Volume 298, number 2,3, 203-705 FEBS 10743 0 I992 Federation of European Biochemical Societies 00145793/9Y$5.OO
Conservation
February 1992
of binding site specificity of three yeast DNA binding proteins G.H.J. Pretorius and HE Muller
D~purttncrrr of Microbiologyandl3fochtistry, Univer.dyoJrfteOrattgeFreeSme. PO Box 339, Bloenrfo,~tein 9300. Soutir Africa Received 23 December 1991 Sequence specific binding of protein extracts from I3 different yeast spccics to three oligonucleotidc probes and two points muiants derived from Succl~urorr~~ces crweisiuc DNA binding proteins were tested using mobility shift assays. The probes were high affinity binding sites forGRFl/RAPI/ AUF1 and CPl/CPFl. Most yeasts in the genus .Succkuroqms showed specific binding to all thre- probes and also displayed similar sequence requiremcnis when challenged by molar cxccsscs of mutant probes. The affinities for the probes varied amongst the other yeasts tested, but in general, CPFI binding activity was ihe most widespread, while the other two were more limited. Transcription factor; Prodn-DNA
interaction; Mobili& shift assay
1. INTRODUCTION
2. EXPERlMENTAL 2.I * Yeasrs
During the last few years, several abundant sequence specific DNA binding proteins have been identified in Smchurottzycesccrevisiae [I]. These proteins fulfill various regulatory and/or structural functions and are currently the subjects of intense investigation. Although exact structure-function relationships have been established for some of the proteins, others need further analysis. One approach to address this problem could be to investigate the occurence of selected sequence specific binding proteins in extracts from a variety of different yeasts, using as probes binding site sequences derived from S. crrevisiue. We have decided to explore this route and the yeast species were chosen to represent a range of relatedness to S. cerevisiae in order to also gain insight into evolution of DNA binding proteins. We chose three S. cerevisiae DNA binding proteins namely GRFI/RAPl [2,3], ABFl [2] and CPl/CPFl [4,5] on the basis of their abundance and well-characterised sequence requirements. For each protein we used as probe a double-stranded oligonucleotide containing a high affinity binding site (wild-type), while fol the first two proteins we also used point mutants which severaly affect their binding by the corresponding S. cerevisiue proteins. By including molar excesses of unlabeled wild-type or mutant oligonucleotides in mobility shift assays, we hoped to gain information regarding both the presence and specility of corresponding DNA binding proteins in the various yeasts.
The yeast strains used as sourse of protein extracts arc listed in Table 1and are maintained in thcculturccollestion of thisdepartment.
Cixre~porrr/e!tccu&-f~sx G.H.J. Prclorius, Department of iviicrobiolopy and Biochemistry, University ofOrangeFree State, PO Box 339, Bloemfontein 9300, South Africa. Pax: (27) (51) 474 152.
2.2, l2igorilrrfC?oridl!s The oligonucleotides were a kind gift from A.R. Duchman,Pennsylvania State University, USA [6,7]. The yeasts were grown with shaking at 30°C in 100 ml YPD broth to mid-logarithmic phase. Lysis was achieved by vortcxing with glass beads and preparation of SIODcxlracts were done as described [8]. Oligonucleotidc probes were k&ted by filling in the 5’ overhanging ends with Klenow polymurase and [“P]dATP. Fifty pup of protein extract from each yeast was used per assay, done csscntially as in Buchman et al. 121.Assays conhxd Iti cpm (O.OS~+l ng) of labeled probe, I ,~g of herring sperm DNA as non+p&fic competitor and a SO-foldmolar excess of unlabeled oligonucleotide when required. The gels (4% polyacrylamide) were dried after elcctrophorcsis and exposed to X-ray film (Crone%4). Band intensities were quantified by scanning the films with a Hocfcr GS300 dcnsitomctcr and accompanying software. The numbers in Table II wcrc calculated as follows: (S/II)x 100, where s is the fraction of radioactivity in the complexed band in the presence of unlabclcdoligonucleotidecompctitor and n is thcsame fraction in the absence of oligonucleotide competitor.
3. RESULTS AND DlSCUSSION Most yeasts in the genus Saccltarontyces (fig.1, lanes l-5) showed binding to all three targets, albeit with different affinities. In all three cases, S. dairensis, Smtisporus and S. servmzii showed similar shifts in mobility which indicate proteins of similar size, while S. kluyveri displayed a smaller shift in mobility. Competition with unlabelled wild type oligonucleotides (Table II) indicated that the binding of S. kluyveri to the RAP1 binding site (ENOl) and S. dairensis to the CPFl target (GAL2) were non-specific. Based on partial rDNA se203
Volume 298, number 2,3
FEBS LE?TERS
February 1992
Tnble I Yeast speciesand abbreviations Spccics -
Abbreviation
Strain
.Smdtarottt,wes cerevisirre Sfrcr~iturotrl,~ccs klt4yseri Saccltarotrtyces dairetrsis Saccfrf4mttt~ces hsport4s Sficcitarottr_vces serra::ii Kh4yi~erotftyces ttu4rsiutf44s Debnrytntyces ltatrsetlii Debqwntyces po~,rttorpitrrs Drbutyotttyces rttefissophiltrs Picltb sripitis Cmdida tt1ilis Schi:osrrcchoront~ccs portrbr Gnkrotqvces geotridrm Scltwattttiotttvces occitienlaiis
S. cercviskte S. khvveri S. rhi;o~sis S. trttisportrs S. ser vu:: ii K. tttar.~ifrttfrs D. htrsertii D. pol,otrorplttrs D. ttreiissopltiltrs P. stipiris C. d/is Sdti:. potttbe G. gcotricituttr Scitw. arcif~etttctlis
CSIR Y2 CBS 308X CBS 421 T CBS 398T CBS d3t IT CBS 1556 CSIR Y510 CSlR Y587 CSIR Y903 CBS 5773T A TCC 9256 CBS 356T CBS 772.71 CSIR 1’993
1
2
3
4
5
6
7
8
9 10 11 12 13 14
RAP1
ABFI
quences, Kurtzman and Robnett [9] constructed a phylogenetic tree of SaccI1arumycr3, Deboryotr~yces, Sclz~lmlnion1yce.s and Schi~osaccl!u~on?),ces.On the Sacclrrrron~ycesbranch of this tree, S. scrva:ziI’ and S. uniS,*.WMS clustered close together, with S. ciuirensis somewhat further away, while S. kluyser’i stood away from the rest of the genus. The same tendency was observed using chromosome separations [IO]and total DNA hybridization within the genus [l I]. As jugcd from the binding and competition results, K. nmxiunus is the closest relative to Saccitaronzyces, followed by D. Iwzsetzii(Fig. 1. and TRYaleII, lanes 6,7). In a recent study, Dorsman et al. [12J reported the existence of proteins in K. fads that recoignised targets for GFl and GF2 (which have subsequently been proved to be identica! to ABFl and CPF I, r[:spectively [13]). K. lcrcris and K. tmrxicmrs are so closely related that they have been synonymised in the past, although they have been separated again [14]. All three proteins analyzed in this study are multifunctional factors involved in activation/repression of transcription, regulation of replication ASwell as having structural roles. Our results indicate that the binding sites for RAP1 and ABE1 are less we!1 conserved than that of CPFl, since virtually all yeasts tested contain
CPFI
Fig. 1. Mobility=shift assays of yeast crude extracts. Three different probes were used in the horizontal panels: EN01 was used for dctecm tion of KAPl, SPT2 for ABFI and GAL2 for CPFI. F denotes the position of the free probes,
activities binding to the GAL2 oligonucleotide. Bram and Kornberg [ 151found an activity in human cells that recognised the consensus CPFl binding site at the S. cereuisiae CDEI element, which forms part of the centromere. It is therefore all the more surprising that S. khyveri and Schiz. powbe showed very little binding (Fig. 1) to the CPFI binding site, and that S. dairensis bound the target non-specifically. Very little is known about the nuclear organization of yeasts other than S. ce~cvisiue, an exception being Sdzh. powhe [16,17]. The Scltiz. ponzbe centromere is much larger and more complex than that of S. cerevisiae, and its binding site for structural and regulatory proteins may thus also be different. It is significant that the sequence preferences as judged by com-
Table II Competitive binding of yeast protein extracts to oligonucleotidcs
Yeast strain* Probe
ENOI ENOlmtA SP?‘, SPT2mtA GAL2
I
2
3
44 100 20
loo
37
99 44
98 46
100 100 66 ND NB IOU
4
5
6
7
8
23
5s
100 28
96 31
31 32 NB 100 LOO NB 33 NB 87
100 36
86 22
100 38
NB 77
95 86
9
IO
II
I2
I3
NB NB
NB NB
NB NB
NB NB
NB
70
NB
100
NB NB 54
NB NB 88
NB NB 53
NB NB 55
NB ND 72
NB NB 72
NB = no binding. * The strain numbers arc the same as in Fig. I.
204
14
Volume 298, number 2,3
FEBS LETTERS
petition with mutant oligonucleotides (Table II), are generally closely related to that found in S. cerevikue. The results obtained in this study may lead to the identification and cloning if genes coding for DNA binding proteins in a variety of yeasts, which in turn could shed light on the mechanisms involved in the evolution of these proteins and their cognate binding sites. REFERENCES [I] Verdicr, J,-M. (1990) Yeast 6, 271-297. [2] Buchmanm A.R.. Kimmcrly, W.J,, Rinc, J. and Kornbcrg, R.D. (1988) Mol. Cell Biol. 8, 210-225. [3] Shore, D. and Nasmyth. K. (1987) Cell 51, 721-732. [4] Baker, R.E., Fitzgerald-Hayes, M. and O’Brien. T.C. (1989) J. Biol. Chcm. 264, 10843-10850. [5] Mcllor, J., Jiang, W., Funk, M., Rathjcn, J., Barnes, CA., Hint, T., Hegemann, J.H. and Phillipsen, P. (1990) EMBO J. 9.40174026. [G] Buchman, A.R., Luc, N.F. and Kornbcrg, R.D. (1988) Mol. Cell Biol. 8, 5086-5099.
February 1992
[7] Buchman, A.R. and Komberg, R.D. (1990) Mol. Cell Biol. 10, 887-897. [8] Ausubcl. F.M., Brent, R., Kingston, R.E., Moore, D.D., Smith, J.A., Scidman, J.G. and Struhl, K. (1987) Current Protoeols in Molecular Biology, Wiley Interscience. New York/Chichater/ BrisbnnflotontolSingapore. [9] Kurtzman. C.P. and Robnett, C.J. (1991) Yeast 7, U-72. [IO] Coctzee, D.J., Kctck, J.L.F. and Pretorius, G.H.J. (1987) J. Microbiol. Methods 7, 219-225. [I I] Martini. A.V. and Kurtzman, C.P. (1985) fnt. J. Sys, Baet. 35, 508-5 I I, [I21 Dorsman, J.C., van Hmwyk, WC. and Grivell. L.A. (1990) Nucleic Acids Res. 18, 2769-277G. 1131Dorsman, J.C., Gozdzicka-Jozefiak, van Hceswijk, W.C. and Grivcil, L.A. (1991) Ycas~ 7, 401-412. 1141Banxtt. f.A., Payne, R.W.. and Yarrow, D. (1990) Yeasts: Characteristics and Identification, Cambridge University Press. Cambridge/New YorWPort Chcner/Mclboume/Sydney. [IS] Bram. R.J. and Kornberg, R.D. (1987) Mol. Cell Biol. 7. 403[I61 %iui. C. and Clarke, L. (1991) J. Cell Biol. 112, 191-201. [l7] Maundrcl. K. Hutchinson, A. and Shall, S. (1988) EMBO I. 7, 2203-2209.
205
Conservation
February 1992
of binding site specificity of three yeast DNA binding proteins G.H.J. Pretorius and HE Muller
D~purttncrrr of Microbiologyandl3fochtistry, Univer.dyoJrfteOrattgeFreeSme. PO Box 339, Bloenrfo,~tein 9300. Soutir Africa Received 23 December 1991 Sequence specific binding of protein extracts from I3 different yeast spccics to three oligonucleotidc probes and two points muiants derived from Succl~urorr~~ces crweisiuc DNA binding proteins were tested using mobility shift assays. The probes were high affinity binding sites forGRFl/RAPI/ AUF1 and CPl/CPFl. Most yeasts in the genus .Succkuroqms showed specific binding to all thre- probes and also displayed similar sequence requiremcnis when challenged by molar cxccsscs of mutant probes. The affinities for the probes varied amongst the other yeasts tested, but in general, CPFI binding activity was ihe most widespread, while the other two were more limited. Transcription factor; Prodn-DNA
interaction; Mobili& shift assay
1. INTRODUCTION
2. EXPERlMENTAL 2.I * Yeasrs
During the last few years, several abundant sequence specific DNA binding proteins have been identified in Smchurottzycesccrevisiae [I]. These proteins fulfill various regulatory and/or structural functions and are currently the subjects of intense investigation. Although exact structure-function relationships have been established for some of the proteins, others need further analysis. One approach to address this problem could be to investigate the occurence of selected sequence specific binding proteins in extracts from a variety of different yeasts, using as probes binding site sequences derived from S. crrevisiue. We have decided to explore this route and the yeast species were chosen to represent a range of relatedness to S. cerevisiae in order to also gain insight into evolution of DNA binding proteins. We chose three S. cerevisiae DNA binding proteins namely GRFI/RAPl [2,3], ABFl [2] and CPl/CPFl [4,5] on the basis of their abundance and well-characterised sequence requirements. For each protein we used as probe a double-stranded oligonucleotide containing a high affinity binding site (wild-type), while fol the first two proteins we also used point mutants which severaly affect their binding by the corresponding S. cerevisiue proteins. By including molar excesses of unlabeled wild-type or mutant oligonucleotides in mobility shift assays, we hoped to gain information regarding both the presence and specility of corresponding DNA binding proteins in the various yeasts.
The yeast strains used as sourse of protein extracts arc listed in Table 1and are maintained in thcculturccollestion of thisdepartment.
Cixre~porrr/e!tccu&-f~sx G.H.J. Prclorius, Department of iviicrobiolopy and Biochemistry, University ofOrangeFree State, PO Box 339, Bloemfontein 9300, South Africa. Pax: (27) (51) 474 152.
2.2, l2igorilrrfC?oridl!s The oligonucleotides were a kind gift from A.R. Duchman,Pennsylvania State University, USA [6,7]. The yeasts were grown with shaking at 30°C in 100 ml YPD broth to mid-logarithmic phase. Lysis was achieved by vortcxing with glass beads and preparation of SIODcxlracts were done as described [8]. Oligonucleotidc probes were k&ted by filling in the 5’ overhanging ends with Klenow polymurase and [“P]dATP. Fifty pup of protein extract from each yeast was used per assay, done csscntially as in Buchman et al. 121.Assays conhxd Iti cpm (O.OS~+l ng) of labeled probe, I ,~g of herring sperm DNA as non+p&fic competitor and a SO-foldmolar excess of unlabeled oligonucleotide when required. The gels (4% polyacrylamide) were dried after elcctrophorcsis and exposed to X-ray film (Crone%4). Band intensities were quantified by scanning the films with a Hocfcr GS300 dcnsitomctcr and accompanying software. The numbers in Table II wcrc calculated as follows: (S/II)x 100, where s is the fraction of radioactivity in the complexed band in the presence of unlabclcdoligonucleotidecompctitor and n is thcsame fraction in the absence of oligonucleotide competitor.
3. RESULTS AND DlSCUSSION Most yeasts in the genus Saccltarontyces (fig.1, lanes l-5) showed binding to all three targets, albeit with different affinities. In all three cases, S. dairensis, Smtisporus and S. servmzii showed similar shifts in mobility which indicate proteins of similar size, while S. kluyveri displayed a smaller shift in mobility. Competition with unlabelled wild type oligonucleotides (Table II) indicated that the binding of S. kluyveri to the RAP1 binding site (ENOl) and S. dairensis to the CPFl target (GAL2) were non-specific. Based on partial rDNA se203
Volume 298, number 2,3
FEBS LE?TERS
February 1992
Tnble I Yeast speciesand abbreviations Spccics -
Abbreviation
Strain
.Smdtarottt,wes cerevisirre Sfrcr~iturotrl,~ccs klt4yseri Saccltarotrtyces dairetrsis Saccfrf4mttt~ces hsport4s Sficcitarottr_vces serra::ii Kh4yi~erotftyces ttu4rsiutf44s Debnrytntyces ltatrsetlii Debqwntyces po~,rttorpitrrs Drbutyotttyces rttefissophiltrs Picltb sripitis Cmdida tt1ilis Schi:osrrcchoront~ccs portrbr Gnkrotqvces geotridrm Scltwattttiotttvces occitienlaiis
S. cercviskte S. khvveri S. rhi;o~sis S. trttisportrs S. ser vu:: ii K. tttar.~ifrttfrs D. htrsertii D. pol,otrorplttrs D. ttreiissopltiltrs P. stipiris C. d/is Sdti:. potttbe G. gcotricituttr Scitw. arcif~etttctlis
CSIR Y2 CBS 308X CBS 421 T CBS 398T CBS d3t IT CBS 1556 CSIR Y510 CSlR Y587 CSIR Y903 CBS 5773T A TCC 9256 CBS 356T CBS 772.71 CSIR 1’993
1
2
3
4
5
6
7
8
9 10 11 12 13 14
RAP1
ABFI
quences, Kurtzman and Robnett [9] constructed a phylogenetic tree of SaccI1arumycr3, Deboryotr~yces, Sclz~lmlnion1yce.s and Schi~osaccl!u~on?),ces.On the Sacclrrrron~ycesbranch of this tree, S. scrva:ziI’ and S. uniS,*.WMS clustered close together, with S. ciuirensis somewhat further away, while S. kluyser’i stood away from the rest of the genus. The same tendency was observed using chromosome separations [IO]and total DNA hybridization within the genus [l I]. As jugcd from the binding and competition results, K. nmxiunus is the closest relative to Saccitaronzyces, followed by D. Iwzsetzii(Fig. 1. and TRYaleII, lanes 6,7). In a recent study, Dorsman et al. [12J reported the existence of proteins in K. fads that recoignised targets for GFl and GF2 (which have subsequently been proved to be identica! to ABFl and CPF I, r[:spectively [13]). K. lcrcris and K. tmrxicmrs are so closely related that they have been synonymised in the past, although they have been separated again [14]. All three proteins analyzed in this study are multifunctional factors involved in activation/repression of transcription, regulation of replication ASwell as having structural roles. Our results indicate that the binding sites for RAP1 and ABE1 are less we!1 conserved than that of CPFl, since virtually all yeasts tested contain
CPFI
Fig. 1. Mobility=shift assays of yeast crude extracts. Three different probes were used in the horizontal panels: EN01 was used for dctecm tion of KAPl, SPT2 for ABFI and GAL2 for CPFI. F denotes the position of the free probes,
activities binding to the GAL2 oligonucleotide. Bram and Kornberg [ 151found an activity in human cells that recognised the consensus CPFl binding site at the S. cereuisiae CDEI element, which forms part of the centromere. It is therefore all the more surprising that S. khyveri and Schiz. powbe showed very little binding (Fig. 1) to the CPFI binding site, and that S. dairensis bound the target non-specifically. Very little is known about the nuclear organization of yeasts other than S. ce~cvisiue, an exception being Sdzh. powhe [16,17]. The Scltiz. ponzbe centromere is much larger and more complex than that of S. cerevisiae, and its binding site for structural and regulatory proteins may thus also be different. It is significant that the sequence preferences as judged by com-
Table II Competitive binding of yeast protein extracts to oligonucleotidcs
Yeast strain* Probe
ENOI ENOlmtA SP?‘, SPT2mtA GAL2
I
2
3
44 100 20
loo
37
99 44
98 46
100 100 66 ND NB IOU
4
5
6
7
8
23
5s
100 28
96 31
31 32 NB 100 LOO NB 33 NB 87
100 36
86 22
100 38
NB 77
95 86
9
IO
II
I2
I3
NB NB
NB NB
NB NB
NB NB
NB
70
NB
100
NB NB 54
NB NB 88
NB NB 53
NB NB 55
NB ND 72
NB NB 72
NB = no binding. * The strain numbers arc the same as in Fig. I.
204
14
Volume 298, number 2,3
FEBS LETTERS
petition with mutant oligonucleotides (Table II), are generally closely related to that found in S. cerevikue. The results obtained in this study may lead to the identification and cloning if genes coding for DNA binding proteins in a variety of yeasts, which in turn could shed light on the mechanisms involved in the evolution of these proteins and their cognate binding sites. REFERENCES [I] Verdicr, J,-M. (1990) Yeast 6, 271-297. [2] Buchmanm A.R.. Kimmcrly, W.J,, Rinc, J. and Kornbcrg, R.D. (1988) Mol. Cell Biol. 8, 210-225. [3] Shore, D. and Nasmyth. K. (1987) Cell 51, 721-732. [4] Baker, R.E., Fitzgerald-Hayes, M. and O’Brien. T.C. (1989) J. Biol. Chcm. 264, 10843-10850. [5] Mcllor, J., Jiang, W., Funk, M., Rathjcn, J., Barnes, CA., Hint, T., Hegemann, J.H. and Phillipsen, P. (1990) EMBO J. 9.40174026. [G] Buchman, A.R., Luc, N.F. and Kornbcrg, R.D. (1988) Mol. Cell Biol. 8, 5086-5099.
February 1992
[7] Buchman, A.R. and Komberg, R.D. (1990) Mol. Cell Biol. 10, 887-897. [8] Ausubcl. F.M., Brent, R., Kingston, R.E., Moore, D.D., Smith, J.A., Scidman, J.G. and Struhl, K. (1987) Current Protoeols in Molecular Biology, Wiley Interscience. New York/Chichater/ BrisbnnflotontolSingapore. [9] Kurtzman. C.P. and Robnett, C.J. (1991) Yeast 7, U-72. [IO] Coctzee, D.J., Kctck, J.L.F. and Pretorius, G.H.J. (1987) J. Microbiol. Methods 7, 219-225. [I I] Martini. A.V. and Kurtzman, C.P. (1985) fnt. J. Sys, Baet. 35, 508-5 I I, [I21 Dorsman, J.C., van Hmwyk, WC. and Grivell. L.A. (1990) Nucleic Acids Res. 18, 2769-277G. 1131Dorsman, J.C., Gozdzicka-Jozefiak, van Hceswijk, W.C. and Grivcil, L.A. (1991) Ycas~ 7, 401-412. 1141Banxtt. f.A., Payne, R.W.. and Yarrow, D. (1990) Yeasts: Characteristics and Identification, Cambridge University Press. Cambridge/New YorWPort Chcner/Mclboume/Sydney. [IS] Bram. R.J. and Kornberg, R.D. (1987) Mol. Cell Biol. 7. 403[I61 %iui. C. and Clarke, L. (1991) J. Cell Biol. 112, 191-201. [l7] Maundrcl. K. Hutchinson, A. and Shall, S. (1988) EMBO I. 7, 2203-2209.
205
