Molecular Biology of the Cell
Vol. 13, 1709–1721, May 2002
Repression and Activation Domains of Rme1p
Structurally Overlap, but Differ in Genetic
Anna Blumental-Perry,* Weishi Li,†Giora Simchen,*‡and
Aaron P. Mitchell†
*Department of Genetics, The Hebrew University, Jerusalem 91904, Israel; and†Institute of Cancer
Research and Department of Microbiology, Columbia University, New York, NY 10032
Submitted September 24, 2001; Revised January 3, 2002; Accepted January 24, 2002
Monitoring Editor: Keith R. Yamamoto
Rme1p, a repressor of meiosis in the yeast Saccharomyces cerevisiae, acts as both a transcriptional
repressor and activator. Rme1p is a zinc-finger protein with no other homology to any protein of
known function. The C-terminal DNA binding domain of Rme1p is essential for function. We find
that mutations and progressive deletions in all three zinc fingers can be rescued by fusion of RME1
to the DNA binding domain of another protein. Thus, structural integrity of the zinc fingers is not
required for the Rme1p-mediated effects on transcription. Using a series of mutant Rme1 proteins,
we have characterized domains responsible for repression and activation. We find that the
minimal transcriptional repression and activation domains completely overlap and lie in an
88-amino-acid N-terminal segment (aa 61–148). An additional transcriptional effector determinant
lies in the first 31 amino acids of the protein. Notwithstanding the complete overlap between
repression and activation domains of Rme1p, we demonstrated a functional difference between
repression and activation: Rgr1p and Sin4p are absolutely required for repression but dispensable
Control of transcription is central to the regulation of cell
achieved through the activities of two types of site-specific
DNA binding proteins: activators and repressors, and
through the action of the Mediator complex of RNA poly-
merase II, which is implicated in positive as well as negative
regulation of transcription (Myers et al., 1999). Some proteins
act as activators in one context and as repressors in another.
The precise mechanism of action of these proteins is un-
Rme1p can exert either a positive or negative effect on
gene expression. Rme1p blocks meiosis in haploid yeast cells
in response to starvation by preventing transcription of
IME1, which encodes a positive regulator of several early
meiotic genes (Kassir and Simchen, 1976; Mitchell and Her-
skowitz, 1986; Kassir et al., 1988; Kupiec et al., 1997). Rme1p
is a zinc-finger protein with no other similarity to known
repressor proteins. Rme1p binds to two sites that lie at
?2030 and ?1950 bp upstream of the IME1 gene (Covitz and
Mitchell, 1993; Shimizu et al., 1998). The binding sites are
contained within a 404-bp DNA segment called the repres-
sion cassette (RC). The RC confers Rme1p-dependent repres-
sion to the heterologous CYC1 promoter when inserted ad-
jacent to the CYC1 upstream activating sequence (UAS)
(Covitz and Mitchell, 1993; Shimizu et al., 1997b). It has been
proposed that Rme1p represses transcription through an
activator exclusion mechanism. Transcriptional activators
Hap1p and Hap2p are unable to bind to their DNA recog-
nition sites at a Rme1p-repressed hybrid promoter (Shimizu
et al., 1997b).
Repression by RME1 depends on Rgr1p and Sin4p (Covitz
et al., 1994). These proteins are best known as subunits of the
Mediator complex of RNA polymerase II (Li et al., 1995;
Carlson, 1997). These subunits of the Mediator were identi-
fied in several additional genetic screens as negative effec-
tors of transcription (Sakai et al., 1990; Stillman et al., 1994;
Jiang et al., 1995). However, they are also required for max-
imal induction of particular sets of genes. In addition, mu-
tations in RGR1 and SIN4 confer phenotypes common to
histone and spt mutations, namely, decreased plasmid su-
perhelicity and activation of UAS-less promoters (Jiang and
Stillman, 1992; Jiang et al., 1995), suggesting that the genes
are involved in determining chromatin structure.
‡Corresponding author. E-mail address: firstname.lastname@example.org.
Abbreviations used: RC, repression cassette; UAS, upstream
activating sequence; GBD, GAL4 DNA binding domain; 3-AT,
© 2002 by The American Society for Cell Biology1709
When an Rme1p binding site is situated 5? of CLN2 or
other reporter genes, it can also activate transcription (Toone
et al., 1995). Activation or repression depends on the context
and flanking regions of the binding site: the presence of RC
causes repression, and the absence of RC causes activation
(Covitz and Mitchell, 1993).
All rme1 mutations obtained so far affect zinc fingers and
confer deficiencies in both repression and activation (Covitz,
1993). Thus, it is not clear whether zinc-finger function is
restricted to DNA binding or whether zinc fingers partici-
pate in repression/activation as well. Mammalian YY1 pro-
tein is a precedent for a later model: it is capable of either
activating or repressing transcription. The repression do-
main of YY1 is embedded within the zinc-finger regions,
although the normal structure of zinc fingers is not required
for repression (Bushmeyer et al., 1995).
The following structure-functional analysis of Rme1p was
performed to delineate the repression and activation do-
mains of Rme1 to see whether or not they overlap and to test
whether the role of the zinc fingers is restricted to DNA
binding. We show that Rme1p can be dissected into two
domains: a minimal transcriptional effector domain, which
resides in an 88-amino-acid segment (aa 61–148) at the N-
terminus of the protein; and the C-terminal DNA binding
domain, which can be replaced by other DNA binding do-
mains. Thus, zinc-finger integrity is not required for either
activation or repression by the effector domain. Additional
effector determinants exist within the first 31 amino acids of
Rme1p. Thus, the Rme1p effector domain is composed of
multiple subdomains that contribute synergistically to effi-
cient repression/activation. Although the repression and
activation domains of Rme1p overlap, only repression, not
activation, depends on Rgr1p (Covitz et al., 1994) and Sin4p.
MATERIALS AND METHODS
Growth Media, Strains, and RME1 Alleles
Yeast cells were grown and media were prepared according to
standard techniques (Rose et al., 1990). Yeast strains were isogenic to
SK-1 (Kane and Roth, 1974); genotypes are listed in Table 1.
The mutations gal80::LEU2, rme1?5::LEU2, IME2-lacZ-URA3, and
(Neigeborn and Mitchell, 1991; Covitz and Mitchell, 1993; Su and
All lexA-RME1 and truncated RME1 alleles were generated by
integrating the corresponding plasmid at the TRP1 locus of strain
AMP1615 or AMP714. The integration plasmid was digested with
Bsu36I to target the integration at the chromosomal TRP1 locus. The
resulting transformants were purified as single colonies. All inte-
grations were confirmed by Southern analysis.
Expression of the lexA-Rme1p derivatives was scored by Western
analysis with anti-lexA antibodies. By this criterion, all lexA-Rme1p
derivatives were expressed at similar levels.
Expression of N-terminal deletions of Rme1p was confirmed by
comparing the phenotypes of GAL80 strains, in which there is no
expression of rme1 alleles, and gal80::LEU2 strains, in which the rme1
alleles are expressed.
The plasmid carrying PGAL1-lexA-RME1 (pWL126) was constructed
as follows. The RME1 coding sequence flanked by BamHI sites was
amplified by PCR using oligos RME1–5?-Bam and RME1–3?-Bam
(see below). The PCR product was ligated into the EcoRV site in
pBS-SK to generate pWL105. A BamHI-BamHI fragment containing
RME1 ORF was excised from pWL105 and inserted into the BamHI
site in pBTM116 (Ruden et al., 1991) to generate pWL107. This
resulted in a fusion of lexA (1–200) to the Rme1p start codon. A
HindIII-SalI fragment containing lexA-RME1 was inserted between
the HindIII-SalI site in pSV150 (Vidan and Mitchell, 1997) to gener-
ate pWL123 with lexA-RME1 driven by the Gal1 promoter. A PstI-
SalI fragment containing PGAL1-lexA-RME1 from pWL123 was in-
serted into PstI-SalI site in pRS304 (Sikorski and Hieter, 1989).
The PGAL1-lexA plasmid (pWL137) was constructed as follows. A
HindIII-SalI fragment containing lexA (1–200) from pBTM116 (Ru-
den et al., 1991) was cloned between the HindIII-SalI sites in pSV150
to produce pWL133. Then, PGAL1-lexA from pWL133 was excised
and moved into the TRP1 integration vector pWL131 (pRS304 with
the BamHI site removed by filling in).
The PGAL1-lexA-rme1–213 plasmid (pWL139) carries the previ-
ously characterized RME1 zinc-finger mutation (Covitz et al., 1991).
The plasmid was derived from pWL126 by site-directed mutagen-
esis using oligos RMESER213 (Covitz et al., 1991).
ClaX plasmids were generated by site-directed mutagenesis using
pWL126 as a template. Clones were screened by ClaI digest, and the
candidates were sequenced to confirm the existence of the mutation.
Deletions between ClaI sites were used to create deletion derivatives
such as PGAL1-LexA-rme1?14148–300.
The plasmids carrying N-terminal deletions of rme1 were con-
structed as follows. To obtain the truncated version of Rme1p under
control of the PGAl1promoter, pWL126 derivatives with the appro-
priate ClaI site were digested with the restriction enzymes PstI and
ClaI. This removed PGAL1-lexA and unwanted parts of RME1. Next,
the remaining plasmids were ligated to the PstI-HindIII fragment of
plasmid pSV150 (Vidan and Mitchell, 1997), carrying PGAL1. The
ligation was performed using a HindIII/ClaI, which introduced
Ser-Pro-His6at the beginning of each protein. The amino acids
serine and proline were chosen because they are the first amino acids
GTCACCGCACCACCACCATCATCATAT-3?; HIII-ClaI antisense: 5?-
Plasmid pLS312S?SS carries the ?UAS-CYC1-lacZ reporter gene
(Guarente and Mason, 1983). Plasmids PAC153–4 and PAC110–6
carry RC-CYC1-lacZ and RRE-CYC1-lacZ reporter genes, respec-
tively (Covitz and Mitchell, 1993), and plasmid pSV152 carries lexA
sites inserted upstream of the ?UAS-CYC1-lacZ (Vidan and Mitch-
ell, 1997) (Figure 1B).
The plasmids to test repression were pBS8, pBS9, and pBS9-LexA
(Figure 1B). They were derived from PAC153–4 by site-directed
mutagenesis. The plasmid pBS8 was created using the bottom-
strand oligonucleotide IME1-R23 (Covitz and Mitchell, 1993). This
resulted in a deletion of sequences from ?2044 to ?2025 bp and
inserted a C to generate a SalI restriction site instead of the Rme1
binding site at ?2030 bp. The plasmid pBS9, which carries muta-
tions of both Rme1 sites, was created using oligonucleotides IME1-
R23 and IME1-BS1 5?-ATTTTATGCTCCCGGGGTACAGC-3?. The
IME1-BS1 replaces the Rme1 binding site at ?1956 to ?1951 bp
upstream of IME1 with 5?-CCGGGA-3? and generates a SmaI re-
striction site instead of an Rme1 site.
The pBS9-LexA reporter plasmids were created by site-directed
mutagenesis of pBS9 using an oligonucleotide containing the lexA
binding site: 5?-TCGAGTACTGTATGTACATACAGTAC-3? (Brent
and Ptashne, 1984). The resulting plasmids pBS9-LexA1 and pBS9-
LexA3 contain one or two lexA binding sites in place of the Rme1
binding site at ?1950.
Modifications in the IME1 Upstream Region
Three types of modification were done. In all cases, the URA3 gene
was placed upstream of ?2146 bp (Figure 1A). In the first case, the
RC was left intact. In the second case, both Rme1 binding sites were
destroyed within the RC, generating an IME1 allele that was not
A. Blumental-Perry et al.
Molecular Biology of the Cell1710
repressible by Rmep1 (nonRme1-IME1 allele). In the third, both Rme1
binding sites were destroyed, and the ?1950 binding site was replaced
by the lexA site, generating the lexO-IME1 allele (Figure 1A).
The modifications were introduced by transforming yeast with
PCR products. The PCR product contained the URA3 gene, fol-
lowed by the desired type of RC. These were flanked by sequences
homologous to the native sequences flanking the RC in the IME1-
upstream region (60 bp on each side of the PCR product). The
plasmids PAC153–4 (Covitz and Mitchell, 1993), pBS9, and pBS9-
LexA were templates for PCR, using primers ime1–2207-yep2 5?-
and ime1–1747 5?-GGCCAAAAAATAGTTCAAATT-3?. Correct in-
tegration was confirmed by Southern analysis and by PCR using
URA3 and IME1-R6 (Covitz, 1993) as primers. PCR products were
digested with the restriction enzymes SmaI and SalI to verify the
presence of the RC with both Rme1 sites destroyed and with SspBI to
verify the presence of the lexA site at position ?1950 bp (Figure 1B).
To test different PGAL1-lexA-rme1 derivatives for their ability to
repress through the lexA binding site, strains marked by the URA3
gene and bearing modified RC were crossed to strain AMP108
GAL80? ura3. Diploids were sporulated, spores were dissected, and
Table 1. Yeast strains used in this study
? GAL80 RME1
a his3 met4
? his4-G IME2-lacZ-URA3
? rme1::PGAL1-S53-RME1::TRP1 sin4?::TRP1 arg6
? rme1::PGAL1-S53-RME1::TRP1 his3?SK rgr1-100
a/? trp1::PGAL1-lexA-rme1-213::TRP1/trp1 lexO-IME1/ime1?12-TRP1 IME2-lacZ-URA3/IME2 his3/HIS3 met4/MET4
a/? trp1::PGAL1-lexA-RME1::TRP1/trp1 lexO-IME1/ime1?12-TRP1 IME2-lacZ-URA3/IME2 his3/HIS3 met4/MET4
a/? trp1::PGAL1-lexA::TRP1/trp1 lexO-IME1/ime1?12-TRP1 IME2-lacZ-URA3/IME2 his3/HIS3 met4/MET4
a/? trp1::PGAL1-lexA-rme1-?179-300::TRP1/trp1 met4/MET4 lexO-IME1/ime1?12-TRP1 IME2-lacZ-URA3/IME2 his3/HIS3
a/? trp1::PGAL1-lexA-rme1-?148-179::TRP1/trp1 met4/MET4 lexO-IME1/ime1?12-TRP1 IME2-lacZ-URA3/IME2 his3/HIS3
a/? trp1::PGAL1-lexA-rme1-?120-179::TRP1/trp1 met4/MET4 lexO-IME1/ime1?12-TRP1 IME2-lacZ-URA3/IME2 his3/HIS3
a/? trp1::PGAL1-lexA-rme1-?148-300::TRP1/trp1 met4/MET4 lexO-IME1/ime1?12-TRP1 IME2-lacZ-URA3/IME2 his3/HIS3
a/? trp1::PGAL1-lexA-rme1-?31-90::TRP1/trp1 met4/MET4 lexO-IME1/ime1?12-TRP1 IME2-lacZ-URA3/IME2 his3/HIS3
a/? trp1::PGAL1-lexA-rme1-?31-120::TRP1/trp1 met4/MET4 lexO-IME1/ime1?12-TRP1 IME2-lacZ-URA3/IME2 his3/HIS3
a/? trp1::PGAL1-lexA-rme1-?210-300::TRP1/trp1 met4/MET4 lexO-IME1/ime1?12-TRP1 IME2-lacZ-URA3/IME2 his3/HIS3
a his3 met4 trp1::PGAL1-rme1?1-31::TRP1
a/? trp1::PGAL1-rme1?1-31::TRP1/trp1 IME2-lacZ-URA3/IME2 his3/HIS3 his4-G/HIS4 met4/MET4
a his3 met4 trp1::PGAL1-rme1?1-61::TRP1
a/? trp1::PGAL1-rme1?1-61::TRP1/trp1 IME2-lacZ-URA3/IME2 his3/HIS3 his4-G/HIS4 met4/MET4
a his3 met4 trp1::PGAL1-rme1?1-90::TRP1
a/? trp1::PGAL1-rme1?1-90::TRP1/trp1 IME2-lacZ-URA3/IME2 his3/HIS3 his4-G/HIS4 met4/MET4
a his3 met4 trp1::PGAL1-rme1?1-120::TRP1
a/? trp1::PGAL1-rme1?1-120::TRP1/trp1 IME2-lacZ-URA3/IME2 his3/HIS3 his4-G/HIS4 met4/MET4
a his3 met4 trp1::PGAL1-rme1?1-148::TRP1
a/? trp1::PGAL1-rme1?1-148::TRP1/trp1 IME2-lacZ-URA3/IME2 his3/HIS3 his4-G/HIS4 met4/MET4
a/? trp1::PGAL1-lexA-rme1-Cla179::TRP1/trp1 met4/MET4 lexO-IME1/ime1?12-TRP1 IME2-lacZ-URA3/IME2 his3/HIS3
a/? trp1::PGAL1-lexA-rme1-Cla239,269::TRP1/trp1 met4/MET4 lexO-IME1/ime1?12-TRP1 IME2-lacZ-URA3/IME2 his3/HIS3
a trp1-901 leu2-3,112 ura3-52 his3-2000 gal4? gal80? LYS::GAL1-HIS3 GAL2-ADE2 GAL7-lacZ RME1 (James et al., 1996)
All the strains are SK-1 derivatives and carry the mutations leu2::hisG, trp1::hisG, lys2 ura3, ho::LYS2, gal80::LEU2, and rme1?5::LEU2, except
where indicated otherwise.
Effector Domains of Rme1p
Vol. 13, May 2002 1711
GAL80? URA3 segregants with the modified RC were obtained.
These were used to transfer RC modification to any other desired
strain. The presence of the modified RC was verified by PCR after
The plasmid pGBDU-C1 (James et al., 1996), which contains the
GAL4 DNA binding domain, was used to check the activation
ability of different domains of Rme1p. The plasmids pGBDU-C1-
rme1–90-210, pGBDU-C1-rme1–120-210, pGBDU-C1-rme1–148-210
were constructed by the following steps.
The desired part of the RME1 coding sequence, flanked by a
BamHI site at its 5? end and by SalI at its 3? end, was amplified by
PCR. Oligonucleotides for the upper strand were BamHI-rme1–90:
5?-GCAGGATCCGGTACAGCACCTCAATTACGG-3?, for pGBDU-
C1-rme1–90-210; BamHI-rme1–120: 5?-GCAGGATCCAATTATG-
TG-3?, for pGBDU-C1-rme1–148-210.
The rme1–210-SalI 5?-GTCGTCGACTTGCTCTATGGGACACT-
TACA-3? primer was used as a bottom-strand oligonucleotide.
Next, the PCR product was ligated into the EcoRV site in pBS-SK.
Third, a BamHI-SalI fragment containing part of the RME1 ORF was
excised from pBS-SK-rme1 and inserted between the BamHI-SalI
sites in pGBDU-C1 to generate an in-frame fusion of the GAL4
binding domain and part of RME1.
To construct plasmid pGBDU-C1-rme1–61-148, the rme1–61-148
was removed from the appropriate pWL126 derivative by ClaI
digestion and then ligated into the ClaI site of plasmid pGBDU-C1.
To create in-frame fusions of Rme1-?31–90p and Rme1?31–120p
to the GAL4 DNA binding domain, the RME1-?31–90 and RME1-
?31–120 alleles were amplified from plasmid templates by PCR
using the following primers, which introduced BamHI sites on both
sides of RME1: RME-5?-Bam primer: 5?-GCAGGATCCTTATGT-
CACCGTGTTATGG-3?; RME-3?-Bam primer: 5?-ACAGGATCCA-
The PCR products were cloned into vector pGEM-T (Promega).
BamHI fragments containing rme1-?31–90 and rme1-?31–120 were
excised from pGEM-T-rme1 and were then ligated into pGBU-C2 at
the BamHI site, resulting in in-frame fusion of rme1-?31–90 and
rme1-?31–120 to the GAL4-DNA binding domain.
?-Galactosidase and Sporulation Assays
The liquid ?-galactosidase assays were conducted as described else-
where (Covitz and Mitchell, 1993). For liquid sporulation assays,
cells were grown for 24–29 h at 30°C in synthetic medium contain-
ing 0.5% glucose and lacking uracil, filtered, washed once in water,
and transferred at the same cell density to 2% potassium acetate
supplemented with lysine. After 24 h at 30°C, half of each culture
was taken for IME2-lacZ assays to monitor IME1 regulation (Smith
et al., 1990). The level of sporulation was scored by counting the
number of asci per 200 cells after 2 d in 2% potassium acetate liquid
medium or on standard Spo plates (Rose et al., 1990). The reported
values are the average of at least three determinations from three
independent transformants. ?-Galactosidase was measured in per-
meabilized cells as previously described (Smith et al., 1990).
Rme1p Effector Domain Acts Independently from the
The Rme1p zinc fingers are located in the C-terminal part of
the protein. To study the repression and activation functions
of Rme1p independently of its DNA binding activity, RME1
was fused to the lexA DNA binding domain (1–200 bp). The
ability of fusion protein lexA-Rme1p to repress and to acti-
vate transcription was tested via both the Rme1 binding site
and the lexA binding site using different reporter genes that
carried wild-type or altered RC from the 5? region of the
tural modifications of the IME1
upstream region (A) and plas-
mids used in this study (B). The
diagram shows Rme1p binding
sites, the UAS, IME1, and URA3
coding regions (labeled rectan-
gles), and RNA start sites (?280
to ?210). Small white rectangles
indicate the Rme1 binding sites.
Small black rectangles indicate
the mutations in the Rme1 bind-
ing sites. Arrow indicates the
lexA operator. End points are
numbered with respect to the
IME1 translation start.
Schematic of struc-
A. Blumental-Perry et al.
Molecular Biology of the Cell 1712
IME1 gene (Figure 1). To repress via the Rme1 binding site,
lexA-Rme1p needs both the repression and DNA-binding
domain from Rme1p. To test repression via the Rme1 site,
we compared ?-galactosidase activity expressed from plas-
mid pBS9, which lacks Rme1p sites, and plasmid pBS8,
which has an intact RC. To repress via the lexA site, lexA-
Rme1p needs only the repression domain of Rme1p. To test
repression via the lexA site, we compared ?-galactosidase
activity from a plasmid that lacks the lexA sites, pBS9, and
pBS9-lexA, which contains the lexA site within the RC (Fig-
ure 1B). Figure 2A shows that lexA-Rme1p can repress via
both the Rme1 and lexA sites. Next, ClaI restriction site
insertion mutations, which disrupt the integrity of the zinc
fingers, were created at four places in the C terminus of the
RME1 coding region by site-directed mutagenesis of lexA-
RME1. We will refer to these alleles as lexA-RME1-ClaX,
where X indicates the number of the amino acid after which
the insertion occurred. lexA-RME1-Cla179, lexA-RME1-
Cla210, and lexA-RME1-Cla269 carry insertions that disrupt
zinc-finger structures, lexA-RME1-Cla239 carries the inser-
tion between the second and the third zinc fingers. All these
mutant derivatives (Figure 2A) failed to repress via the
Rme1p sites located in the promoter of reporter plasmid
pBS8. Nevertheless, all four Cla mutants retained the wild-
type ability to repress via the lexA site located in the pro-
moter of reporter plasmid pBS9-lexA. In addition, Rme1–
213p, the zinc-finger mutant that has been shown to be
incapable of binding to the Rme1 sites (Covitz and Mitchell,
1993; Shimizu et al., 1997b), and Rme1?210–300p, with the
last two zinc fingers deleted, display wild-type levels of
repression through the lexA site.
To test whether repression of reporter plasmids by lexA-
Rme1p reflects repression by Rme1p in the natural context of
its DNA binding site, we examined repression of both the
wild-type and a modified IME1 chromosomal region. The
wild-type IME1 has the natural Rme1p binding sites, so
lexA-Rme1p derivatives must have both functional DNA
binding and repression domains from Rme1p to exert re-
pression of IME1. The modified IME1 has a deletion of the
two Rme1p sites and an insertion of a single lexA site at
position ?1950 bp. We call this altered allele lexO-IME1
(Figure 1A). Thus, lexA-Rme1p derivatives need to have
only a functional repression domain from Rme1p to repress
lexO-IME1. We assayed expression of IME1 and lexO-IME1
by sporulation ability and by expression of an ime2-lacZ
meiotic reporter gene (Smith et al., 1990). To permit expres-
sion of lexA-Rme1p hybrid proteins in sporulating cells, the
proteins were expressed from the GAL1 promoter in dip-
loids homozygous for a gal80 mutation. This genotype
causes high-level GAL1 promoter activity in sporulation me-
dium even without the addition of galactose. Our assays of
sporulation in strains expressing lexA-Rme1p derivatives
are shown in Figure 3. None of the mutant lexA-Rme1p
derivatives repressed the natural IME1 locus. However, all
of the insertion and deletion derivatives with perturbations
of the zinc-finger region repressed lexO-IME1 and thus
blocked sporulation and ime2-lacZ expression (Figure 3A).
Although lexA-Rme1–213p and lexA-Rme1-Cla239-Cla269p
repress less efficiently than wild-type, the Rme1p derivative
carrying the deletion of the two last zinc fingers represses
very efficiently. Therefore, these mutations might interfere
with the protein secondary structure and not with repres-
sion itself. In conclusion, this analysis confirms that the
structural integrity of Rme1p zinc fingers and the last two
zinc fingers are needed only for DNA binding, not for re-
Activation ability of the same lexA-Rme1p derivatives
was assayed by the ability to activate transcription of an
UAS-less reporter gene with the Rme1 site (pAC110–6) or a
reporter with the lexA site replacing the UAS (pSV152)
(Figure 1B). All the mutants were able to activate only via
the lexA site (Figure 4). We conclude that structural integrity
of the Rme1p zinc-finger region is not required for either
repression or activation.
Rme1p Effector Domain Lies in the N-Terminus of
C-Terminal Extent of the Effector Domain
date the role of the N-terminal region of Rme1p in repres-
sion and/or activation, a lexA-Rme1p derivative lacking
amino acids 4–179 was constructed. This mutant protein
was defective in both activation and repression in assays of
either the Rme1 or lexA binding sites (Figures 2B and 4).
Therefore, the N-terminal region of Rme1p is required for
both activation and repression. To determine the C-terminal
boundary of the repression domain, we created increasingly
large deletions of the C-terminal part of the protein and
various deletions internal to the zinc fingers. These deletions
were tested for their ability to repress expression from re-
porter plasmid and to repress sporulation (Figures 2B and
3B). All these alleles failed to repress expression from the
reporter plasmid and sporulation via the Rme1 site. Via the
lexA site, lexA-Rme1?210–300p repressed efficiently (it
showed 8.9-fold repression and allowed only 1.2% sporula-
tion). However, lexA-Rme1?179–300p was partially defec-
tive in repression of lexA-IME1 (permitting 16% sporulation,
with 2.9-fold repression), whereas lexA-Rme1?148–300p
showed a significant reduction in repression ability (46%
sporulation, with 1.4-fold repression). Therefore, residues
179–210, which lie within the first zinc-finger domain, may
contribute to repression. This region is composed of charged
and hydrophobic amino acids. It contains a long stretch of
hydrophobic residues at 187–197 (FATLVEFAAHL). More-
over, this region is predicted to form an ?-helix with very
prominent clusters of hydrophobic amino acids at both sides
of the helix.
To elucidate the role of amino acids 120–179 in repression,
two additional deletion derivatives were tested for their
ability to repress via the lexA site. Figures 2B and 3B show
that lexA-Rme1-?148–179p repressed very efficiently (5.6-
fold repression, 2.6% sporulation), whereas a protein with a
deletion of 28 more amino acids (lexA-Rme1-?120–179p)
repressed only weakly (1.7-fold repression, 23% sporula-
tion). These results indicate that the amino acid residues
120–148 are required for repression activity, whereas amino
acids 148–179 are not.
To determine the C-terminal boundary of the activation
domain, the same lexA-Rme1p derivatives were tested for
their ability to activate transcription of the reporter plas-
Rme1?210–300, lexA-Rme1?179–300, and lexA-Rme1?148–
300 were unable to activate transcription of the reporter gene
via the Rme1 site, whereas they activated via the lexA site.
Effector Domains of Rme1p
Vol. 13, May 2002 1713
(B). A schematic of lexA-Rme1p is shown at the top. The shaded regions represent zinc-finger like domains. Numbers below the Rme1p
derivatives indicate positions (in amino acids) of insertion mutation or site of other alteration. Rme1 site repression fold was calculated as
?-galactosidase units produced by cells with pBS9 (carrying RC that lacks Rme1 sites) divided by ?-galactosidase units of cells with pBS8
(carrying RC with Rme1 site). lexA site repression was calculated as ?-galactosidase units of pBS9 divided by ?-galactosidase units of
pBS9-lexA (carrying RC with lexA site in place of Rme1 site). (C) Western blot showing the expression of lexA-Rme1p derivatives in
transformed yeast cells.
Properties of lexA-Rme1p derivatives carrying different alterations in the zinc fingers (A) and in regions proximal to zinc fingers
A. Blumental-Perry et al.
Molecular Biology of the Cell1714
This again demonstrates that amino acids 148–300 are re-
quired for DNA binding and not for activation. Activation of
transcription was more efficient by lexA-Rme1?210–300 and
lexA-Rme1?179–300 than by lexA-Rme1?148–300. This can
mean that amino acids 148–179 contribute to but are not
necessary for activation. Thus, the C-terminal boundary of
the minimal activation domain lies proximal to amino acid
N-Terminal Extent of the Effector Domain
N-terminal boundary of the region required for repression,
we constructed six ClaI restriction site insertion mutations at
30 codon intervals in the N-terminus. Deletions between
these insertions were then constructed and assayed for re-
pression of IME1 by sporulation and by ime2-lacZ expression
(Figure 5). Rme1?1–31p and Rme1?1–61p repressed almost
as efficiently as wild-type Rme1p. However, Rme1?1–90p,
Rme1?1–120p, and Rme1?1–148p had little or no ability to
repress. Therefore, amino acids 1–60 of Rme1p are dispens-
able for repression, and the region between amino acids 61
and 89 contains the N-terminal boundary of the repression
To map the
domain. Together with data from the previous section, our
results indicate that amino acids 61–148 of Rme1p are re-
quired for full repression. In agreement, the protein deleted
for 61–148 amino acids is completely incapable of repression
(Figure 2B). Repression and activation may be entirely inde-
pendent functions, involving different domains of Rme1p.
Alternatively, they may be interdependent and reside in the
same region of the protein. To distinguish between these
alternatives, we tested the series of N-terminal Rme1p dele-
tion derivatives for activation ability (Figure 5). We saw a
gradual reduction in activation ability of different N-termi-
nal deletions of Rme1p. Proteins deleted for 30 or 60 resi-
dues retained a strong ability to activate transcription; pro-
teins deleted for more than 90 residues did not possess
activation ability. Therefore, the minimal activation and re-
pression functions of Rme1p are independent of amino acids
1–60 and depend on residues between 61 and 89. Together
with the results presented above, our data show that both
activation and repression by Rme1p depend on amino acids
fingers (A) and alterations in the regions of RME1 that are proximal to zinc-finger regions (B). The Rme1 site repression was tested by the
ability of different rme1 derivatives to repress sporulation in gal80? diploid strains carrying the wild-type RC. The lexA site repression was
tested by the ability of different rme1 derivatives to repress sporulation via the lexA site placed at position ?1950 bp in RC. Sporulation was
scored after 48 h in liquid sporulation medium. IME2-LacZ expression was calculated after 24 h in sporulation medium.
The effect on repression of the IME1 chromosomal region by lexA-Rme1p derivatives carrying different alterations in the zinc
Effector Domains of Rme1p
Vol. 13, May 20021715
To confirm the mapping of the Rme1p minimal activation
domain, we assayed activation by fusions of RME1 N-ter-
minal segments to the GAL4 DNA binding domain (GBD).
Activation ability was determined by expression of GAL1-
HIS3 and GAL1-ADE2 reporter genes (James et al., 1996),
which allowed the cells to grow on media lacking histidine
or adenine, respectively. Figure 6 shows that Rme1p amino
acids 61–148 were sufficient to activate both reporter genes,
whereas residues 90–210 activated transcription of only one
reporter gene (HIS3), and did so weakly. The addition of 5
mM 3-aminotriazole (3-AT) completely inhibited the ability
of cells carrying amino acids 90–210 of Rme1p to grow on
synthetic medium lacking histidine. The shorter segments,
with amino acids 120–210 and 148–210, did not activate
either reporter. These results indicate that Rme1p residues
61–148 comprise a transcriptional activation domain, which
can be transferred to a heterologous DNA-binding domain.
Thus, we have demonstrated that the 88 amino acids 61–148
(B). Activation ability was determined by liquid ?-galactosidase assays using pAC110–6 as a reporter for the Rme1 site and pSV152 as a
reporter for the lexA site activation.
Activation by lexA-Rme1p derivatives carrying different alterations in the zinc fingers (A) and in regions proximal to zinc fingers
A. Blumental-Perry et al.
Molecular Biology of the Cell1716
of Rme1p contain a potent effector of transcription function
that confers activation and, possibly in cooperation with
amino acids 179–210, repression of high-level transcription.
For certain activators, the minimal activation domain is
not always the only region of the protein that possesses the
ability to affect transcription (Hope et al., 1988; Drysdale et
al., 1995; Jackson et al., 1996). In such cases, high-level acti-
vation can occur with only a portion of minimal activation
domain if other parts of the protein contain functionally
redundant activation subdomains (Hardwick et al., 1992;
Regier et al., 1993; Walker et al., 1993; Blair et al., 1994;
Jackson et al., 1996). The progressive N-terminal deletions of
such proteins show gradual reduction in activation ability
(Hope et al., 1988). Moreover, the effect of mutation in resi-
dues, which are important to activation function, often can
be seen only when several regions important for activation
are mutated (Jackson et al., 1996). Progressive N-terminal
deletions of Rme1p display a gradual reduction in repres-
The Rme1 site repression was tested by the ability of gal80? diploids carrying different Rme1p derivatives to repress sporulation in strains
with wild-type RC. Sporulation was scored after 48 h in liquid sporulation medium. IME2-LacZ expression was determined after 24 h in
sporulation medium. Activation ability was determined by liquid ?-galactosidase assays using pAC110–6 as a reporter.
Effect of a series of N-terminal deletions on the ability to repress the endogenous IME1 gene and to activate a reporter plasmid.
to the indicated Rme1p derivatives. The ability to activate transcription of ADE2 and HIS3 reporter genes in strain PJ69–4A was tested on
SC medium (Rose et al., 1990) lacking adenine and on SC medium lacking histidine with or without addition of 3-AT.
Transcriptional activation by Gal4-Rme1p fusion derivatives. The GAL4 DNA-binding domain was expressed as a fusion protein
Effector Domains of Rme1p
Vol. 13, May 2002 1717
sion/activation ability rather than a sudden complete loss of
activity (Figure 5). Moreover, we were unable to obtain a
single mutation that affects Rme1p function as an effector of
transcription, except mutations that interfere with binding
to DNA (Covitz, 1993). These results suggest that additional
activation/repression determinants could exist in the N-
terminal region of Rme1p. To test this possibility, we con-
structed two additional fusion proteins on the basis of
Rme1?1–90p and Rme1?1–120p alleles, adding back the first
31 amino-acid residues to each of these proteins. These two
proteins were tested for their ability to repress sporulation
and to activate transcription of the reporter gene CYC1-lacZ
via the Rme1 site (Figure 5). In addition, these two proteins
were tested for their ability to activate transcription of ADE2
and HIS3 reporter genes via the GAL4-DNA binding site
(Figure 6). The addition of the first 31 amino acids to
Rme1?1–90p renders it a potent effector of transcription.
Rme1?31–90p efficiently activates transcription of all re-
porter genes. The addition of the first 31 residues to the
effector-deficient Rme1?1–120p converts it to a weak tran-
scriptional effector. This lowers sporulation of strains carry-
ing this allele from 79.5% to 46% and is associated with a
very weak activation of CYC1-lacZ gene (Figure 5).
Rme1?31–120 fused to the GAL4 DNA binding domain ac-
tivated transcription of the HIS3 reporter gene, but not the
ADE2 reporter gene. The activation of the HIS3 transcription
is known to require weaker interactions between the activa-
tion domain and basic transcription machinery compared
with other reporter genes (James et al., 1996). This weak
ability to activate transcription of the HIS3 was inhibited by
the addition of 5 mM of 3-AT (Figure 6). Thus, the first 31
amino acids of Rme1p represent an additional effector mod-
ule, which can restore activation/repression when the min-
imal effector domain of Rme1p is altered. Accordingly, lexA-
Rme1?4–31 repression fold is lower then that of the wild-
type lexA-Rme1p (Figure 2B). Moreover, it is possible that
amino acids 31–60 of Rme1p also contribute redundantly to
repression and activation, because the progressive deletion
of these amino acids results in an additional reduction in
activation ability. The significance of this region is also sug-
gested by the comparison of the activation and repression
abilities of proteins Rme1?1–31 and Rme1?1–61 in Figure 5.
The progressive C-terminal deletions of Rme1p also display
a gradual reduction in repression/activation ability. Indeed,
compare activation and repression abilities of proteins lexA-
Rme1?210–300, lexA-Rme1?179–300, and lexA-Rme1?148–
300 via the lexA sites in Figures 2B, 3B, and 4B. Moreover,
the amino acids 148–179 are dispensable for repression in
the otherwise wild-type protein but contribute to the repres-
sion in lexA-Rme1?179–300 (Figure 2B and 3B). These data
donot sporulate, and
suggest that Rme1p may include multiple redundant deter-
minants that can contribute synergistically to repression.
In summary, the data presented above demonstrated that
structural integrity of zinc fingers of Rme1p is not required
for Rme1p-mediated effects on transcription. Amino acids
61–148 of the N-terminus comprise a minimal activation
domain, which efficiently activates transcription when trans-
ferred to a heterologous DNA binding domain. Moreover,
these amino acids are absolutely required for repression as
well. However, it seems that the efficient repression depends
on additional amino acids of the C terminus (179–210).
Additional effector determinants exist within the first 31
amino acids of the N-terminal part of the Rme1 protein.
These additional effector determinants contribute both to
repression and to activation.
Sin4p and Rgr1p Are Not Required for Rme1p-
The finding that Rme1p repression and activation domains
overlap suggests that Rme1p may have a single biochemical
activity or interaction that influences transcription. If so,
other gene products must determine whether that activity
results in repression or activation. This model predicts that
such gene products will be required only for activation or
repression, but not for both activities. Covitz et al. (1994)
showed previously that the rgr1–100 mutation causes a de-
fect in repression of the IME1 and reporter genes, but not in
activation of reporter genes. However, rgr1–100 is not a
simple loss-of-function mutation: whereas the rgr1? muta-
tion is lethal (Stillman et al., 1994), the rgr1–100 is not, and is
partially dominant (Covitz et al., 1994). This could mean that
rgr1–100 mutation causes a defect only in repression,
whereas rgr1? may be defective in both repression and
activation. It was also found previously that a sin4? muta-
tion causes a defect in repression (Covitz et al., 1994). Thus,
using reporter genes, we quantified activation and repres-
sion activities of Rme1p in sin4?, rgr1–100, and control
strains (Figure 1B). It has been shown that rgr1 and sin4
mutations permit some expression of genes lacking UAS
regions (Jiang and Stillman, 1992; Stillman et al., 1994). In-
deed, the expression of all reporter plasmids was higher in
the rgr1–100 and the sin4? strains (Table 2). The rgr1–100
and the sin4? strains were both defective in repression of
reporter plasmids, as expected. On the other hand, the acti-
vation fold was 53 for the wild-type strain and 89 and 47 for
sin4? and rgr1–100 strains carrying the wild-type RME1
gene, respectively. In conclusion, Sin4p is not required for
Rme1p-dependent activation, just as Rgr1p is not required
for this mode of transcriptional activation.
Table 2. Effects of rgr1-100 and sin4? on Rme1p-mediated repression and activation
pBS9 pBS8Fold pLG312S?SSpAC110-6Fold
A. Blumental-Perry et al.
Molecular Biology of the Cell1718
Rme1p functions both as a repressor and an activator of
transcription. The three zinc fingers in the Rme1p C-termi-
nal region are required for DNA binding (Figure 7) (Covitz
and Mitchell, 1993; Shimizu et al., 1997a; Shimizu et al., 2001).
We have shown here that the Rme1p N-terminal region is
necessary and sufficient for both repression and activation
(Figures 2 and 4). Thus, the role of Rme1p as an effector of
transcription is not simply to displace proteins that bind to
overlapping DNA sites.
The minimal regions required for repression and activa-
tion overlap completely and lie between amino acids 61 and
148 (Figure 7). It is unusual for a protein to have a single
effector region that directs both repression and activation.
For example, Ume6p has a central repression domain that
interacts with Sin3p and Rdp3p (Kadosh and Struhl, 1997)
and an N-terminal activation domain that interacts with
Rim11p and Ime1p (Bowdish et al., 1995; Rubin-Bejerano et
al., 1996; Malathi et al., 1997). Rap1p has neighboring but
separable repression and activation domains (Sussel and
Shore, 1991). It is possible that the single Rme1p transcrip-
tional effector domain interacts with a single target protein
whose activity—repression or activation—is dictated by
neighboring proteins or the chromatin environment. A sec-
ond possibility is that the N-terminal region of Rme1p can
interact with two different proteins or complexes that indi-
vidually yield exclusively repression or activation.
The Rme1p N-terminal effector domain shows no exten-
sive homology to known activation or repression domains,
and has no significant primary sequence identity to other
proteins in current databases. However, the effector do-
mains, does contain residues and possible secondary struc-
tures that are implicated in activation or repression by other
transcription factors. For example, it contains stretches of
bulky hydrophobic amino acids and charged residues (Fig-
ure 7). Two regions (85–98 and 116–122) are predicted to
form ?-helices, which may facilitate protein–protein interac-
tions. These patches of similarity to other repressors and
activators are consistent with the possibility that Rme1p has
interdigitated residues that contribute only to repression or
activation. This model predicts that mutational alteration of
specific Rme1p N-terminal residues may impair only repres-
sion or activation, in contrast to the broad effects of the
deletions studied here. However, we note that random mu-
tagenesis of RME1 has yielded numerous mutations that
impair DNA binding, but none that specifically impair re-
pression. It is possible that the individual Rme1p effector
segments function redundantly, so that multiple point mu-
tations would be necessary to inactivate a specific effector
function. Our deletion analysis here is consistent with such
a model, in that regions flanking the effector domain can
augment repression and activation (Figure 7).
The observation that Rme1p activation and repression
domains overlap brings to the foreground the question of
whether identical protein complexes form at Rme1p-re-
pressed and Rme1p-activated promoters. One simple possi-
bility is that the Mediator or a smaller Rgr1p-Sin4p complex
is the Rme1p-interacting target, because these proteins act as
both positive and negative regulators of transcription (Still-
man et al., 1994). However, we have shown clearly that Sin4p
and Rgr1p are not required for Rme1p-mediated activation.
Thus, if RNA polymerase II holoenzyme subcomplexes are
direct Rme1p targets, there must be distinct subcomplexes
that are brought to the Rme1p-repressed and Rme1p-acti-
vated promoters (Myers et al., 1999). Perhaps recruitment of
a Mediator subcomplex lacking Sin4p and Rgr1p prompts
RNA polymerase II to activate transcription, as occurs when
the Rme1p-binding site is situated in place of a UAS.
We have favored the model that Rgr1p-Sin4p is recruited
by Rme1p at repressed promoters because it explains genetic
relationships simply. However, we have recently observed
that lexA-Rgr1p does not repress the lexO-IME1 test gene
(Blumental-Perry, 2001), whereas lexA-Rme1p derivatives
are effective repressors. In addition, the fact that Rme1p
repression excludes nearby transcriptional activators from
DNA (Shimizu et al., 1997b) is not an expected consequence
of direct interaction between Rme1p and the Mediator.
Thus, more complex biochemical relationships must be con-
sidered. One possibility, discussed previously (Shimizu et
al., 1997b), is that Rme1p repression depends on a nucleo-
some structure or density that is unachievable in rgr1 or sin4
mutants. This model predicts that other mutations with
similar effects on nucleosome structure will also impair
Rme1p repression specifically. A second possibility is that a
gene specifying the hypothetical Rme1p corepressor is not
expressed in rgr1 or sin4 mutants. Candidate corepressor
genes may then be identified through genome-wide expres-
sion surveys. A direct approach to this question is to identify
proteins that interact with the Rme1p effector domain. Over-
lap between Rme1p repression and activation regions pre-
cludes the use of conventional two-hybrid cloning, but we
expect that biochemical identification of Rme1p effector re-
summary of Rme1p. The C-ter-
minal part of Rme1p contains
three zinc fingers (ZF boxes) and
C-terminal segment (CTR) with
properties of ?-helix. This part of
DNA- binding (Shimizu et al.,
2001). The N-terminal part of
Rme1p contains minimal effector
domain (hatched region), which
spans between amino acids 61 and 148, and additional effector subdomains (dotted regions), which augment repression and activation. The
minimal effector domain contains stretches of bulky hydrophobic amino acids at positions 62–64,66; 72–77; 92–93; and 127–132 and stretches
of charged residues at positions 96–101 and 112–117 (underline). The regions 85–98 and 116–122 are predicted to form ?-helixes. Amino acids
are presented by their one-letter codes.
Effector Domains of Rme1p
Vol. 13, May 2002 1719
gion–interacting proteins, combined with chromatin immu-
noprecipitation of the RC, will provide a direct route to
address these mechanistic questions.
We thank Dr. D. Zenvirth and Dr. S. Klein for comments on the
manuscript and Dr. R. Kornberg for fruitful discussion. This work
was supported by Human Frontier Science Program grant RG0379/
1997-M and National Institutes of Health grant GM-39531 to A.P.M.
and by a research grant from the Israel Science Foundation to G.S.
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Effector Domains of Rme1p
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