The ESS1 Prolyl Isomerase and Its Suppressor BYE1 Interact ... · induced by the Ess1 peptidyl-prolyl cis/trans isomerase. Here, we examined the role of ESS1 in transcription by studying

Copyright 2003 by the Genetics Society of America

The ESS1 Prolyl Isomerase and Its Suppressor BYE1Interact With RNA Pol II to Inhibit Transcription

Elongation in Saccharomyces cerevisiae

Xiaoyun Wu,*,† Anne Rossettini*,‡ and Steven D. Hanes*,†,1

*Molecular Genetics Program, Wadsworth Center, New York State Department of Health and†Department of Biomedical Sciences, School of Public Health, State University of New York,

Albany, New York 12208 and ‡Wagner College, Staten Island, New York 10301

Manuscript received June 10, 2003Accepted for publication August 13, 2003

ABSTRACTTranscription by RNA polymerase II (pol II) requires the ordered binding of distinct protein complexes

to catalyze initiation, elongation, termination, and coupled mRNA processing events. One or more proteinsfrom each complex are known to bind pol II via the carboxy-terminal domain (CTD) of the largest subunit,Rpb1. How binding is coordinated is not known, but it might involve conformational changes in the CTDinduced by the Ess1 peptidyl-prolyl cis/trans isomerase. Here, we examined the role of ESS1 in transcriptionby studying one of its multicopy suppressors, BYE1. We found that Bye1 is a negative regulator of transcrip-tion elongation. This led to the finding that Ess1 also inhibits elongation; Ess1 opposes elongation factorsDst1 and Spt4/5, and overexpression of ESS1 makes cells more sensitive to the elongation inhibitor 6-AU.In reporter gene assays, ess1 mutations reduce the ability of elongation-arrest sites to stall polymerase. Wealso show that Ess1 acts positively in transcription termination, independent of its role in elongation. Wepropose that Ess1-induced conformational changes attenuate pol II elongation and help coordinate theordered assembly of protein complexes on the CTD. In this way, Ess1 might regulate the transition betweenmultiple steps of transcription.

ESS1 encodes a highly conserved, peptidyl-prolyl iso- 1997). Ess1 and its homologs have a substrate specificitymerase (PPIase; Hanes et al. 1989; Hani et al. 1995). that is distinct from that of other PPIases and has been

Ess1 and its homologs (Dodo and Pin1) have been impli- defined as Ser-Pro or Thr-Pro, where the residues pre-cated in such diverse biological functions as checkpoint ceding proline are phosphorylated (Yaffe et al. 1997;control in mitosis (Lu et al. 1996), signaling during Fischer et al. 1998).embryonic development (Hsu et al. 2001), and regula- The first major clue that Ess1 is important for tran-tion of gene transcription (Wu et al. 2000, 2001; Shaw scription came from results of a multicopy suppressor2002). ESS1 was discovered in Saccharomyces cerevisiae and screen, in which five out of six genes identified werewas shown to be essential in this organism (Hanes et transcription related (Wu et al. 2000). Among thoseal. 1989) and in the human fungal pathogen Candida identified was FCP1, which encodes a phosphatase spe-albicans (Devasahayam et al. 2002). It is present in all cific for the carboxy-terminal domain (CTD) of Rpb1,eukaryotic organisms that have been examined, but is the largest subunit of RNA polymerase II (pol II; Arch-not essential in some other fungi (Crenshaw et al. 1998; ambault et al. 1997; Kobor et al. 1999). ESS1 was thenHuang et al. 2001; P. Ren, A. Rossettini and S. D. shown to interact genetically with RPB1 and, in particu-Hanes, unpublished observations) or in metazoans lar, with alleles that alter the CTD (Wu et al. 2000, 2001;(Maleszka et al. 1996; Fujimori et al. 1999). As do other C. B. Wilcox, A. Rossettini and S. D. Hanes, unpub-PPIases, e.g., cyclophilins and FKBPs (Dolinski and lished observations). ESS1 also interacts with genes in-Heitman 1997), Ess1 likely functions by the cis-trans volved in CTD function, such as SRB2 and SRB10, whichconversion of the peptide bond preceding prolines suppress the phenotype of CTD truncations (Wu et al.within target proteins, thus altering protein conforma- 2000, 2001; C. B. Wilcox, A. Rossettini and S. D.tion (Fischer et al. 1998; Hani et al. 1999). This might

Hanes, unpublished observations), and Ess1 binds theoccur in newly synthesized proteins to aid in folding or

CTD in vitro (Morris et al. 1999; Wu et al. 2000). Thesein mature proteins to control their activity or associationand other data suggest that the interaction of Ess1 withwith other protein complexes (Dolinski and Heitmanthe CTD is important for RNA pol II transcription invivo (Wu et al. 2000, 2001).

The CTD consists of multiple copies of the heptad re-1Corresponding author: Wadsworth Center, New York State Depart- peat, YSPTSPS, which undergoes reversible phosphoryla-ment of Health, 120 New Scotland Ave., Albany, NY 12208.

E-mail: [email protected] tion during transcription (Corden et al. 1985; Dahmus

Genetics 165: 1687–1702 (December 2003)

1688 X. Wu, A. Rossettini and S. D. Hanes

1996). This covalent modification regulates the binding during transcription. Our results are consistent with themodel that Ess1 coordinates the binding of accessoryof numerous transcriptional accessory proteins to the

CTD and might, in part, control the transition from one proteins to the CTD to regulate the transition betweendiscrete steps of transcription.step of transcription to the next (McCracken et al. 1997a;

Patturajan et al. 1998; Morris and Greenleaf 2000;Barilla et al. 2001; Dichtl et al. 2002b; Licatalosi et al.

MATERIALS AND METHODS2002). Phosphorylation of Ser2 and Ser5 in the heptadrepeat also generates Ess1 target sites (phospho-Ser- Yeast strains: Strains used in this article are listed in Table 1.Pro) that might trigger the binding of Ess1 to the CTD W303 1A (wild type) and W303 1B were gifts from Rod(Morris et al. 1999). As previously proposed, the bound Rothstein. ess1ts strains YGD-ts8W (ess1A144T ) and YGD-ts22W

(ess1H164R) were described previously (Wu et al. 2000). YXW21Ess1 would isomerize the Ser-Pro peptide bonds, alter-and YXW20 are MAT� versions of YGD-ts8W and YGD-ts22W,ing the CTD conformation, and thus provide an addi-respectively, obtained in backcrosses with W303 1B. In alltional, noncovalent means to regulate the binding and crosses involving ess1ts strains, here and below, ESS1 allele

release of accessory proteins to the CTD (Wu et al. 2000). status was confirmed by allele-specific PCR. YXW85-a was gen-Since Ess1 binds the CTD, it could potentially act erated by mating CBW1 (Wu et al. 2000) with YXW21 and

selecting for G418R segregants (containing rpb1�) that wereduring any step in which the CTD is involved. The CTDtemperature sensitive (ts; containing ess1 A144T). YXW86-a waswas initially shown to be required for initiation (Carl-derived in the same way, except that CBW1 was mated withson 1997), and we have obtained genetic evidence that YXW20. YXW84-a, which has the same genotype as CBW1, was

Ess1 is required at this step (C. B. Wilcox, A. Rosset- regenerated in these crosses by selecting for G418R non-tstini and S. D. Hanes, unpublished observations). The segregants. YXW95, YXW96, and YXW97 were made by plas-

mid shuffling in YXW84-a using plasmids pYF1866, pYF1869,CTD also appears to play a role in elongation, termina-and pYF1864, as appropriate.tion, and mRNA processing (including 5� capping, splic-

A bye1� strain, YXW35-a, was made by transforming W303ing, and 3�-end processing; Hirose and Manley 2000; 1B with a bye1�LEU2 fragment derived from pXW13 (Wu etHowe 2002; Proudfoot et al. 2002). The CTD’s involve- al. 2000) followed by backcrossing correct deletion isolatesment in elongation is suggested by the changes in the (as demonstrated by PCR) with W303 1A and identifying Leu�

segregants. A dst1� strain, YXW52-�, was made by trans-pattern of CTD phosphorylation that occur during elon-forming W303 1B with a dst1�URA3 fragment generated bygation (Payne et al. 1989; O’Brien et al. 1994; Komar-PCR using URA3-specific primers containing 45 nucleotidesnitsky et al. 2000; Schroeder et al. 2000) and by the homologous to DST1 flanking sequence. Deletions were con-

genetic interactions observed between the CTD and the firmed by PCR, and two independent isolates were used.elongation factors Dst1 (yeast TFIIS) and Spt4-Spt5 YXW56 and YXW57-� (ess1ts dst1� double-mutant strains) were

generated by mating YXW52-� with YGD-ts8W and YGD-ts22W,(Lindstrom and Hartzog 2001). The CTD’s role inrespectively, and obtaining Ura� (carrying dst1�) ts segre-termination may be direct, or it may be related to itsgants. YAR1–YAR3 were derivatives of YXW52-�, YXW56-a, androle in 3�-end processing (McCracken et al. 1997b; Lic- YXW57-� in which the URA3 in dst1�URA3 was replaced by

atalosi et al. 2002). The requirement for the CTD in HIS3 using a dst1�HIS3 PCR fragment. Correct recombinantselongation and termination raises the possibility that were confirmed by PCR. Two independent isolates were used

in each study. A dst1� bye1� double-mutant strain, YXW103-�,Ess1 may also be important for these postinitiation steps.was made by mating between YAR1 and YXW35-a and selectingIn fact, work by Hani et al. (1995) has implicated Ess1His� (carrying dst1�) Leu� (carrying bye1�) segregants.in termination.

GHY180 (spt4�), OY96 (spt5-194), GHY92 (spt5-242), andTo identify in which step(s) in transcription Ess1 func- a wild-type control strain OY100 were gifts from Grant Hartzog

tions, we first characterized a potent multicopy sup- (Hartzog et al. 1998), as was OY175 (spt5� � pSPT5; G.pressor of ESS1, the yeast open reading frame (ORF) Hartzog, unpublished data). YXW77, sharing the same geno-

type as OY175, was a meiotic segregant derived from matingYKL005C, which in this article we have renamed BYE1OY175 with YXW21. YXW82-a (spt5� � pspt5-194) was made(Bypass of ESS1). We studied BYE1 because it encodesby plasmid shuffling in OY175 using pBM24 and then pspt5-a protein that contains a TFIIS-like domain and was 194. The final shuffle isolates were backcrossed to YXW21 and

therefore suspected to be an elongation factor. Indeed, meiotic segregants with the correct genotype were obtained.we found that Bye1 interacts with Rpb1 and acts nega- YXW79-a and YXW80-a were ess1ts spt5� � pSPT5 strains gener-

ated by mating YXW77 with YXW21 and YXW20, respectively,tively in elongation, a result that also implicated Ess1and selecting for Leu� (bearing spt5�) Ura� (bearing pSPT5)in elongation, since high levels of Bye1 eliminate thets segregants from the resultant diploid.requirement for Ess1. Using genetic interaction tests, Plasmids: Yeast expression vector pJGS-4 (2�, TRP1) was

chemical inhibitor experiments, and reporter gene used to express hemagglutinin (HA)-tagged wild-type and mu-assays, we showed that Ess1 is involved in elongation and tant Bye1 proteins. It contains an ADH1 promoter and an

ADH1 terminator, between which there are unique EcoRI andacts to oppose the positive effects of known elongationXhoI sites followed by stop codons in all three reading framesfactors Dst1 and the Spt4-Spt5 complex. The results(Zhu and Hanes 2000). pWTN, expressing N-terminally HA-also indicate that the termination readthrough defectstagged Bye1, was constructed in three steps. First, an EcoRI

previously reported in ess1 mutants (Hani et al. 1999) PCR fragment corresponding to the N-terminal portion ofmay be unrelated to the role of Ess1 in elongation, Bye1 (arbitrarily defined by the unique EcoRI site at the begin-

ning of the TFIIS-like domain) was inserted into pJGS-4, givingsuggesting that Ess1 acts in multiple, sequential steps

1689A PPIase Involved in Elongation

TABLE 1

Yeast strains used in this study

Strain Genotype Source

W303 1A MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 Rod RothsteinYGD-ts8W MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 ess1A144T Wu et al. (2000)YGD-ts22W MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 ess1H164R Wu et al. (2000)YXW21 MAT� ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 ess1A144T This studyYXW20 MAT� ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 ess1H164R This studyCBW1 MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 rpb1�G418 � pRP112 Wu et al. (2000)

(RPB1, CEN, URA3)YXW84-a Same as CBW1, derived from backcrosses to YXW21 This studyYXW85-a MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 ess1A144T rpb1�G418 � This study

pRP112 (RPB1, CEN, URA3)YXW86-a MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 ess1H164R rpb1�G418 � This study

pRP112 (RPB1, CEN, URA3)YXW95 MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 rpb1�G418 � pYF1866 This study

(RPB1, CEN, TRP1)YXW96 MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 rpb1�G418 � pYF1869 This study

(rpo21-18, CEN, TRP1)YXW97 MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 rpb1�G418 � pYF1864 This study

(rpo21-24, CEN, TRP1)YXW35-a MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 bye1�LEU2 This studyYXW52-� MAT� ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 dst1�URA3 This studyYXW56-� MAT� ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 ess1A144T dst1�URA3 This studyYXW56-a MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 ess1A144T dst1�URA3 This studyYXW57-� MAT� ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 ess1H164R dst1�URA3 This studyYAR1 MAT� ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 dst1�HIS3 This studyYAR2 MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 ess1A144T dst1�HIS3 This studyYAR3 MAT� ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 ess1H164R dst1�HIS3 This studyYXW103-� MAT� ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 dst1�HIS3 bye1�LEU2 This studyOY100 MAT� his4-912� lys2-128� leu2�1 ura3-52 Hartzog et al. (1998)GHY180 MAT� his4-912� lys2-128� leu2�1 ura3-52 spt4�2::HIS3 Hartzog et al. (1998)OY96 MATa his4-912� lys2-128� leu2�1 ura3-52 spt5-194 Hartzog et al. (1998)GHY92 MAT� his4-912� lys2-128� leu2�1 ura3-52 spt5-242 Hartzog et al. (1998)OY175 MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 spt5�2::LEU2 � pMS4 G. Hartzog, unpublished data

(pSPT5, CEN, URA3)YXW77-a Same as OY175, derived from backcrosses to YXW21 This studyYXW82-a MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 spt5�2::LEU2 � This study

pspt5-194 (spt5-194, CEN, URA3)YXW79-a MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 ess1A144T spt5�2::LEU2 � This study

pMS4 (SPT5, CEN, URA3)YXW80-a MATa ura3-1 leu2-3,112 trp1-1 can1-100 ade2-1 his3-11,15 ess1H164R spt5�2::LEU2 � This study

pMS4 (SPT5, CEN, URA3)

rise to pA(untag). Second, a pair of annealed oligos encoding encoding either the TFIIS-like domain plus the tagged C-termi-nal region of Bye1 or just the tagged C-terminal region of Bye1,the HA epitope and SV40 nuclear localization sequence (NLS)

was inserted into the BglII site at the beginning of Bye1. This respectively. All plasmids containing PCR-generated frag-ments were sequenced and found to be free of mutations.intermediate construct encodes a deletion mutant of Bye1

(pA). Finally, the remainder of Bye1 was reconstituted into Bye1 point mutants were made using PCR overlap extension(Horton et al. 1990). The PCR fragment containing the de-pA as an EcoRI-XhoI fragment from pXW11 (Wu et al. 2000).

pWTC, expressing the C-terminally tagged Bye1, was made in sired point mutations in the TFIIS-like domain was insertedinto the AvrII and XhoI sites of pA. Subsequently, the C-termi-a similar way, except that the annealed HA-NLS oligos were

inserted into the BglII site at the C-terminal end of Bye1. pB nal region of Bye1 was reconstituted as a KpnI fragment frompWTN. This KpnI fragment also contained an ADH1 termina-was made by inserting an AvrII-XhoI PCR fragment encoding

the TFIIS-like domain into the same sites in pA. pC was made tor, replacing that in the intermediate plasmids. The presenceof desired mutations was confirmed by DNA sequencing,by replacing the BglII-XhoI fragment in pA(untag) with a BglII-

XhoI PCR fragment encoding the TFIIS-like domain, followed which also verified the absence of undesired mutations.pJGS-DST1, expressing Dst1, was made by insertion of theby insertion of the annealed HA-NLS oligos into the BglII

site. pE was made by inserting an AvrII-XhoI PCR fragment Dst1 coding sequence as a PCR fragment into the XhoI sitein pJGS-4. pXW14 was made by insertion of an XhoI-BamHIencoding the tagged C-terminal region of Bye1 into the same

sites in pA(untag). pD and pF were also derived from pA(un- restriction fragment expressing BYE1 from pXW11 (Wu et al.2000) into the same sites in pRS423 (Christianson et al.tag) by replacing the BglII-XhoI fragment with a PCR fragment


1992). YEpESS1 and YEpHESS1 were previously described tions. BYE1 was identified as a multicopy suppressor of(Hanes et al. 1989), as well as pRS413-ESS1 (Wu et al. 2000) temperature-sensitive ess1 mutations, and it also sup-and pRS424-ESS1 (Wu et al. 2001).

presses an ess1� mutation (Wu et al. 2000). WhereasPlasmids expressing wild-type RPB1 (pYF1866) or mutantdeletion of BYE1 had no obvious phenotype on its own,rpb1 (pYF1869 and pYF1864) were gifts from James Friesen

(Archambault et al. 1992). pRP112, which expresses wild- it showed a synthetic-enhancing interaction with ess1ts

type RPB1 on a URA3 plasmid, was provided by Jeff Cordon mutations. For example, bye1� ess1ts double mutants(West and Corden 1995). Plasmids expressing wild-type SPT5 showed severe slow-growth phenotypes at 30�, a temper-(pMS4 and pBM24) and mutant spt5 (pspt5-194 and pspt5-

ature at which both single mutants grow normally (data242) were provided by Grant Hartzog (Swanson et al. 1991;not shown). Both the suppression and the syntheticHartzog et al. 1998; G. Hartzog, unpublished data). The

artificial arrest site (ARTAR) elongation reporter and control phenotype suggest that ESS1 and BYE1 have partiallyplasmids, pDK12 and pDK1, were gifts from Kevin Struhl (Kul- overlapping functions or act in the same or a parallelish and Struhl 2001). Termination reporter and control pathway.plasmids, pL101 and pD16, were obtained from Linda Hyman

The TFIIS-like domain of Bye1 is required for sup-(Tulane University; Hyman et al. 1991).pression of ess1 mutants: Bye1 contains two obviousYeast media and manipulations: Rich medium (YPD) and

selective complete synthetic medium (CSM) were prepared sequence motifs that suggest a role in transcription (Fig-according to standard protocols (Adams et al. 1997). Yeast ure 1A). The first is a PHD finger [amino acids (aa)plates containing 5-fluoroorotic acid (5-FOA) or G418 were 72–133], which has been found in proteins involvedmade according to standard protocol (Wach et al. 1994;

in chromatin remodeling (Aasland et al. 1995). TheAdams et al. 1997). Plates containing 6-azauracil (6-AU) weresecond is a TFIIS-like region (aa 177–354), which sharesprepared by adding 6-AU to desired final concentrations into

CSM minus ura medium or CSM medium lacking uracil and 43% overall similarity to a conserved sequence in theanother amino acid for plasmid selection. For 6-AU sensitivity Drosophila elongation factor, TFIIS (Wind and Reinesassays, pRS306 (CEN, URA3; Sikorski and Hieter 1989) was 2000). The TFIIS-like region in Bye1 is only weakly simi-introduced into all ura� strains to confer ura prototroph.

lar to that of the yeast TFIIS homolog, Dst1. In addition,Spot-test growth assays were performed by growing yeast cellsBye1 contains two bipartite NLSs at its N terminus (aato midlog phase at 30� and then diluting each culture to �107

cells/ml (OD600 0.5). Serial dilutions were then carried out 30–47; Dingwall and Laskey 1991) and was reportedand 2–3 �l of each was spotted onto desired media. Each to localize to the nucleus (TRIPLES project; Kumar etexperiment was carried out several times, using multiple iso- al. 2000).lates, with relevant genotypes spotted on the same plate and

To determine which sequence motif within Bye1 isincubated for the same time.required for suppression of ess1 mutants, we generatedStructure modeling: The TFIIS-like domain of Bye1 was

modeled using the nuclear magnetic resonance (NMR) struc- a series of deletion constructs (Figure 1A). Each dele-ture of domain II of Dst1 (PDB ID: 1ENW) as a template. tion was engineered to carry an SV40 NLS to ensureModeling was done both through the SwissModel server nuclear localization of the mutant protein and an HA(http://www.expasy.org/swissmod) and by the MolScript soft-

epitope to allow protein levels to be monitored. Addi-ware (Kraulis 1991). These two gave similar results.tion of the NLS and HA tag to full-length Bye1 at eitherWestern blotting: HA-tagged Bye1 proteins in whole cell

lysates were detected using anti-HA mouse monoclonal anti- the amino terminus (WTN) or the carboxy terminusbody 12CA5 (Roche, Indianapolis) as primary antibody at 1 (WTC) did not affect Bye1’s ability to suppress ess1 mu-�g/ml, and HRP-conjugated goat anti-mouse Ig (Amersham, tants (Figure 1B). Each Bye1 deletion mutant was testedArlington Heights, IL) as secondary antibody at 1:3000. Results

for suppression activity in both ess1A144T and ess1H164R mu-were revealed using an enhanced chemiluminescence detec-tants (Figure 1B and data not shown). Identical resultstion kit.

Reporter assays: Cells were grown in raffinose medium to were obtained using either allele.midlog phase at 25�, prior to galactose induction. -Gal activity The results are summarized in Figure 1A and demon-was measured as previously described (Hanes and Brent strate that the TFIIS-like domain is necessary for sup-1989). For the ARTAR-based reporter assays, in agreement

pression (mutants A, E, and F), although it does notwith Kulish and Struhl (2001), we found that ARTAR causedappear to be sufficient (mutant C). By contrast, thea decrease in -gal activity only under low-galactose conditions

(0.02%). However, in our assays, this decrease was subtle PHD finger and the C-terminal region are individually(about twofold after 2 hr of induction) and transient (not dispensable for suppression (mutants D and B). Westerndetectable after 4 hr of induction). Further reduction of the analyses confirm that differences in the ability of thegalactose concentration or the induction time resulted in -gal

deletion mutants to suppress were not due to differ-activities that were too low to be quantitated. These differencesences in protein levels in either ess1A144T (Figure 1C,may be due to the fact that we used a high-copy plasmid

version of the reporter system, rather than the integrated right) or ess1H164R mutants (data not shown), since levelsversion used previously (Kulish and Struhl 2001). of mutant proteins were equal to or greater than those

of wild-type Bye1. The fact that the TFIIS-like domainis required suggests that an elongation-related activity

RESULTSmay be important for the suppression of ess1 mutants.

We also observed that deletion mutants that retainedPositive interactions between ESS1 and its suppressor,BYE1: One approach to understanding the role of Ess1 the PHD finger but lacked the TFIIS-like domain (mu-

tants A and E) exhibited dominant negativity, in whichin transcription is to study genes whose mutation oroverexpression suppresses the lethality of ess1 muta- they enhanced the growth defects of ess1ts mutants (Fig-


Figure 1.—The TFIIS-likedomain of Bye1 is required forsuppression of ess1 mutants.(A) Schematic of Bye1 deletionmutants and summary of theirphenotypes. The black bar de-notes the HA epitope tag andSV40 NLS. WTN and WTC arefull-length Bye1 carrying N-ter-minal or C-terminal tags, re-spectively. Sup, suppression ofgrowth defect of ess1ts mutantsat restrictive temperatures;Dom Neg, dominant negative,i.e., growth inhibition of ess1ts

mutants at permissive tempera-ture. (B) Growth of ess1A144T

mutant cells overexpressingBYE1 deletion constructs. Cellswere grown on selective me-dium at the indicated tempera-ture for 3 days. (C) Westernanalysis of HA-tagged Bye1 pro-teins from the wild-type andess1A144T mutant cells. Cells ex-pressing the indicated Bye1protein were grown to midlogphase at 30� and shifted to 37�for 4 hr before harvesting. Ar-rowheads point to the HA-tagged wild-type and mutantBye1 proteins. v, empty vectorcontrol (pJGS-4).

ure 1B, 32�). This might be explained by the fact that (56 residues with 27% identity and 43% similarity; Fig-ure 2A). This region in Dst1 mediates binding to thePHD fingers are thought to mediate protein-protein

interactions and dominant negativity might occur be- Rpb1 subunit of pol II and four residues in this region(K196, R198, R200, and K209) form a basic patch thatcause the mutant proteins form inactive complexes that

interfere with wild-type Bye1 function. is critical for Rpb1 binding (Awrey et al. 1998). Bye1also contains several basic residues in this region, sug-The TFIIS-like domain of Bye1 likely binds Rpb1 to

suppress ess1 mutants: Although there is little overall gesting that it, too, might bind Rpb1 (Figure 2A).To investigate this possibility, we first generated asequence similarity between Bye1 and the elongation

factor Dst1, a short region in the C-terminal portion of structural model of the conserved region of Bye1 (resi-dues 292–347) using the NMR structure of the TFIIS-the TFIIS-like domain of Bye1 appears to be conserved


Figure 2.—Predicted Rpb1-binding residues in Bye1 are important for suppression. (A) Alignment of the most conservedregion of Bye1 (within the TFIIS-like domain) with Dst1. Four residues in Dst1 known to contact Rpb1 are shown in red, andthe three that have conserved counterparts in Bye1 are shaded. Residues in Bye1 shown in green are proposed to contact Rpb1.(B) Structural model of the TFIIS-like domain of Bye1 using domain II of Dst1 as template. See text for details. Side chains ofrelevant basic residues are labeled. (C) Suppression phenotype of bye1 mutants carrying Ala substitutions in positions predictedto contact Rpb1. ess1A144T cells carrying vector (pJGS-4) or plasmids expressing wild-type or mutated Bye1 were grown on selectivemedium at 37� for 5 days. (D) Western analysis to detect wild-type and mutant Bye1 proteins. Wild-type and ess1A144T mutant cellsexpressing the indicated Bye1 protein were grown to midlog phase at 30� and shifted to 37� for 4 hr before harvesting. Epitope-tagged Bye1 proteins were detected using anti-HA monoclonal antibodies.

signature domain of Dst1 as a template (Figure 2B). and K305A was not due to an insufficient expression ofthe mutant proteins (Figure 2D). These data suggestThe two structures superimposed well, with an RMS

deviation of only 0.16 A. The comparison clearly identi- that Bye1 binds Rpb1 using its TFIIS-like domain andthat this interaction is required for suppression.fies side chains of three of the four key residues in Dst1

(R198, R200, and K209) as having spatial equivalents To support this idea, we tested the converse, usingtwo Rpb1 alleles (rpo21-18 and rpo21-24) that fail to bindin Bye1 (K305, R307, and K316), with the possibility of a

fourth (K196 in Dst1 similar to K299 in Bye1). Together, Dst1 due to a small insertion or a substitution in thecognate binding site of Rpb1 (I1237TRARV andthese residues in Bye1 might form a basic patch capable

of binding Rpb1. E1230K, respectively; Archambault et al. 1992; Wu et al.1996). If Bye1 also binds this site, then these mutationsWe tested this idea by generating alanine substitutions

in three of the four conserved residues in Bye1 (K305, should abolish the Bye1-Rpb1 interaction, and suppres-sion of ess1ts mutants should be lost. For this experiment,R307, and K316). Since BYE1 mutations (bye1�) have

no discernible phenotype, we measured their ability to ess1H164R rpb1 double-mutant strains containing eithervector alone or a multicopy BYE1 plasmid were testedsuppress ess1ts mutants. The R307A mutation lost its ability

to suppress, while the K305A mutation was significantly for growth at 34� (nonpermissive for ess1ts mutants). Asa control, we first showed that the rpo21 alleles, whichreduced for suppression (Figure 2C). K316A, however,

suppressed almost as well as the wild type. This could are known to be ts at 37� (Archambault et al. 1992),were, in fact, able to grow normally at 34� (Figure 3A,be due to the presence of other basic residues (K314

and K317) adjacent to K316, which might supply the top). Once this was established, we showed that BYE1overexpression could no longer suppress ess1H164R in therequisite contact. The loss of suppression for K307A


Figure 3.—Dst1-binding residues in Rpb1 areimportant for BYE1 to suppress ess1ts mutants. (A)BYE1 does not suppress ess1ts in cells that expressan Rpb1 with reduced affinity for Dst1. Cells ofthe indicated genotype carrying plasmids with se-lected rpb1 alleles were streaked for single colo-nies on selective medium (CSM �his �trp) andincubated for 3 days (top) and 7 days (bottom)at 25� or 34�. (Top) Controls showing that rpo21-18 and rpo21-24 cells are not ts for growth at 34�.Plasmids used were: vector (pRS423; 2�, HIS3),pBYE1 (pXW14; 2�, HIS3), pRPB1 (pYF1866;CEN, TRP1), prpo21-18 (pYF1869; CEN, TRP1),and prpo21-24 (pYF1864; CEN, TRP1). (B) Rpb1mutations with reduced binding to Dst1 show asynthetic growth defect with ess1ts mutations atpermissive temperature. Yeast patches (YXW84-a,YXW85-a, and YXW86-a) with the indicated geno-type carrying the indicated plasmid were grownon selective medium containing 5-FOA to selectagainst the wild-type RPB1 plasmid (pRP112; CEN,URA3). The plate was incubated at 25� for 5 days.Plasmids used were the same as in A. pRS314(CEN, TRP1; Sikorski and Hieter 1989) was usedas vector control.

rpb1 mutant backgrounds (rpo21-18 and rpo21-24; Figure mutants (using rpo21-18 or rpo21-24 alleles) were syn-thetic lethal and that ess1H164R rpb1 mutants grew poorly.3A, bottom), indicating that the Rpb1 residues required

for interaction with Dst1 are also required for suppres- This was demonstrated by the inability to cure a pRPB1(URA3) plasmid from ess1A144T rpb1 double-mutant cells onsion by BYE1. These results are consistent with the idea

that Bye1 binds to Rpb1. Biochemical experiments will 5-FOA medium or, in the case of the ess1H164R rpb1, by areduced curing rate (Figure 3B). Although ess1H164R rpb1be needed to confirm a physical interaction between

Bye1 and Rpb1 and to demonstrate that this binding is cells were viable, they were slow growing (Figure 3A, 25�plate). These phenotypes mimicked those observed fordirect.

The importance of the Bye1-Rpb1 interaction is re- ess1ts bye1� mutants. Thus, it appears that loss of theBye1 binding site on Rpb1 is functionally equivalent tovealed when Ess1 function is compromised. In the course

of our experiments, we found that ess1A144T rpb1 double deletion of BYE1. The simplest interpretation is that the


Figure 4.—6-AU sensitivity of BYE1 mutants isconsistent with an inhibitory role in elongation.(A) (Top) Deletion of BYE1 (bye1�) makes cellsless sensitive to 6-AU. Wild-type (WT), bye1�(YXW35-a), dst1� bye1� (YXW103-�), and controldst1� (YAR1) cells were assayed for sensitivity to6-AU. An elevated temperature (32�) was used todemonstrate the effect of BYE1 deletion sincewild-type cells show mild 6-AU sensitivity at thistemperature. (Bottom) Overexpression of DST1partially rescued this increased 6-AU sensitivityin wild-type cells. (B) Overexpression of BYE1 indst1� mutant cells makes them more sensitive to6-AU. dst1� cells (YXW52-�) carrying the indi-cated plasmid were used. In A and B, threefoldserial dilutions were spotted on CSM �ura me-dium (or CSM �ura �trp medium to select forthe TRP1 plasmids) with or without 6-AU. Plateswere incubated at the indicated temperatures for3–5 days. Plasmids used were vector (pJGS-4),pDST1 (pJGS-DST1; 2�, TRP1), and pBYE1(pWTN; 2�, TRP1).

Bye1-Rpb1 interaction is needed for Bye1 to augment rendered dst1� mutant cells more sensitive to 6-AU (Fig-ure 4B). These results suggest that Bye1 has a negativeEss1’s role in transcription, and this interaction be-

comes critical when Ess1 activity is compromised. role in elongation, opposite to that of Dst1, consistentwith an Rpb1 binding-site competition model.Finally, since our experiments suggested that Bye1

and Dst1 might bind the same site on Rpb1, it then We also tested the 6-AU sensitivity of the bye1 dst1double mutant (Figure 4A), thinking that perhaps theseemed possible that a bye1 dst1 double mutant might

have the same phenotype as the rpo21 mutants (ts for bye1 mutation might reverse the sensitivity caused bydst1�. It did not, however, as results showed that dst1�growth at 37�; 6-AU sensitive, see below). However,

growth of the bye1 dst1 double mutant was not ts; growth is epistatic to bye1� since the double mutant did notgrow on 100 �g/ml 6-AU.was similar to that of the single mutants at all tempera-

tures tested (25�, 30�, 34�, and 37�; data not shown). This DST1-ESS1 genetic interactions suggest a negative rolefor Ess1 in elongation: Our original goal was to studyresult suggests that perhaps binding of other proteins to

this site might also affect Rpb1 function. the BYE1 suppressor as a means to understand ESS1function. Thus far, we have provided evidence for aBYE1 mutants are less sensitive to 6-AU, suggesting a

negative role in elongation: The mutational and genetic negative role of Bye1 in elongation. This implies a corre-sponding role for Ess1, because ess1 mutations are sup-analyses described above (Figures 2C and 3, A and B)

suggest that Bye1 and Dst1 bind to the same site on pressed by overexpression of Bye1. If Ess1 functionsnegatively in elongation, then Ess1 and Dst1 shouldRpb1. Bye1 might therefore compete with Dst1 and

antagonize its role in elongation. To determine whether oppose one another, just as Bye1 and Dst1 do. In fact,deletion of DST1 partially rescued the defects in ess1tsBYE1 has a negative role in elongation, we employed the

elongation inhibitor 6-AU, which is thought to promote mutant cells (ess1ts dst1�), allowing growth at nonper-missive temperature (35�; Figure 5A). Moreover, overex-pausing and arrest by limiting the intracellular pools of

GTP and UTP (Exinger and Lacroute 1992). Muta- pression of DST1 enhanced the defects in ess1ts mutantcells, resulting in slow growth at permissive temperaturetions in genes that promote elongation are often associ-

ated with hypersensitivity to 6-AU (e.g., dst1�; Losson (32�; Figure 5B). This effect was reversed by reintroduc-ing wild-type ESS1 (Figure 5B). These results show thatand Lacroute 1981; Wind and Reines 2000).

If Bye1 functions negatively in elongation, then dele- ESS1 and DST1 genetically oppose one another and,since Dst1 stimulates elongation, the implication is thattion of BYE1 should render cells less sensitive to 6-AU.

Indeed, we observed a reduced sensitivity to 6-AU in Ess1 inhibits elongation.ess1 mutants are less sensitive to 6-AU: To test whetherbye1� cells, especially at elevated temperature (32�), at

which wild-type cells were 6-AU hypersensitive (Figure Ess1 inhibits elongation, we measured the 6-AU sensitiv-ity of ess1 mutant cells. We found that ess1H164R mutant4A). As controls for 6-AU efficacy, DST1 deletion and

overexpression strains (dst1�; pDST1) were used. De- cells were more resistant to 6-AU than wild-type cellswere (Figure 6A), especially when compared to growthtails of the 6-AU experiments are described in materi-

als and methods. Although overexpression of BYE1 without the drug. Results with the ess1A144T mutantshowed the same trend but were more subtle, perhapshad no effect on wild-type cells (data not shown), it


a negative role for ESS1 in elongation, overexpressionof ESS1, which does not affect the growth of wild-typecells (Hanes et al. 1989; Figure 7A), enhanced thegrowth defects of spt4 and spt5 mutants, two of which arets (spt4� and spt5-194) and one of which is cold sensitive(spt5-242; Figure 7A). The effects of ESS1 on spt mutantswere most pronounced at semipermissive temperatures,at which spt mutants carrying vector alone are still viable.This effect of ESS1 overexpression resembled the effectpreviously reported for dst1� on these mutants (Hartzoget al. 1998), again indicating a negative role for ESS1 inelongation (summarized in Figure 7B).

Since overexpression of ESS1 enhanced spt5 mutantphenotypes, mutation of ESS1 might be expected tosuppress them. However, we were unable to generatestable ess1ts spt5-194 or ess1ts spt5-242 double mutants bytetrad dissection (where the spt5 mutations were carriedon plasmids), despite repeated attempts (Table 2 anddata not shown). This suggests that mutation of ESS1did not suppress spt5 alleles, but instead that ess1ts spt5double mutants are synthetic lethal. This was confirmedfor one spt5 allele, using a plasmid-shuffle assay in whichess1ts spt5� cells carrying an spt5-242 plasmid failed tolose a URA3-based SPT5 plasmid on 5-FOA (Figure 7C).This synthetic lethality was somewhat surprising, butmight be explained by the fact that both ESS1 and SPT5are essential genes thought to have multiple effects ontranscription (Lindstrom et al. 2003; C. B. Wilcox, A.Rossettini and S. D. Hanes, unpublished observations).Indeed, human Spt5 has been shown to have both posi-

Figure 5.—DST1 and ESS1 oppose one another. (A) Dele- tive and negative effects on transcription in vitro (Wadation of DST1 suppresses the growth defect of ess1ts mutant et al. 1998).cells. Cells (W303 1B, YXW21, YXW20, YXW52-�, YXW56-�, Physical interactions between Ess1 and Spt5 may alsoand YXW57-�) with the indicated genotype were tested for

occur as suggested by their common presence withingrowth on rich medium (YPD) at 35� for 3 days. (B) Overex-protein complexes (Ho et al. 2002). In addition, thepression of DST1 enhances the growth defect of ess1ts mutant

cells. ess1A144T and ess1H164R mutant cells carrying the indicated human orthologs (Pin1 and hSpt5) have been shownplasmids were streaked on selective medium (CSM �trp �his) to interact in vitro via a Ser-Pro rich region (the CTR)and incubated at 32� for 4 days. Plasmids used were: vector within hSpt5 (Lavoie et al. 2001). However, the CTR is(pJGS-4), pDST1 (pJGS-DST1), and pESS1 (pRS413-ESS1;

not found in yeast Spt5, so any potential interactionsCEN, HIS3) or a control plasmid (pRS413; CEN, HIS3; Sikor-between Ess1 and Spt5 are likely to be different. Func-ski and Hieter 1989).tional consequences of potential Ess1-Spt5 (or Pin1-hSpt5) interactions have yet to be demonstrated.

ess1ts causes a reduced sensitivity to an ARTAR: Onebecause its growth is generally less robust (data notshown). As expected, overexpression of ESS1 had the way in which Ess1 and/or Bye1 could inhibit elongation

is by promoting polymerase pausing or arrest. To testopposite effect, rendering dst1� mutant cells more sensi-tive to 6-AU (Figure 6B). These two results suggest that this idea, we employed a reporter gene system that has

been reported to monitor elongation arrest (KulishEss1 inhibits elongation. Accordingly, loss of Ess1 func-tion may cause hyperelongation by pol II and, if so, this and Struhl 2001). In this system (Figure 8A), the

ARTAR sequence (containing three artificial arrest sites)effect should be reversed by 6-AU. Remarkably, as pre-dicted, low concentrations of 6-AU rescued growth of is inserted into the coding region of lacZ, driven by

the GAL1-inducible promoter. Elongation arrest at theess1H164R mutants at restrictive temperature (34�; Figure 6C).ESS1 interacts genetically with elongation factors SPT4 ARTAR would decrease lacZ expression, as measured

by -galactosidase activity.and SPT5: Genetic interactions reported between SPT4and SPT5 and elongation factors including DST1 indi- If ESS1 or BYE1 promotes arrest, then mutation of

either of them should result in a pol II that is less likelycate that SPT4 and SPT5 function positively in elonga-tion and that elongation is compromised in spt4 and to arrest and thus result in a reduced ARTAR activity.

ARTAR activity was determined as the ratio of -galspt5 mutant cells (Hartzog et al. 1998). Consistent with


Figure 6.—The 6-AU sensitivity of ESS1 mu-tants is consistent with an inhibitory role in elon-gation. (A) ess1ts mutations make cells less sensitiveto 6-AU. Wild-type (WT), ess1H164R, and controldst1� (YAR1) mutant cells were assayed for sensi-tivity to 6-AU. (B) Overexpression of ESS1 makesdst1� mutant cells more sensitive to 6-AU. dst1�cells (YXW52-�) carrying vector (pRS424; 2�,TRP1) or pESS1 (pRS424-ESS1; 2�, TRP1) wereused. (C) The growth defect of ess1H164R mutantcells at restrictive temperature (34�) is suppressedby low concentration of 6-AU. Strains used werethe same as in A. In A–C, threefold serial dilutionswere spotted onto medium with or without 6-AU.Plates were incubated at the indicated tempera-tures for 3–5 days. In A, cells were spotted on thesame plate and incubated for the same time, buta composite figure was generated to eliminateduplicate isolates.

activity of (�)ARTAR:(�)ARTAR reporters. In our the fact that ESS1 was reisolated as PTF1 (processing/termination factor 1) in a screen for mutations thatassays, the ARTAR activity was rather low (see materials

and methods), resulting in only a 2.1-fold decrease in resulted in transcription readthrough and 3�-end pro-cessing defects using cryptic or weak terminators (Hani-gal activity in wild-type cells (Table 3A). In ess1A144T

and ess1H164R mutant cells, ARTAR activity was reduced et al. 1995, 1999). To confirm that Ess1 functions intermination, we employed a reporter gene system thatto 1.6- and 1.9-fold, respectively (Table 3A). Although

this reduction was modest, the effect was reproducible uses the ADH2 terminator (ADH2T ; Hyman et al. 1991).In this system (Figure 8B), the ADH2T is inserted inand could be reversed by addition of plasmids express-

ing wild-type ESS1 (Table 3A). The results are consistent the mini-intron of rp51, which is fused to lacZ. Correcttermination at the ADH2T results in a truncated tran-with the idea that ess1 mutants cause hyperelongation.

No effect on ARTAR activity was detected in bye1� mu- script and no -gal activity. However, in the case of atermination defect, polymerase reads through the ADH2T,tant cells.

A better demonstration of an effect of ess1ts on ARTAR synthesizing the rp51-lacZ fusion transcript, which willbe spliced and translated and give rise to -gal activity.activity was provided using dst1� cells. Consistent with

the earlier work (Kulish and Struhl 2001), we found Note that this assay is an indirect measure of read-through, since it actually monitors overall reporter genethat reporter gene expression is generally lower in dst1�

mutant cells. However, more important was the finding expression.We estimated transcription readthrough by compar-that deletion of DST1 increased ARTAR activity (to 3.5-

fold; Table 3B), but that the increase in ARTAR activity ing -gal activity reporters with or without the termina-tor. In this assay, ess1H164R mutant cells showed a signifi-was reversed (to 1.8-fold) by ess1A144T and ess1H164R in dou-

ble mutants (Table 3C). Thus, ess1 is epistatic to dst1� cant readthrough (almost 20%), consistent with earlierstudies (Hani et al. 1999). Readthrough was not ob-with respect to ARTAR activity, suggesting that hyper-

elongation caused by ess1 mutations counteracts the served in either wild-type cells or ess1H164R cells expressingwild-type ESS1 on a plasmid (�3%; Table 4). Theseinefficient elongation in dst1� cells. If the ARTAR does,

in fact, cause elongation arrest in vivo, then our results results suggest that Ess1 is important for termination.The roles of ESS1 in elongation and termination maysuggest that Ess1 promotes pausing or arrest and that

loss of this function causes hyperelongation, rendering be independent. Or they may be interdependent so thatchanges in elongation might also affect termination; forpol II resistant to arrest.

Mutations in ESS1 cause defects in transcription ter- example, in ess1 mutant cells, hyperelongating pol IImight fail to recognize or respond to the cis-actingmination: In addition to inhibiting elongation, ESS1 may

also promote termination. This was first suggested by poly(A) and 3� cleavage sequences and, thus, be unable


Figure 7.—ESS1 interacts genetically with SPT4and SPT5. (A) Overexpression of ESS1 enhancesthe growth defects of spt4 and spt5 mutants. Wild-type cells (OY100) and spt4 or spt5 mutants car-rying a multicopy ESS1 plasmid (YEpESS1) or acontrol vector (pRS426) were plated on selectivemedium using fivefold serial dilutions and incu-bated at the indicated temperatures for 2–3 days.The spt4� (GHY180) and spt5-194 (OY96) mu-tants are ts at 37�, while spt5-242 (GHY92) is cs.(B) The effect of ESS1 overexpression on spt4 andspt5 mutant cells is summarized and compared tothe effect of dst1� (Hartzog et al. 1998). sl gr,slow growth. (C) ess1ts mutations are syntheticallylethal with the spt5-242 mutation. Yeast cells(YXW77-a, YXW79-a, and YXW80-a) with the indi-cated genotype carrying an empty vector(pRS314) or plasmids expressing SPT5 (pBM24)or spt5-242 (pspt5-242) were grown on selectivemedium containing 5-FOA to select against thewild-type SPT5 plasmid (pMS4; CEN, URA3). Theplate was incubated at 25� for 6 days.

to terminate. If the observed termination defect is a mutant cells (data not shown). These results show thatEss1 functions in sequential postinitiation steps in tran-consequence of the elongation defect, then we expect it

to be rescued by the elongation-related ess1 suppressors, scription.BYE1 and dst1�. However, overexpression of BYE1 didnot prevent transcription readthrough, nor did the

DISCUSSIONdst1� mutation (Table 4). Moreover, overexpression ofDST1 did not enhance this readthrough. These results We have shown that Ess1 and Bye1 are likely negativeindicate that the defects in termination and elongation regulators of elongation. This is the first described func-in ess1 cells are not interdependent, suggesting that the tion for Bye1 (formerly known as YKL005C). Althoughfunctions of Ess1 in termination and elongation are Ess1 and Bye1 both seem to inhibit elongation, they

probably act by different mechanisms. Ess1 binds phos-separable. Similar results were obtained with ess1A144T


TABLE 2

Synthetic lethality between ess1ts mutations and spt5-194 by segregation analysis

No. of No. of spt5� segregants Segregation ofDiploid parent Plasmid tetrads analyzed bearing plasmid ESS1 alleles

ess1A144T/ESS1 spt5�/SPT5 pSPT5 20 21 14 ESS1:7 ess1A144T

pspt5-194 40 18 18 ESS1:0 ess1A144T

ess1H164R/ESS1 spt5�/SPT5 pSPT5 20 14 7 ESS1:7 ess1H164R

pspt5-194 40 20 16 ESS1:4a ess1H164R

ess1A144T (YXW21) or ess1H164R (YXW20) cells were mated to either spt5� � pSPT5 (YXW77) or spt5� � pspt5-194 (YXW82-a) cells, and the resulting diploids were sporulated at 25�. Tetrads were dissected, and sporeswere allowed to grow on YPD for 7 days at 25�. The number of Leu� (containing spt5�2::LEU2) and Ura�

(containing pSPT5 or pspt5-194) were counted. The ESS1 allele status of these Leu� Ura� colonies wasdetermined by allele-specific PCR. Sequences of primers used for PCR are available upon request. Plasmidsused were pSPT5 (pMS4; SPT5, CEN, URA3) and pspt5-194 (spt5-194, CEN, URA3).

a These four ess1H164R spt5-194 segregants were extremely slow growing and fast reverting and hence were notstable.

pho-Ser-Pro sites within the CTD of Rpb1 and is likely sensitivity and on the growth of ess1 mutant cells suggestthat these proteins function in the same process, butto isomerize the CTD, coordinating the exchange of

proteins required for elongation and termination. Bye1 perform opposite functions. This might be explainedby the fact that Bye1 and Dst1 share a similar domainprobably binds Rpb1, but elsewhere within the protein

(�aa 1230–1237), and might act by competing with Dst1 and may bind to the same site on Rpb1. This wouldresult in a competition for Rpb1 binding in which Bye1for binding to the elongating pol II complex. These

results lead to the idea that, in yeast, as in mammals displaces Dst1, reducing the ability of pol II to elongate.However, two findings suggest that Bye1 does more(Yamaguchi et al. 1999; Wada et al. 2000; Ping and Rana

2001), elongation by RNA pol II proceeds as a result of than simply displace Dst1, but that it has an inherentnegative activity. First, overexpression of BYE1 increasedthe balance between positive and negative elongation

factors. 6-AU sensitivity, suggesting an inhibition of elongation,even when no Dst1 was present (in dst1� mutants).Bye1 is a negative regulator of transcription elonga-

tion: The opposite effects of Bye1 and Dst1 on 6-AU Second, the TFIIS-like domain alone, which containsthe Rpb1 binding site and should displace Dst1, failedto suppress ess1 mutants. The simplest explanation isthat in addition to displacing Dst1, Bye1 actively inhibitselongation. For example, Bye1 might induce a changeeither in the structure of the elongating pol II complexor in the association of other factors with this complex.This negative elongation activity of Bye1 might dependon sequences outside the Rpb1 binding site, which differfrom those in Dst1. Biochemical experiments areneeded to test the negative role for Bye1 in elongationand to help elucidate the underlying mechanism.

Ess1 is a negative regulator of transcription elonga-tion: Several lines of evidence indicated that Ess1 inhib-its elongation. These include the genetic interactionsobserved among ESS1 and DST1, SPT4, SPT5, the effects

Figure 8.—Schematic of the elongation and termination of Ess1 on 6-AU sensitivity, and the effects of ess1 muta-reporter genes used. (A) The ARTAR reporter gene used tions on elongation through an artificial arrest site. Inhi-to monitor transcription elongation. The ARTAR sequence

bition of elongation by Ess1 may be an important means(three artificial arrest sites) is inserted in the coding regionto reduce the expression of certain genes. Alternatively,of lacZ and is designed to allow more lacZ expression when

pausing/arrest fails to occur (Kulish and Struhl 2001). (B) we favor the idea that Ess1 may be required for theThe ADH2T reporter gene used to monitor transcription termi- proper expression of certain genes by attenuating polnation. The ADH2 terminator is a 327-bp region from the II elongation to a degree sufficient for closely coupled3�-end of ADH2 (bp 1220 to bp 1546 from the start of the

events such as mRNA capping, splicing, and chromatinADH2 ORF) and is inserted in the ribosomal protein 51 mini-remodeling (Hartzog et al. 2002; Howe 2002;intron and designed to express lacZ when termination fails to

occur (Hyman et al. 1991). Proudfoot et al. 2002) to occur in an orderly manner.


TABLE 3 be difficult to elucidate in vivo, given the artificial natureof ARTAR and the lack of information about naturalAnalysis of elongation pausing/arrest in ess1 mutantspause or arrest sites in yeast. Future work will benefitusing an artificial arrest reporterfrom the use of in vitro elongation systems (Christieet al. 1994).Strain �ARTAR �ARTAR �:� ratio

An additional role for Ess1 in termination and 3�-endA. Mutations in ESS1 reduced ARTAR activity processing: In addition to its role in elongation, ADH2TWT 154 � 16 72.2 � 3.8 2.13

reporter assays strongly suggest a role for Ess1 in termi-bye1� 196 � 8.3 94.7 � 2.1 2.07nation. Such a dual role has also been found for CHD1ess1A144T 70.4 � 5.0 43.4 � 2.4 1.62and SSU72, which had been described as terminationess1H164R 92.1 � 9.9 49.0 � 3.5 1.88factors but also seem to affect elongation (Woodage et

WT � vector 299 � 2 160 � 19 1.87 al. 1997; Alen et al. 2002; Dichtl et al. 2002a; KroganWT � pESS1 135 � 18 97.2 � 11 1.39 et al. 2002). In the case of Ess1, we have shown theess1A144T � vector 157 � 15 118 � 9 1.33 functions to be separable, because the termination de-ess1A144T � pESS1 129 � 27 69.3 � 8.2 1.86

fect in ess1 mutants was not rescued by the elongation-ess1H164R � vector 239 � 24 146 � 12 1.64related suppressors (BYE1 and dst1�). Thus, Ess1 seemsess1H164R � pESS1 154 � 7 82.5 � 5.2 1.87to act independently in these two processes.

B. Mutation in DST1 increased ARTAR activity Are the functions of Ess1 in elongation and termina-dst1� � vector 12.2 � 1.0 3.52 � 0.15 3.47 tion required for viability? Deletion of ESS1 is lethal,dst1� � pDST1 111 � 10 50.9 � 8.1 2.18 probably due to an essential role in transcription. The

work of Hani et al. (1999) suggested that terminationC. Mutations in ESS1 reversed the increasemight be the critical step, since the ess1 mutants isolatedof ARTAR activity caused by dst1�in their transcription readthrough screen were also tsWTa 319 � 33 191 � 10 1.67for growth. However, we do not think this is the casedst1� 35.1 � 2.1 10.8 � 1.2 3.25

ess1A144T 146 � 19 115 � 6 1.27 because neither BYE1 nor dst1�, which restore cell via-ess1A144T dst1� 36.8 � 3.5 20.3 � 1.7 1.81 bility, rescued transcription readthrough of ADH2T (Ta-

ble 4), nor did any of the previously described (Wu etWTb 422 � 60 269 � 27 1.57 al. 2000) multicopy suppressors of ess1 mutants (datadst1�b 11.0 � 2.5 3.64 � 0.36 3.02

not shown). In addition, ess1 mutations caused read-ess1H164R b 165 � 18 115 � 6 1.43through only at weak terminators (Hani et al. 1999),ess1H164R dst1�b 10.0 � 1.0 5.58 � 0.35 1.79and we found that efficiency of termination by ADH2T

Cells (W303 1B, YXW35-a, YGD-ts8W, YGD-ts22W, YAR1, was only reduced rather than abolished in ess1 mutants.YAR2, and YAR3) were grown to midlog phase in raffinoseFor example, the levels of ADH2T reporter gene expres-medium at 25� and then induced with 0.02% galactose for 2sion suggested that termination was still �80% effectivehr. �:� ratios were calculated using the mean values of -gal

units. Plasmids used were vector (pJGS-4) and pDST1 (pJGS- in ess1H164R cells vs. �97% effective in wild-type cellsDST1). (Table 4). Thus, it is possible that only a subset of genes

a Data for WT were obtained by normalizing results of assays will be affected by loss of Ess1 and that these effects ondone on different days (using -gal activities of ess1A144T cells).termination may be modest.b Assays were done on cells grown at 30�.

We suggest that instead, elongation may be the criticalstep since reducing elongation genetically or with 6-AUrestores viability. However, for both elongation and ter-Ess1 may inhibit elongation by increasing the likeli-

hood of pol II pausing or arrest rather than by reducing mination, it is possible that suppression occurs as a resultof a total bypass of the lethal defect, for instance by increas-the overall rate of elongation. An effect of Ess1 on paus-

ing or arrest is suggested by the fact that ess1 mutations ing the rates of other steps in transcription. Therefore, itremains a formal possibility that defects in either processincreased reporter gene expression in the ARTAR assay

(implying reduced pol II arrest; Table 3) and is consis- may cause the lethality. Isolation of elongation- or termi-nation-specific suppressors that do not rescue thetent with genetic interactions observed between ESS1

and SPT5. Previous studies showed that while the growth growth defects may be useful to narrow the possibilities.Ess1 may coordinate the binding of elongation anddefects of spt5-242 mutant cells are rescued by 6-AU

(presumably by reducing the rate of elongation) and termination factors to the CTD: Coordinating the bind-ing of accessory proteins to the pol II complex is crucialby an Rpb2 mutation that results in a “slow” polymerase

(Hartzog et al. 1998), they are exacerbated by pol II for the multistep process of transcription. For example,the exchange of initiation factors with elongation fac-pausing or arrest (Hartzog et al. 1998). We found that

overexpression of ESS1 also exacerbated the growth de- tors is needed for pol II to begin elongation (Pokholoket al. 2002). A similar exchange of elongation with termi-fects of spt5-242 mutant cells (Figure 7A), consistent

with a role for Ess1 in pol II pausing or arrest. An nation factors is likely to be required. Since many ofthese factors bind to the CTD, Ess1 may play a directexact role of Ess1 in pausing or arrest, however, may


TABLE 4

Mutations in ESS1 result in transcription readthrough of ADH2 terminator,which is not rescued by elongation-related suppressors

Strain �Terminator �Terminator % “readthrough”a

WTb � vector-1 41.3 � 6.45 1.09 � 0.46 2.6WTb � pESS1-1 25.9 � 3.58 1.16 � 0.35 4.4ess1H164R � vector-1 74.5 � 1.6 13.8 � 0.5 18.5ess1H164R � pESS1-1 45.3 � 1.0 1.24 � 0.16 2.7

ess1H164R � pBYE1 53.5 � 1.4 11.1 � 0.8 20.8

WTb 55.5 � 4.84 1.36 � 0.27 2.5dst1� 6.38 � 0.46 0.21 � 0.03 3.3ess1H164R 103 � 12 19.3 � 1.1 18.7ess1H164R dst1� 8.29 � 0.32 2.37 � 0.01 28.6

ess1H164R � vector-2 60.1 � 16.0 16.6 � 1.8 27.6ess1H164R � pESS1-2 64.0 � 4.6 2.19 � 0.12 3.4ess1H164R � pDST1 70.2 � 0.7 15.2 � 0.8 21.7

Cells (W303 1A, YGD-ts22W, YAR1, and YAR3) were grown to midlog phase in liquid raffinose medium at25� and then induced with 2% galactose for 4 hr at 37�. Plasmids used were vector-1 (pRS423), pESS1-1(YEpHESS1), pBYE1 (pXW14), vector-2 (pJGS-4), pESS1-2 (pRS424-ESS1), and pDST1 (pJGS-DST1).

a The % “readthrough” is calculated as mean -gal units of (�) terminator/(�) terminator 100, but isonly suggestive of actual levels of readthrough transcription, since it is based on overall expression levels ratherthan on a direct measurement.

b Data for WT were obtained by normalizing results of assays done on different days (using -gal activitiesof ess1H164R � vector-1 cells or ess1H164R cells, as appropriate).

role in coordinating protein exchange by altering the individual repeats may be isomerized independently. Asa result, Ess1 could generate a variety of CTD isomers,conformation of the CTD. The elongation and termina-

tion defects observed in ess1 mutant cells may result from each attracting a distinct spectrum of pol II accessorya loss of this coordination. proteins.

Ess1 may also play an indirect role in coordinating Moreover, CTD isomerization may act together withprotein exchange. Ess1-induced conformational changes CTD phosphorylation to constitute an autoregulatoryin the CTD may alter its affinity for CTD-specific kinases loop that governs CTD interactions. In this loop, Ess1-and phosphatases. This will lead to changes in the CTD dependent isomerization would control the CTD phos-phosphorylation state, which would in turn affect the phorylation state. Since Ess1 binds only to phosphory-binding of other proteins. During elongation, the CTD lated CTD, this would in turn control Ess1 binding andhas been observed to undergo changes in phosphoryla- CTD isomerization. Such a phosphorylation-isomeriza-tion (Komarnitsky et al. 2000; Schroeder et al. 2000), tion regulatory loop could control the binding and re-and it is possible that these changes are initiated by lease of individual protein complexes to pol II and helpEss1. Finally, Ess1 may have additional effects on gene drive the transcription cycle.regulation as a result of interactions with other tran- We thank Jeff Cordon, James Friesen, Grant Hartzog, Linda Hyman,scription-related proteins, such as members of histone and Kevin Struhl for gifts of plasmids and yeast strains. We thankdeacetylase complexes (e.g., Arevalo-Rodrıguez et al. Hongmin Li for help with structural modeling, the Wadsworth Center

Molecular Genetics Core Facility for oligonucleotide synthesis and2000).DNA sequencing, and Joan Curcio, Chuck Lowry, Randy Morse, DerekIsomerization of the CTD may generate a diverse andScholes, and members of the Hanes lab for helpful discussions and/dynamic platform for protein binding: The transitionor critical comments on the manuscript. A.R. was supported by a

between discrete steps in transcription, such as that be- National Science Foundation Research Experience for Undergradu-tween elongation and termination, is likely to be a com- ates grant BIR-9987844. This work was supported by National Institutes

of Health grant R01-GM55108 (S.D.H.).plicated process involving the exchange of many differ-ent proteins, perhaps multiple copies of each protein,and this exchange may not occur in an all-or-none fash-ion. Ess1-dependent conformational changes might be LITERATURE CITEDcapable of coordinating such complicated exchanges,

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The ESS1 Prolyl Isomerase and Its Suppressor BYE1 Interact ... · induced by the Ess1 peptidyl-prolyl cis/trans isomerase. Here, we examined the role of ESS1 in transcription by studying