Vol. 10, No. 5
MOLECULAR AND CELLULAR BIOLOGY, May 1990, p. 2437-2441
0270-7306/90/052437-05$02.00/0 Copyright C) 1990, American Society for Microbiology
The ABF1 Factor Is the Transcriptional Activator of the L2 Ribosomal Protein Genes in Saccharomyces cerevisiae FLAVIO DELLA SETA,1'2 SILVIA-ANNA CIAFRE,2 CHRISTIAN MARCK,1 BINA SANTORO 2 CARLO PRESUTTI,2 ANDRE SENTENAC,1 AND IRENE BOZZONI2* Department de Biologie, Service de Biochemie, Centre d'Etudes Nucleaires de Saclay, 91191 Gif-sur-Yvette, France,' and Dipartimento di Genetica e Biologia Molecolare, Centro di Studio degli Acidi Nucleici del Consiglio Nazionale delle Ricerche, Universita "La Sapienza," 00185 Rome, Italy2 Received 19 September 1989/Accepted 26 January 1990
The same factor, ABF1, binds to the promoters of the two gene copies (L2A and L2B) coding for the ribosomal protein L2 in Saccharomyces cerevisiae. In vitro binding experiments and in vivo functional analysis showed that the different affinities of the L2A and L2B promoters for the ABF1 factor are responsible for the differential transcriptional activities of the two gene copies. The presence of ABF1-binding sites in front of many housekeeping genes suggests a general role for ABF1 in the regulation of gene activity.
competed almost 5- to 10-fold less efficiently than L2A for complex formation on the L2A probe, suggesting that the affinity of the L2B promoter for ABF1 is lower than that of the L2A promoter (Fig. lb and c, lanes 5 through 10). Using the probes shown in Fig. 1, we analyzed, by methylation interference and DNase footprinting, the interaction of the ABF1 factor with both the coding (probe 2) and the noncoding (probes 1 and 3) strands of the L2A and L2B promoters. For methylation interference, 60,000 cpm of the DNA probes and 3 ,ug of pSP65 DNA were incubated with 2 jig of proteins from fraction 80 and treated as described by Carnevali et al. (5); for DNase I footprinting, 10,000 cpm of the DNA probes and 3 ,ug of pSP65 DNA were incubated with 3 jig of proteins from fraction 80 and treated as described by Huet et al. (14). On the coding strand of the L2A promoter, a guanine at position -179 was a major contact point of the factor; a second guanine at position -170 was a minor contact point (Fig. 2a, lane 2). On the noncoding strand of the L2A promoter, there was another contact point at the guanine at position -169 (Fig. 2b, lane 2). Therefore, two regions 10 nucleotides apart made specific contacts with the factor, as indicated by the sequence shown in Fig. 2 (bottom left). DNase footprinting performed on complexes with probe 1 (Fig. 2a, lane 6) revealed a region of protection that included the sites identified by the methylation interference assay. These experiments confirmed the binding of ABF1 to the sequence (from positions -181 to -168) on the L2A promoter that disclosed a consensus ABF1-binding site (Table 1). A second region of DNase protection was observed around position -70 (Fig. 2a, lane 6), although no specific contacts were detected at the same position in the methylation interference analysis (lane 2). This DNase footprint was observed also when the purified ABF1 factor was used (data not shown). Figure 2c shows the DNase footprint and methylation interference analyses performed on the coding strand of the L2B promoter (probe 3; Fig. 1). A guanine residue at position -174 (lane 2) made specific contacts with the protein factor, and this position corresponded to the DNase-protected region (lane 6). The protected region is reported on the sequence shown in Fig. 2 (bottom right). This sequence was lacking an important C residue at position -184 (substituted by an A) that was always conserved in all the ABF1-binding sites identified so far (Table 1). The affinity-purified ABF1 factor interacted
The gene coding for the ribosomal protein (r-protein) L2 is present in the Saccharomyces cerevisiae genome in two copies, L2A and L2B (21), which are differentially expressed like many other duplicated yeast r-protein genes (1, 18). The L2A copy contributes 72% of the L2 mRNA present in the cell, whereas the L2B copy makes the remaining 28% (17). To define the molecular basis of the differential expression of the L2 gene copies, we undertook a comparative analysis of the cis- and trans-acting elements involved in their transcriptional activation. ABF1 factor binds to the L2A and L2B promoters. A yeast protein extract was separated on a heparin-Sepharose column (F. Della Seta et al., manuscript in preparation), and the different fractions were assayed for specific interaction with the L2A promoter. With probe 1, a specific binding activity was identified in fraction 80 (Fig. la, lane 2). This activity cofractionated with the previously characterized and purified ARS-binding factor ABF1 (7, 24; Della Seta et al., in preparation). A similar shift was obtained with the heparinSepharose fraction 80 and with the affinity-purified ABF1 factor (Fig. la, lanes 2 and 5). The affinity-purified factor used in this work (Della Seta et al., in preparation) had the characteristic electrophoretic migration properties of ABF1 and was recognized by anti-ABF1 antibodies (kindly provided by J. F. X. Diffley and B. Stillman). Competition with a 20-fold molar excess of an oligonucleotide (GTCAC TATAAACG; oligonucleotide 1) containing the ABF1 target site from the RPC40 promoter (Della Seta et al., in preparation) resulted in a total inhibition of binding (Fig. la, lane 3); no competition was observed with a 20-fold molar excess of an oligonucleotide (GTTACTATAAACG; oligonucleotide 2) containing a point mutation in the same ABF1-binding site (lane 4) known to decrease the affinity for the factor (Della Seta et al., in preparation). Upon titration of the L2A probe with increasing amounts of protein, only one retarded band was obtained (Fig. lb, lanes 2 through 4), suggesting the existence of only one binding site. Specific binding to the L2B promoter (probe 3) was obtained with the same heparinSepharose protein fraction (Fig. lc, lanes 2 through 4). Again, the same retarded band was obtained with the affinity-purified ABF1 factor (data not shown). Competition experiments between the two promoters showed that L2B *
Corresponding author. 2437
2438
MOL. CELL. BIOL.
NOTES
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PROBE t PROBE: 2
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PROBE
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FIG. 1. Band shift analysis of protein complexes with L2A and L2B promoters. A schematic representation of the different probes utilized is given at the top. Probes 1 and 2 identify the L2A promoter, and probe 3 identifies the L2B promoter. Wavy lines represent linker sequences of the vectors in which the different fragments were cloned, and the asterisks indicate the labeled end, which was the 3' terminus in all three cases. The filled box represents the region showing protein-binding activity. (a) Lanes: 1, probe 1 alone; 2, probe 1 plus 400 ng of proteins from fraction 80 of the heparin-Sepharose column and 600 ng of unspecific competitor DNA; 3, same as lane 2 plus 20-fold molar excess of oligonucleotide 1 (see the text); 4, same as lane 2 plus 20-fold molar excess of oligonucleotide 2; 5, probe 1 plus 1 ,ul of the affinity-purified ABF1 fraction. (b and c) Probes 1 and 3, respectively, were tested for binding affinity to ABF1. Probes were incubated with no protein (lanes 1) or with 20 ng (lanes 2), 200 ng (lanes 3), or 400 ng (lanes 4) of proteins from fraction 80 of the heparin-Sepharose column. The binding with 400 ng of proteins was tested for competition with 10- to 20- and with 50-fold molar excesses of cold L2A promoter (lanes 5 through 7) and L2B promoter (lanes 8 through 10).
with the two promoters to give methylation interference and DNase protection patterns that were the same as those of the partially purified factor (data not shown). The identification, as ABF1, of the factor recognizing the two ribosomal promoters was then firmly established by its binding specificity: (i) the L2 promoter binding was efficiently competed for by a well-characterized ABF1-binding site but not by the same site mutated in an important conserved base; (ii) the contact points identified by methylation interference analysis corresponded to the critical C G pairs of the consensus sequence (Table 1). A number of distinct factors, named successively SBF-B (23), ABF1 (3, 7), TAF (13), GFI (9), OBF1 (10), SUF (8), and BAFi (12), appear to recognize the same type of sequence. Based on their general sequence specificity they -
probably correspond to the same factor, although several cases of multiple factors recognizing the same sequence motif have been reported (15, 22). In vivo analysis of the L2A and L2B promoters. To investigate whether the lower affinity of the L2B promoter for ABF1 could be responsible for the lower transcriptional activity of the gene and account for the observed low L2B mRNA levels, the same DNA fragments utilized in the in vitro binding experiments (probes 1 and 3) were cloned in front of the lacZ reporter gene (Fig. 3) in plasmid YEp354ATG (obtained by insertion of SphI linkers [CATG CATGCATG] in the repaired HindIII site of plasmid YEp354 [19] to have an ATG in frame with the lacZ coding region) and assayed for transcriptional activity in vivo. Northern (RNA) blot analysis (Fig. 3) of total RNA ex-
NOTES
VOL. 10, 1990
3
1
2439
1 2 3 4 5 6
2
A 70 i
4
-175 -80 -
a
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a)
b) -181
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a"^ FIG. 2. DNase footprint and methylation interference analyses. Protein complexes obtained with probes 1 (a), 2 (b), and 3 (c) were analyzed for DNase footprinting (lanes 5 and 6) and methylation interference (lanes 1 and 2). Lanes 1 and 5 represent the free probes, and lanes 2 and 6 represent the probes reacted with the protein fraction; lanes 3 and 4 show the G and G+A sequencing reactions of the probes. The schemes alongside the footprints indicate the regions that show protection from the DNase (hashed boxes) and their positions relative to the ATG; the arrowheads indicate the positions of the G residues whose methylation interferes with the binding of the factor. These regions are reported on the sequences shown at the bottom. The asterisks indicate the labeled strands; the letters refer to the panel in which the corresponding probe was used. Positions -181 and -186 refer to the location of the ABF1 consensus sequence with respect to the ATG. Brackets indicate the DNase-resistant regions, and the arrowheads indicate the contact points.
tracted from yeast strains transformed with vector DNA (p354ATG, lane a) or with constructs containing the L2A (p354L2A, lane b) and L2B (p354L2B, lane c) promoters showed that a strong expression of lacZ was obtained with the L2A promoter; in contrast, a lower level of lacZ transcription was obtained with the L2B promoter. A difference of almost 7 times was estimated by densitometric scanning and normalization to the URA3 signals. This value almost corresponds to the difference between the binding affinities of the two promoters for the ABF1 factor in vitro (5 to 10 times). A point mutation in one of the most conserved positions of the ABF1-binding site (the C at position 3 substituted by a T), which was already known to reduce the affinity for ABF1 (3; Della Seta et al., in preparation) and which represents the major difference between the L2A and L2B ABF1-binding sites (Table 1), was introduced in plas-
mid p354L2A (p354L2AATT; Fig. 3). The level of lacZ RNA produced by this construct was significantly reduced with respect to that obtained with the wild-type promoter (Fig. 3; compare lanes b and d). The fact that a point mutation known to decrease the binding affinity for ABF1 also decreases promoter activity strongly supports the idea that ABF1 is involved in transcriptional activation of the L2A gene. By extension it is likely that ABF1 is required for transcription of the L2B gene, whose transcriptional level is severalfold lower than that of L2A. Indeed, in competition experiments the L2B ABF1-binding site was much weaker than the L2A site. Remarkably, both the L2B and the mutated L2A (L2AATT) promoters show lower transcriptional activity with respect to the wild-type L2A, and both have an ABF1 site that deviates from the consensus by a change of the C at position +3 (an A in L2B and a T in
NOTES
2440
MOL. CELL. BIOL. TABLE 1. ABF1 sites upstream yeast genes
Translation YSCRGL2 YSCRP13 SCTIF1 YSCEIF4E YSCSUP2 YSCMES1 YSCATS
YSCGDHN
-181 -186 -150 -232 -199 -223 -672 -184 -190 -173
-> ->
YSCHTS1
-169 -200 -> -318 -> -231 -277 -> -386 ->
ATCGCCTTACAG
Transcription / nucleic acids YSCPABPG
RXCDYNNNNNACG
ABF1 consensus
RKCDYNNNNNACG
ABF1 consensus
ATCACTTCGGACG GTCACTACCGACG ATCGCTGCAGACG GTCGCTTGACACG GTCACCGTATACG GTCTTCATTAACG GXCGTCGCTAACG
SCARG1
PABP
J04096 YSCCBP6 YSCCOX6 YSCCOXCH2
RPB150 RAD3
RAD5 2
YSCMSS51 YSCOMPMI1
RPC40 RPC160
YSCRIP1 SCMAS1 SCPET54
SPT2 SPT3 SSB1 PPA
-117 -301 -200 -192 -357 -220 -135 -112
-> ->
MOLECULAR AND CELLULAR BIOLOGY, May 1990, p. 2437-2441
0270-7306/90/052437-05$02.00/0 Copyright C) 1990, American Society for Microbiology
The ABF1 Factor Is the Transcriptional Activator of the L2 Ribosomal Protein Genes in Saccharomyces cerevisiae FLAVIO DELLA SETA,1'2 SILVIA-ANNA CIAFRE,2 CHRISTIAN MARCK,1 BINA SANTORO 2 CARLO PRESUTTI,2 ANDRE SENTENAC,1 AND IRENE BOZZONI2* Department de Biologie, Service de Biochemie, Centre d'Etudes Nucleaires de Saclay, 91191 Gif-sur-Yvette, France,' and Dipartimento di Genetica e Biologia Molecolare, Centro di Studio degli Acidi Nucleici del Consiglio Nazionale delle Ricerche, Universita "La Sapienza," 00185 Rome, Italy2 Received 19 September 1989/Accepted 26 January 1990
The same factor, ABF1, binds to the promoters of the two gene copies (L2A and L2B) coding for the ribosomal protein L2 in Saccharomyces cerevisiae. In vitro binding experiments and in vivo functional analysis showed that the different affinities of the L2A and L2B promoters for the ABF1 factor are responsible for the differential transcriptional activities of the two gene copies. The presence of ABF1-binding sites in front of many housekeeping genes suggests a general role for ABF1 in the regulation of gene activity.
competed almost 5- to 10-fold less efficiently than L2A for complex formation on the L2A probe, suggesting that the affinity of the L2B promoter for ABF1 is lower than that of the L2A promoter (Fig. lb and c, lanes 5 through 10). Using the probes shown in Fig. 1, we analyzed, by methylation interference and DNase footprinting, the interaction of the ABF1 factor with both the coding (probe 2) and the noncoding (probes 1 and 3) strands of the L2A and L2B promoters. For methylation interference, 60,000 cpm of the DNA probes and 3 ,ug of pSP65 DNA were incubated with 2 jig of proteins from fraction 80 and treated as described by Carnevali et al. (5); for DNase I footprinting, 10,000 cpm of the DNA probes and 3 ,ug of pSP65 DNA were incubated with 3 jig of proteins from fraction 80 and treated as described by Huet et al. (14). On the coding strand of the L2A promoter, a guanine at position -179 was a major contact point of the factor; a second guanine at position -170 was a minor contact point (Fig. 2a, lane 2). On the noncoding strand of the L2A promoter, there was another contact point at the guanine at position -169 (Fig. 2b, lane 2). Therefore, two regions 10 nucleotides apart made specific contacts with the factor, as indicated by the sequence shown in Fig. 2 (bottom left). DNase footprinting performed on complexes with probe 1 (Fig. 2a, lane 6) revealed a region of protection that included the sites identified by the methylation interference assay. These experiments confirmed the binding of ABF1 to the sequence (from positions -181 to -168) on the L2A promoter that disclosed a consensus ABF1-binding site (Table 1). A second region of DNase protection was observed around position -70 (Fig. 2a, lane 6), although no specific contacts were detected at the same position in the methylation interference analysis (lane 2). This DNase footprint was observed also when the purified ABF1 factor was used (data not shown). Figure 2c shows the DNase footprint and methylation interference analyses performed on the coding strand of the L2B promoter (probe 3; Fig. 1). A guanine residue at position -174 (lane 2) made specific contacts with the protein factor, and this position corresponded to the DNase-protected region (lane 6). The protected region is reported on the sequence shown in Fig. 2 (bottom right). This sequence was lacking an important C residue at position -184 (substituted by an A) that was always conserved in all the ABF1-binding sites identified so far (Table 1). The affinity-purified ABF1 factor interacted
The gene coding for the ribosomal protein (r-protein) L2 is present in the Saccharomyces cerevisiae genome in two copies, L2A and L2B (21), which are differentially expressed like many other duplicated yeast r-protein genes (1, 18). The L2A copy contributes 72% of the L2 mRNA present in the cell, whereas the L2B copy makes the remaining 28% (17). To define the molecular basis of the differential expression of the L2 gene copies, we undertook a comparative analysis of the cis- and trans-acting elements involved in their transcriptional activation. ABF1 factor binds to the L2A and L2B promoters. A yeast protein extract was separated on a heparin-Sepharose column (F. Della Seta et al., manuscript in preparation), and the different fractions were assayed for specific interaction with the L2A promoter. With probe 1, a specific binding activity was identified in fraction 80 (Fig. la, lane 2). This activity cofractionated with the previously characterized and purified ARS-binding factor ABF1 (7, 24; Della Seta et al., in preparation). A similar shift was obtained with the heparinSepharose fraction 80 and with the affinity-purified ABF1 factor (Fig. la, lanes 2 and 5). The affinity-purified factor used in this work (Della Seta et al., in preparation) had the characteristic electrophoretic migration properties of ABF1 and was recognized by anti-ABF1 antibodies (kindly provided by J. F. X. Diffley and B. Stillman). Competition with a 20-fold molar excess of an oligonucleotide (GTCAC TATAAACG; oligonucleotide 1) containing the ABF1 target site from the RPC40 promoter (Della Seta et al., in preparation) resulted in a total inhibition of binding (Fig. la, lane 3); no competition was observed with a 20-fold molar excess of an oligonucleotide (GTTACTATAAACG; oligonucleotide 2) containing a point mutation in the same ABF1-binding site (lane 4) known to decrease the affinity for the factor (Della Seta et al., in preparation). Upon titration of the L2A probe with increasing amounts of protein, only one retarded band was obtained (Fig. lb, lanes 2 through 4), suggesting the existence of only one binding site. Specific binding to the L2B promoter (probe 3) was obtained with the same heparinSepharose protein fraction (Fig. lc, lanes 2 through 4). Again, the same retarded band was obtained with the affinity-purified ABF1 factor (data not shown). Competition experiments between the two promoters showed that L2B *
Corresponding author. 2437
2438
MOL. CELL. BIOL.
NOTES
L2A H ine }X
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so
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PROBE t PROBE: 2
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...
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PROBE
1
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2 3 4 5
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2
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to
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b)
a)
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C)
FIG. 1. Band shift analysis of protein complexes with L2A and L2B promoters. A schematic representation of the different probes utilized is given at the top. Probes 1 and 2 identify the L2A promoter, and probe 3 identifies the L2B promoter. Wavy lines represent linker sequences of the vectors in which the different fragments were cloned, and the asterisks indicate the labeled end, which was the 3' terminus in all three cases. The filled box represents the region showing protein-binding activity. (a) Lanes: 1, probe 1 alone; 2, probe 1 plus 400 ng of proteins from fraction 80 of the heparin-Sepharose column and 600 ng of unspecific competitor DNA; 3, same as lane 2 plus 20-fold molar excess of oligonucleotide 1 (see the text); 4, same as lane 2 plus 20-fold molar excess of oligonucleotide 2; 5, probe 1 plus 1 ,ul of the affinity-purified ABF1 fraction. (b and c) Probes 1 and 3, respectively, were tested for binding affinity to ABF1. Probes were incubated with no protein (lanes 1) or with 20 ng (lanes 2), 200 ng (lanes 3), or 400 ng (lanes 4) of proteins from fraction 80 of the heparin-Sepharose column. The binding with 400 ng of proteins was tested for competition with 10- to 20- and with 50-fold molar excesses of cold L2A promoter (lanes 5 through 7) and L2B promoter (lanes 8 through 10).
with the two promoters to give methylation interference and DNase protection patterns that were the same as those of the partially purified factor (data not shown). The identification, as ABF1, of the factor recognizing the two ribosomal promoters was then firmly established by its binding specificity: (i) the L2 promoter binding was efficiently competed for by a well-characterized ABF1-binding site but not by the same site mutated in an important conserved base; (ii) the contact points identified by methylation interference analysis corresponded to the critical C G pairs of the consensus sequence (Table 1). A number of distinct factors, named successively SBF-B (23), ABF1 (3, 7), TAF (13), GFI (9), OBF1 (10), SUF (8), and BAFi (12), appear to recognize the same type of sequence. Based on their general sequence specificity they -
probably correspond to the same factor, although several cases of multiple factors recognizing the same sequence motif have been reported (15, 22). In vivo analysis of the L2A and L2B promoters. To investigate whether the lower affinity of the L2B promoter for ABF1 could be responsible for the lower transcriptional activity of the gene and account for the observed low L2B mRNA levels, the same DNA fragments utilized in the in vitro binding experiments (probes 1 and 3) were cloned in front of the lacZ reporter gene (Fig. 3) in plasmid YEp354ATG (obtained by insertion of SphI linkers [CATG CATGCATG] in the repaired HindIII site of plasmid YEp354 [19] to have an ATG in frame with the lacZ coding region) and assayed for transcriptional activity in vivo. Northern (RNA) blot analysis (Fig. 3) of total RNA ex-
NOTES
VOL. 10, 1990
3
1
2439
1 2 3 4 5 6
2
A 70 i
4
-175 -80 -
a
i:i.i.
I
.-.
.b.
i.
5-.l
*
-165
-160
0
-130
I
S
,* ;. I,
-200
.
_ :I
am
-200 - 93
am -
am6
-
-
.)
04 .
a)
b) -181
y.69
C) -186
a"^ FIG. 2. DNase footprint and methylation interference analyses. Protein complexes obtained with probes 1 (a), 2 (b), and 3 (c) were analyzed for DNase footprinting (lanes 5 and 6) and methylation interference (lanes 1 and 2). Lanes 1 and 5 represent the free probes, and lanes 2 and 6 represent the probes reacted with the protein fraction; lanes 3 and 4 show the G and G+A sequencing reactions of the probes. The schemes alongside the footprints indicate the regions that show protection from the DNase (hashed boxes) and their positions relative to the ATG; the arrowheads indicate the positions of the G residues whose methylation interferes with the binding of the factor. These regions are reported on the sequences shown at the bottom. The asterisks indicate the labeled strands; the letters refer to the panel in which the corresponding probe was used. Positions -181 and -186 refer to the location of the ABF1 consensus sequence with respect to the ATG. Brackets indicate the DNase-resistant regions, and the arrowheads indicate the contact points.
tracted from yeast strains transformed with vector DNA (p354ATG, lane a) or with constructs containing the L2A (p354L2A, lane b) and L2B (p354L2B, lane c) promoters showed that a strong expression of lacZ was obtained with the L2A promoter; in contrast, a lower level of lacZ transcription was obtained with the L2B promoter. A difference of almost 7 times was estimated by densitometric scanning and normalization to the URA3 signals. This value almost corresponds to the difference between the binding affinities of the two promoters for the ABF1 factor in vitro (5 to 10 times). A point mutation in one of the most conserved positions of the ABF1-binding site (the C at position 3 substituted by a T), which was already known to reduce the affinity for ABF1 (3; Della Seta et al., in preparation) and which represents the major difference between the L2A and L2B ABF1-binding sites (Table 1), was introduced in plas-
mid p354L2A (p354L2AATT; Fig. 3). The level of lacZ RNA produced by this construct was significantly reduced with respect to that obtained with the wild-type promoter (Fig. 3; compare lanes b and d). The fact that a point mutation known to decrease the binding affinity for ABF1 also decreases promoter activity strongly supports the idea that ABF1 is involved in transcriptional activation of the L2A gene. By extension it is likely that ABF1 is required for transcription of the L2B gene, whose transcriptional level is severalfold lower than that of L2A. Indeed, in competition experiments the L2B ABF1-binding site was much weaker than the L2A site. Remarkably, both the L2B and the mutated L2A (L2AATT) promoters show lower transcriptional activity with respect to the wild-type L2A, and both have an ABF1 site that deviates from the consensus by a change of the C at position +3 (an A in L2B and a T in
NOTES
2440
MOL. CELL. BIOL. TABLE 1. ABF1 sites upstream yeast genes
Translation YSCRGL2 YSCRP13 SCTIF1 YSCEIF4E YSCSUP2 YSCMES1 YSCATS
YSCGDHN
-181 -186 -150 -232 -199 -223 -672 -184 -190 -173
-> ->
YSCHTS1
-169 -200 -> -318 -> -231 -277 -> -386 ->
ATCGCCTTACAG
Transcription / nucleic acids YSCPABPG
RXCDYNNNNNACG
ABF1 consensus
RKCDYNNNNNACG
ABF1 consensus
ATCACTTCGGACG GTCACTACCGACG ATCGCTGCAGACG GTCGCTTGACACG GTCACCGTATACG GTCTTCATTAACG GXCGTCGCTAACG
SCARG1
PABP
J04096 YSCCBP6 YSCCOX6 YSCCOXCH2
RPB150 RAD3
RAD5 2
YSCMSS51 YSCOMPMI1
RPC40 RPC160
YSCRIP1 SCMAS1 SCPET54
SPT2 SPT3 SSB1 PPA
-117 -301 -200 -192 -357 -220 -135 -112
-> ->
