needed for efficient transcription activation of those rp genes which are activated by ABF1. Acknowledgements The authors are indebted to their collaborators at the Biochemisch Laboratorium for their research efforts and stimulating discussions. We thank Mrs. P.G. Brink for expertly preparing the typescript. References 1 Planta, R.J., Mager, W.H., Leer, R.J., Woudt, L.P., Raur, H.A. and EI-Baradi, T.T.A.L. (1986) in Structure, Function and Genetics of Ribosomes (Hardesty, B. and Kramer, G., eds.), pp. 699-718, Springer Verlag New York. 2 Mager, W.H. (1988) Biochim. Biophys. Acta 949,1-15. 3 Planta, R.J. and Mager, W.H. (1988) in Genetics of Translation (Tuite, M.F., Picard, M. and Bolotin-Fukuhara, M., eds.) NATO ASI series H, Vol. 14, pp. 117-129, Springer-Veda& 4 Leer, R.J., Van Raamsdonk-Duin, M.M.C., Mager, W.H. and Planta, R.J. (1985) Curr. Genet. 9, 273-277. 5 Rotenberg, H.O. and Woolford, J.L. (1986) Mol. Cell Biol. 6, 674-687. 6 Woudt, LP., Mager, W.H., Nieuwint, R.T.M., Wassenaar, G.M., Van der Kuyl, A.C., Murre, J.J., Hoekman, M.F.M., Brockhoff, P.G.M. and Planta, R.J. (1987) Nucleic Acids Res. 15, 6037-6048.

355 7 Huet, J., Cottrelle, P., Cool, M., Vignais, M.-L., Thiele, D., Marck, C., Buhler, J.M., Sentenac, A. and Fromageot, P. (1985) EMBO J. 4, 3539-3547. 8 Vignais, M.-L., Woudt, L.P., Wassenaar, G.M., Mager, W.H., Sentenac, A. and Planta, R.J. (1987) The EMBO J. 6, 1451-1457. 9 Shore, D. (1987) Cell 51,721-732. 10 Huet, J. and Sentenac, A. (1987) Proc. Natl. Acad. SCi. USA 84, 3648-3652. 11 Nieuwint, R.T.M., Mager, W.H., Maurer, C.T.C. and Planta, R.J. (1989) CURT.Genet. 15, 247-251. 12 Herruer, M.H., Mager, W.H., Woudt, L.P., Nieuwint, R.T.M., Wassenaar, G.M., Groeneveld, P. and Planta, R.J. (1987) Nucleic Acids Res. 15, 10133-10144. 13 Herruer, M.H., Mager, W.H., Doorenbosch, M.M., Wessels, P.L.M., Wassenaar, G.M. and Planta, R.J. (1989) Nucleic Acids Res. 17, 7427-7439. 14 Hamil, K.G., Nam, H.G. and Fried, H.M. (1988) Mol. Cell. Biol. 8, 4328-4341. 15 Rhode, P.R., Sweder, K.S., Oegema, K.F. and Campbell, J.L. (1989) Genes Dev. 3, 1926-1939. 16 Hairier, H., Kavety, B., Vandekerckhove, J., Kiefer, F. and Gallwitz, D. (1989) EMBO J. 8, 4265-4272. 17 Diffley, J.F.X. and Stillman, B. (1989) Science 246, 1034-1038. 18 Dorsman, J.C., Doorenbosch, M.M., Maurer, C.T.C., De Winde, J.H., Mager, W.H., Planta, R.J~ and Grivell, L.A. (1989) Nucleic Acids Res. 17, 4917-4923.

19 Chambers, A., Tsang, J.S.H., Stanway, C.A., Kingsman, A.J. and Kingsman, S.H. (1989) Mol. Cell. Biol. 9, 5516-5524. 20 Stanway, C.A., Chambers, A., Kingsman, A.J. and Kingsman, S.H. (1989) Nucleic Acids Res. 17, 9205-9218. 21 Brand, A.H., Mieklen, G. and Nasmyth, K. (1987) Cell 51, 709719. 22 Buchman, A.R., Kimmerly, W.J., Rine, J. and Kornberg, R.D. (1988) Mol. Cell. Biol. 8, 210-225. 23 Buchman, A.R., Lue, N.F. and Kornberg, R.D. (1988) Mol. Cell. Biol. 8, 5086-5099. 24 Shore, D., Stillman, D.J., Brand, A.H. and Nasmyth, K. (1987) EMBO J. 6, 461-467. 25 Berman, J., Tachinaba, C.Y. and Tye, B.K. (1986) Proc. Natl. Acad. Sei. USA 83, 3713-3717. 26 Hofmann, J.F., Laroche, T., Brand, G.H. and Gasser, S.M. (1989) Cell 57, 725-737. 27 Bram, R.J. and Kornberg, R.D. (1987) Mol. Cell. Biol. 7, 403-409. 28 Cai, M. and Davis, R.W. (1989) Mol. Cell. Biol. 9, 2544-2550. 29 Santangelo, G.M. and Tornow, J. (1990) Mol. Ceil. Biol. 10, 859-862. 30 Buchman, A.R. and Kornberg, R.D. (1990) Mol. Cell. Biol. 10, 887-897. 31 Dorsman, J.C., van Heeswijk, W.C. and Grivell, L.A. (1988) Nucleic Acids Res. 16, 7287-7301.

Multifunctional DNA-binding proteins mediate concerted transcription activation of yeast ribosomal protein genes.

Transcription activation of ribosomal protein genes (rp genes) in yeast is mediated through two different abundant transacting proteins, RAP1 and ABF1...
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