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Genetics, Vol. 173, 1871-1884, August 2006, Copyright © 2006
doi:10.1534/genetics.106.058834
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* Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, Department of Biochemistry, School of Medicine and Biomedical Sciences, SUNY, Buffalo, NY 14214-3000 and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada
2 Corresponding author: Department of Molecular and Cell Biology, 408 Barker Hall, University of California, Berkeley, CA 94720-3202.
E-mail: kanecm{at}berkeley.edu
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ABSTRACT |
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and taf14, we discovered genetic interactions between PPR2 and both TFG1 and TFG2 encoding the two larger subunits of the TFIIF complex that also contains Taf14p. Mutant alleles of tfg1 or tfg2 that render cells cold sensitive have improved growth at low temperature in the absence of TFIIS. Remarkably, the amino-terminal 130 amino acids of TFIIS, which are dispensable for the known in vitro and in vivo activities of TFIIS, are required to complement the lethality in taf14 ppr2 cells. Analyses of deletion and chimeric gene constructs of PPR2 implicate contributions by different regions of this N-terminal domain. No strong common phenotypes were identified for the ppr2 and taf14 strains, implying that the proteins are not functionally redundant. Instead, the absence of Taf14p in the cell appears to create a dependence on an undefined function of TFIIS mediated by its N-terminal region. This region of TFIIS is also at least in part responsible for the deleterious effect of TFIIS on tfg1 or tfg2 cold-sensitive cells. Together, these results suggest a physiologically relevant functional connection between TFIIS and TFIIF.
The gene encoding Taf14p is nonessential, although disruption of TAF14 renders cells temperature sensitive and alters both constitutive and induced transcription (WELCH and DRUBIN 1994; CAIRNS et al. 1996). In yeast, taf14 creates synthetic growth defects with swi1, snf2, and snf5 mutants (DAVIE 1998), whereas overexpression of Taf14p suppresses synthetic interactions between mutations in the Mediator subunit soh1 and ppr2 (MALAGON et al. 2004). Cells lacking Taf14p require TFIIS for viability, and as we report here, the N-terminal domain of TFIIS is required for this viability. While TFIIS biochemical and genetic associations with transcript elongation are well documented (reviewed in FISH and KANE 2002), no Taf14p link to transcript elongation has been reported previously.
The TFIIF complex is one of six nuclear complexes with which Taf14p is associated (HENRY et al. 1992, 1994; CAIRNS et al. 1996; TREICH and CARLSON 1997; EBERHARTER et al. 1998; STEGER et al. 1998; JOHN et al. 2000; SHEN et al. 2000; KABANI et al. 2005). In addition to the nonessential Taf14 (Tfg3) subunit, yeast TFIIF comprises the essential Tfg1 and Tfg2 polypeptides, which are homologous to the RAP74 and RAP30 subunits of human TFIIF, respectively. Numerous studies of both yeast and mammalian TFIIF have revealed the complex function of this general transcription factor, as it has been implicated in multiple steps of the transcription cycle that include delivery of RNAPII to the preinitiation complex (PIC), transcription start site utilization, wrapping of promoter DNA around the PIC, promoter clearance of the polymerase, enhancing elongation efficiency, and stimulating the activity of the RNAPII C-terminal domain (CTD) phosphatase Fcp1 (summarized and referenced in GHAZY et al. 2004). Its elongation stimulatory mechanism is distinct from that of TFIIS, however, and TFIIF can even interfere with the activity of TFIIS in vitro (ELMENDORF et al. 2001; ZHANG and BURTON 2004). The results from the genetic studies described herein not only implicate TFIIF as the primary candidate of the TAF14:PPR2 interaction, but also support the view that TFIIF and TFIIS functionally interact to control transcription in vivo.
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MATERIALS AND METHODS |
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Plasmids:
Plasmids used in this work are listed in Table 2. Construction details are available upon request. All PPR2-containing constructs were sequenced and tested for their ability to rescue the 6AU sensitivity of CMKy3, a PPR2 disruption strain. The TFIIS constructs are diagrammed in Figure 1. Plasmids (TRP1 containing, pRS314 based) expressing epitope-tagged versions of wild-type Tfg1, Tfg1-E346A, Tfg1-W350A, wild-type Tfg2, Tfg2-L59K, or Tfg2-L303R were described previously (GHAZY et al. 2004). Integration plasmids p306/g2-L59K and p306/g2-L303R contained an 
2800-bp insert containing 800 bp upstream and 1200 bp downstream of the TFG2 coding region.
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Gene disruptions and integrations:
Disruptions of PPR2 were made by replacing codons 40–284 of PPR2 with the hisG-URA3-hisG cassette (ALANI et al. 1987). The disruption was confirmed by Southern blot or PCR analysis on the 6AU-sensitive transformants. To select for uracil auxotrophy, strains containing a ppr2::hisG-URA3-hisG disruption were plated onto 5-FOA medium (ALANI et al. 1987), and surviving colonies were recovered. Strains containing a TRP1 disruption were made by inserting the hisG-URA3-hisG cassette into the middle of the TRP1 gene using pNKY1009 (ALANI et al. 1987). Complete disruptions of TAF14 were made in a diploid PPR2/ppr2 strain (CMKy35) by replacing one copy of the entire target gene with the kanr gene (GULDENER et al. 1996). Transformants were selected on G418-containing solid media and replica plated to new G418 solid media after 48 hr. Correct integrants were identified by PCR. Integrants of tfg2 mutations in strain CH1305 were constructed by standard two-step gene replacement (ROTHSTEIN 1991) using SphI-linearized p306/g2-L59K or p306/g2-L303R (GHAZY et al. 2004). Correct integrations were again confirmed by genomic PCR.
Strain recovery and tetrad analysis:
Synthetic interactions were identified by tetrad analysis of heterozygous diploids. Tetrad analyses required special care to ensure germination and strain recovery. Thus, after sporulation of the TAF14/taf14::kanr PPR2/ppr2::hisG-URA3-hisG TRP1/trp1::hisG diploid (CMKy68), tetrads were dissected on YPD solid media (plate A) and then incubated at 30° for 7 days. The colony size at every spore position was noted, and in the case of extremely small colonies the cells were transferred to fresh YPD media (plate B) and incubated for 1 week at 25° or 30°. Plate A was replica plated to G418, SC-Trp, and SC-Ura media to determine genotypes. The ability of different TFIIS constructs to rescue viability of ppr2 taf14 strains also was assayed by tetrad analysis. After sporulation of the TAF14/taf14::kanr ppr2::hisG/ppr2::hisG diploid (CMKy91) containing a pRS315-derived plasmid, tetrads were dissected on YPD media (plate A) and incubated at 30° for 4–5 days. Plate A was replica plated to G418 and SC-Leu media to determine genotypes. Each spore position was examined to assess whether germination had occurred. Extremely small colonies from plate A were transferred to fresh YPD solid media and to G418 and SC-Leu media to determine genotypes.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Synthetic lethality between null alleles of TAF14 and PPR2:
Strains disrupted in either taf14 or ppr2 are viable at 30°. Haploid strains containing each mutant were mated, the heterozygous TAF14/taf14::kanr, PPR2/ppr2::URA3 diploid was sporulated, and 52 tetrads were dissected. The tetrad analysis clearly demonstrated that the taf14 allele is synthetically lethal in conjunction with a ppr2 mutation (Figure 2). The genotypes were determined by replica plating tetrads to selective media and by genomic PCR. The ppr2 taf14 segregants grew to barely visible microcolonies (1000 cells) after 3 days of incubation, and no further growth was observed after an additional week of incubation. No viable cells could be recovered from these microcolonies. The taf14 strains also grew slowly even at the permissive temperature in a PPR2 background (Figure 2) (WELCH and DRUBIN 1994). These results confirm a synthetic interaction between TAF14 and PPR2 that permits <10 cell divisions before loss of viability.
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and ppr2 strains: and ppr2 strains might highlight a common or interacting function of the two proteins. To investigate this, each individual disruption was tested for growth under a variety of conditions. The taf14 strains are characterized by a temperature-sensitive phenotype, a sensitivity to high osmotic strength medium (WELCH et al. 1993), aberrant morphology, and cytoskeletal defects (WELCH and DRUBIN 1994). The osmotic stress phenotype is most clearly demonstrated on 0.9 M NaCl medium, and modest sensitivity is also observed on 1.8 M sorbitol and 1.2 M KCl medium (WELCH et al. 1993). It has been shown previously that ppr2 strains are slightly temperature sensitive (HEMMING et al. 2000) and that 1 M sorbitol medium suppresses the mild temperature sensitivity of the ppr2 strains. In contrast, growth of a taf14 strain on 1 M sorbitol medium did not suppress the temperature sensitivity of the taf14 strain (data not shown). The ppr2 strain grew much better than the taf14 strain on 0.9 M NaCl medium, but not as well as the wild-type strain (Figure 3A). This observation was extended over three genetic backgrounds that included CH1305, W303, and YPH499. Cells disrupted for PPR2 exhibit slightly aberrant morphology in stationary phase, although the morphology defect is not as severe as for taf14 cells (WELCH et al. 1993; CHRISTIE 1995). The ppr2 strain was very sensitive to 6AU and slightly sensitive to MPA, whereas the taf14 strain exhibited no sensitivity to 6AU (Figure 3B) and a greater sensitivity to MPA than the ppr2 cells (Figure 3C).
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and ppr2 strains have been identified. These results suggest that Taf14p and TFIIS are not functionally redundant, but rather have a functional interaction within a cellular process, most likely transcriptional elongation.
The N-terminal region of TFIIS is necessary, but not sufficient, to complement the TAF14-PPR2 synthetic interaction:
The C-terminal region of TFIIS harbors all of the known in vivo and in vitro functions of this transcription factor (FISH and KANE 2002). As noted earlier, this region alone can rescue the synthetic lethality between disruptions in PPR2 and genes encoding the SWI1, SNF2, or SNF5 components of the Swi/Snf complex (DAVIE and KANE 2000). In addition, a PPR2 gene encoding amino acids 131–309 of yeast TFIIS is sufficient to complement the 6AU sensitivity of a ppr2 strain (DAVIE and KANE 2000). This truncated protein has full activity in cleavage and readthrough assays (CHRISTIE et al. 1994; CHRISTIE 1995), and binds to purified RNAPII as efficiently as full-length TFIIS in vitro (AWREY et al. 1998).
Despite the fact that the C-terminal region of TFIIS contains all of its known activities, a plasmid expressing the C-terminal 178 amino acids surprisingly was unable to complement the synthetic lethality between ppr2 and taf14 (Figure 4). In this experiment, a TAF14/taf14 ppr2/ppr2 diploid strain was transformed with a plasmid containing either full-length PPR2 (pJD27, encoding amino acids 1–309) or the truncated gene (pJD29, encoding amino acids 131–309). The resulting diploids were sporulated and 40 tetrads for each were dissected and analyzed for the presence of ppr2 taf14 haploid strains carrying a plasmid-borne PPR2 gene for viability; 10 dissected tetrads are shown for each transformation (Figure 4). For the 10 tetrads shown from the diploid containing the full-length TFIIS plasmid, 26 of the 40 spores gave rise to viable haploid strains; 8 of these, which grew slowly, were ppr2 taf14 strains carrying the full-length PPR2 gene (Figure 4A). Overall, from the 40 tetrads, 34 spores gave rise to slow-growing ppr2 taf14 strains carrying the full-length PPR2 gene. In significant contrast, no viable double disruption mutants were recovered when the plasmid containing the truncated PPR2 gene was present (Figure 4B). This unexpected outcome also was obtained using the same strategy in a different strain background (data not shown). Thus, these results strongly suggest that TFIIS lacking residues 1–130 is unable to complement the synthetic lethality between ppr2 and taf14, further distinguishing the interaction of PPR2 and TAF14 from that with SWI1, SNF2, or SNF5.
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The "Elongin" and "variable" regions of TFIIS domain I contribute to the genetic interaction between PPR2 and TAF14:
The preceding results indicated that the presence of domain I was required for TFIIS to complement the synthetic lethality between PPR2 and TAF14. To identify determinants within domain I important for the genetic interaction with TAF14, further dissection of this domain was carried out. Initially, two N-terminal deletion variants of PPR2 were constructed and tested for their ability to restore viability to a ppr2 taf14 strain. Construct V35 encodes a TFIIS protein lacking the first two 
-helices of domain I, while all four helices are removed in the K78 construct (Figure 1). In control experiments, both of these deletion constructs were able to rescue the 6AU sensitivity of a ppr2 strain (Table 3) and growth curves in the presence or absence of 6AU indicated that each strain grew equivalently to one carrying a plasmid encoding full-length TFIIS (data not shown). Western blot analysis further demonstrated that the steady-state protein levels for both mutant proteins were equivalent to that of wild-type TFIIS (data not shown). Importantly, however, tetrad analysis of CMKy91 transformants revealed that neither N-terminal deletion variant was able to restore viability to a ppr2 taf14 strain (data not shown).
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TFIIS is species specific in that the yeast and human proteins do not function with each other's RNAPII molecules (reviewed in FISH and KANE 2002). This specificity has been shown to reside in the polymerase-binding domain II (AWREY et al. 1998) and in a flexible linker connecting domains II and III (SHIMASAKI and KANE 2000). Any contribution by domain I to this species specificity has not been tested previously because there were no phenotypes to evaluate. However, requirement for the N-terminal region in taf14 cells allowed a direct test of regions in the N-terminal region of human TFIIS that might substitute for the yeast sequence. Chimeric human–yeast N-terminal regions were fused to sequences encoding amino acids 131–309 of the yeast TFIIS protein (Figure 1) and tested for function in vivo. A construct containing the complete human N-terminal region attached to the yeast C-terminal region (hI-yII-yIII) was able to rescue the 6AU sensitivity of a ppr2 strain (Table 3). This result might have been predicted on the basis of results presented above, wherein the yeast C-terminal region alone is functional in this assay. However, analysis of tetrads resulting from CMKy91 transformed with this same hI-yII-yIII construct revealed no viable ppr2 taf14 double disruption mutants (Figure 5A and Table 3). Thus, this result indicated that there must be species-specific determinants for the genetic interaction between PPR2 and TAF14 within the N-terminal domain of yeast TFIIS.
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taf14 strain. Further, these results, in conjunction with the N-terminal deletion studies, indicate that both regions of the N-terminal domain are required to restore viability to a ppr2 taf14 strain and that the variable region shows species specificity in this requirement.
Genetic interactions between TFIIS and the TFIIF complex:
Taf14p is a subunit of six distinct nuclear complexes that include TFIIF, TFIID, Ino80, SWI/SNF, RSC, and NuA3 (HENRY et al. 1992, 1994; CAIRNS et al. 1996; TREICH and CARLSON 1997; EBERHARTER et al. 1998; STEGER et al. 1998; JOHN et al. 2000; SHEN et al. 2000; KABANI et al. 2005). The role of TFIIF in both transcription initiation and elongation made this complex an attractive candidate for the observed genetic interactions between TAF14 and PPR2. The genes encoding the other two TFIIF subunits, TFG1 and TFG2, are essential. Thus, conditional mutants in each gene were tested for synthetic interactions with ppr2.
Two conditional tfg1 alleles were tested for genetic interactions with ppr2. The tfg1 mutants contained an alanine substitution for glutamic acid 346 (E346A) or an alanine substitution for tryptophan 350 (W350A), and each exhibits reduced growth rates at 16° or 37°, respectively (GHAZY et al. 2004). Both substitutions also confer upstream shifts in transcription start site utilization in vivo and in vitro (GHAZY et al. 2004). When a plasmid shuffle strategy was used, the double tfg1-E346A ppr2 mutants grew somewhat better than the single tfg1-E346A mutant at all temperatures tested (Figure 6B). In contrast, the tfg1-W350A allele, itself conferring temperature and cold sensitivity, was synthetically lethal with ppr2 (Figure 6A).
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or ppr2 for synthetic phenotypes at the permissive temperature. The tfg2 mutants tested contained a lysine substitution for leucine 59 (L59K), or an arginine substitution for leucine 303 (L303R); the mutants exhibit reduced growth rates at 37° or 16°, respectively (GHAZY et al. 2004). The tfg2-L59K mutation confers altered start site utilization in vivo and in vitro (GHAZY et al. 2004), whereas the tfg2-L303R mutant exhibits normal transcription initiation. Strains containing each of these mutations were crossed to taf14 cells (CMKy70), the resulting diploids were sporulated, and tetrads were analyzed for synthetic growth phenotypes. Double mutants were viable, but exhibited a synthetic growth defect even at 26° (data not shown).
Using the plasmid-shuffle strategy, each tfg2 mutant was then tested in combination with ppr2. In the absence of TFIIS, cells containing tfg2-L59K grew less well at 37°; in contrast, the tfg2-L303R ppr2 strain grew better at 16° than the tfg2-L303R strain (Figure 7, A and B). This improvement in growth was even more pronounced when both mutations were carried on the chromosome (Figure 7C).
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cells, also plays a role in the observed genetic interaction between PPR2 and TFG2. To test this, plasmids encoding full-length TFIIS or truncated TFIIS (amino acids 131–309; yIIyIII) were transformed into the ppr2 tfg2-L303R strain (CMKy132) and the transformants were tested for growth at the permissive (30°) and restrictive (16°) temperatures. Compared to the transformant with control vector alone (pRS315), the transformant expressing the N-terminally truncated TFIIS grew slightly less well at the restrictive temperature but significantly better than the transformant expressing full-length TFIIS (Figure 7C and Table 3). Thus, these results demonstrate that the N-terminal region of TFIIS harbors an unidentified activity that is both deleterious to a strain containing the tfg2-L303R allele and essential for the viability of a strain containing a deletion of taf14. |
DISCUSSION |
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and the ability of the C-terminal half of TFIIS to complement the synthetic lethality. Additional studies have also shown that the SWI/SNF complex can influence promoter clearance and elongation, and that SWI/SNF becomes essential in cells lacking TFIIS (SULLIVAN et al. 2001; COREY et al. 2003).
To further investigate the genetic interactions between TFIIS and SWI/SNF, we analyzed the growth properties of cells containing a PPR2 deletion in combination with a deletion of the TAF14 subunit of the SWI/SNF complex. As was observed with deletions of SWI1, SNF2, or SNF5, cells containing a deletion of TAF14 were synthetically lethal in combination with ppr2 (Figure 2). Importantly, however, complementation of the growth defects in ppr2 taf14 cells required full-length TFIIS including the N-terminal domain I (Figure 4), in contrast to the dispensability of domain I for complementation of the growth defects seen in ppr2 cells containing deletions of other SWI/SNF components (DAVIE and KANE 2000). This result, along with additional phenotypic differences, suggested that the TAF14:PPR2 synthetic interaction was distinct from the interactions between TFIIS and other components of the SWI/SNF complex.
In addition to the SWI/SNF complex, Taf14p is a member of five additional distinct nuclear complexes that include Ino80, NuA3, RSC, TFIID, and TFIIF (HENRY et al. 1992, 1994; CAIRNS et al. 1996; TREICH and CARLSON 1997; EBERHARTER et al. 1998; STEGER et al. 1998; JOHN et al. 2000; SHEN et al. 2000; KABANI et al. 2005). To date, no growth defects have been observed in ppr2 cells carrying disruptions in either INO80 or SAS3 [the latter the catalytic subunit of NuA3 (JOHN et al. 2000); data not shown], effectively ruling out these complexes as being responsible for the ppr2 taf14 phenotype. While TFIID and RSC remain to be tested, our results reported here provide evidence for complex genetic interactions between ppr2 and conditional alleles in the genes encoding the two larger TFIIF subunits, TFG1 and TFG2. These included synthetic lethality between ppr2 and temperature-sensitive alleles of TFG1 or TFG2, the ability of the ppr2 allele to partially suppress the cold sensitivity conferred by TFG1 or TFG2 alleles, and the deleterious effect of TFIIS domain I on this suppression (see below).
TFIIS domain I and TFIIF:
Genetic interactions between ppr2 and tfg1 and tfg2 alleles focus attention on TFIIF as the complex responsible for the ppr2 taf14 synthetic lethality. In contrast to the requirement for full-length TFIIS for viability of taf14 cells, the partial suppression of the cold-sensitive phenotype of tfg1 and tfg2 alleles required the absence of TFIIS, and specifically for the tfg2-L303R strain, deletion of domain I. Although the underlying mechanisms are unclear, the fact that domain I is required in the absence of the TFIIF subunit Taf14p, yet domain I is deleterious in the presence of the Tfg2-L303R mutant protein, strongly suggests that this region of the TFIIS protein has functional significance for TFIIF function. As noted earlier, previous work had shown that this region of TFIIS was dispensable for all of the known in vitro activities of TFIIS as well as the ability to complement the 6AU-sensitivity of ppr2 cells (HORIKOSHI et al. 1985; HIRASHIMA et al. 1988; AGARWAL et al. 1991; REINES 1992; GUO and PRICE 1993; CHRISTIE et al. 1994; NAKANISHI et al. 1995). The first 75 amino acids of domain I compose a conserved four-helix bundle and have been referred to as the Elongin (sub)domain (BOOTH et al. 2000). Analysis of chimeric PPR2 constructs demonstrated that either human or yeast sequences in this region were able to complement the ppr2 taf14 strain (Figure 5). The remaining 60 residues in the N-terminal half of TFIIS are more variable among organisms, and a chimeric gene containing human sequences in this region was unable to complement ppr2 taf14 cells (Figure 5). This variable domain of the mouse protein has been suggested to undergo differential phosphorylation in vivo (SEKIMIZU et al. 1981), and the observed variation in sequence and length in tissue-specific isoforms of metazoan TFIIS molecules raises the possibility of isoform-specific TFIIS functions (UMEHARA et al. 1997; LABHART and MORGAN 1998). The variable region might serve as a platform for interaction with other proteins, or it may provide a flexible tether between the four-helix bundle and the C-terminal half of TFIIS that is known to interact with RNA polymerase II (BOOTH et al. 2000; KETTENBERGER et al. 2003).
In human and Xenopus cells, domain I appears to play a role in nuclear targeting of TFIIS (G. MORGAN, University of Nottingham, personal communication). However, yeast TFIIS lacking domain I still localizes to the nucleus (data not shown). Indeed, a specific karyopherin encoded by NMD5 has been shown to be involved in TFIIS transport in yeast (ALBERTINI et al. 1998). This nuclear localization of TFIIS lacking domain I also occurs in taf14 cells, and Taf14p remains nuclear in ppr2 cells (data not shown). Thus, the necessity for domain I in complementing ppr2taf14 synthetic lethality is not due to a role in localizing TFIIS to the nucleus.
Additional physical and genetic interactions involving domain I have also been reported. This region alone can interact with RNAPII-containing complexes during affinity chromatography (PAN et al. 1997), suggesting that domain I may facilitate interactions between TFIIS and other proteins found in one or more holoenzyme complexes. For example, TFIIS is physically associated with a Paf1p-containing RNAPII complex (WADE et al. 1996) and PPR2 exhibits genetic interactions with PAF1 (SQUAZZO et al. 2002). However, the requirement for TFIIS domain I in these physical and genetic interactions remains to be determined. Domain I has also been shown to be essential for viability of a strain disrupted for RPB4 (WERY et al. 2004), a nonessential subunit of RNAPII important for viability at extreme temperatures (WOYCHIK and YOUNG 1989). Moreover, recent two-hybrid screening has revealed physical interactions between yeast TFIIS and both Srb9p and Spt8p that require domain I (WERY et al. 2004). Srb9p is a component of Mediator (HENGARTNER et al. 1995), whereas Spt8p is a subunit of SAGA, a coactivator with histone acetyltransferase activity (STERNER et al. 1999). The SAGA and Mediator complexes function during preinitiation complex formation (CHADICK and ASTURIAS 2005; TIMMERS and TORA 2005), and recent work indicates that TFIIS can associate with both initiation and elongation complexes from yeast (POKHOLOK et al. 2002; PRATHER et al. 2005; S. HAHN, personal communication). Thus, the functional association between TFIIS and Taf14p may reside with TFIIF in complexes during all these phases of transcription, although biochemical evidence demonstrating a role for TFIIS prior to the elongation phase of transcription has yet to be reported.
Complex genetic interactions between PPR2 and the genes encoding the three TFIIF subunits:
The tfg1-E346A and tfg1-W350A substitutions used in this study reside in a region of the protein proposed to be involved in interaction with Tfg2p and RNAPII, and the alleles confer pronounced or moderate upstream shifts in start site usage, respectively (GHAZY et al. 2004). Interestingly, the temperature-sensitive tfg1-W350A allele was synthetically lethal in combination with ppr2, whereas a strain containing the cold-sensitive tfg1-E346A allele grew better in combination with ppr2 (Figure 6). For the Tfg2 subunit, the two alleles used in this study encode mutations located in functionally distinct regions of the protein. The tfg2-L59K substitution is located within the domain reported to interact with Tfg1p, TFIIB, and RNAPII and confers both temperature sensitivity and modest upstream shifts in start site utilization (GHAZY et al. 2004). Cells containing this allele in combination with ppr2 grew less well than tfg2-L59K PPR2 cells at both 30° and 37° (Figure 7). In contrast, cells containing the cold-sensitive tfg2-L303R substitution, which resides within a DNA-binding domain that has similarity to the 
70 family of bacterial factors and does not confer any detectable start site alterations (GARRETT et al. 1992; TAN et al. 1994), grew better when in combination with ppr2 at 16° (Figure 7). Thus, irrespective of their effects on start site utilization, the TFIIF alleles conferring temperature sensitivity grew less well at the restrictive temperature in combination with a TFIIS null allele, whereas strains containing the TFIIF alleles conferring cold sensitivity grew better at 16° upon the loss of TFIIS.
How does one rationalize the positive and negative genetic effects between TFIIS and the subunits of TFIIF? In regard to the growth defects, the loss of TFIIS function was lethal to the taf14 temperature-sensitive strains and sickened cells with temperature-sensitive alleles of either TFG1 or TFG2. Although the role of Taf14p in the S. cerevisiae TFIIF complex remains unclear, it has been reported that the Taf14/Tfg3 homolog in the fission yeast Schizosaccharomyces pombe is involved in transcriptional regulation under stress conditions, most notably at elevated temperatures (KIMURA and ISHIHAMA 2004). Similarly, S. cerevisiae strains containing a TAF14 deletion are temperature sensitive (WELCH et al. 1993) and also exhibit sensitivity to osmotic stress (Figure 3). Thus, one possible explanation for the synthetic lethality between a TFIIS deletion and either a TAF14 deletion or temperature-sensitive TFIIF alleles is a combined deficiency for the proper transcription of stress-related genes, a testable hypothesis.
In contrast to the case with temperature-sensitive TFIIF alleles, deletion of TFIIS improved the growth of strains containing the cold-sensitive tfg1-E346A or tfg2-L303R mutations. Although the precise biochemical defects associated with TFIIF complexes containing either of these mutant subunits remains to be established, cold-sensitive mutations are often found to adversely affect interactions between protein complexes. Thus, these mutant TFIIF complexes might have a diminished interaction with RNAPII elongation complexes that is exacerbated further by the competitive binding of TFIIS to the polymerase. Indeed, tfg1-E346A has a reduced affinity for RNAPII by gel shift analysis (GHAZY et al. 2004). Although both TFIIS and TFIIF can stimulate transcription in vitro, biochemical evidence suggests that they act independently, that they function through distinct mechanisms, and that their relationship may be antagonistic, at least during elongation (UPTAIN et al. 1997; ELMENDORF et al. 2001; SHILATIFARD et al. 2003; ZHANG and BURTON 2004). TFIIF (and mechanistically similar factors Elongin and ELL) increases the overall rate of elongation and suppresses pausing, whereas TFIIS acts upon stalled or arrested RNAPII elongation complexes in which the growing 3' end of the nascent transcript has lost register with the catalytic site of the polymerase (IZBAN and LUSE 1992; REINES 1992; IZBAN and LUSE 1993; GU and REINES 1995). TFIIS stimulates transcript hydrolysis by the polymerase to generate a new 3'-OH aligned with the catalytic center, a process that enables resumption of RNA synthesis (IZBAN and LUSE 1992; REINES et al. 1992). Importantly, however, Elmendorf and co-workers reported that TFIIF inhibited the TFIIS-induced cleavage of nascent transcripts by paused RNAPII in vitro, and proposed that TFIIF maintains the proper alignment of the 3' end of the nascent transcript with the polymerase active site (ELMENDORF et al. 2001). Thus, this property of TFIIF would presumably contribute to increased elongation efficiency by ensuring that nonarrested RNAPII elongation complexes are no longer prone to TFIIS-induced cleavage. Relatedly, Burton and co-workers have suggested that TFIIF stabilizes one of several conformations of the ternary elongation complex and that this stabilized conformation would not be sensitive to TFIIS (ZHANG and BURTON 2004). Determining the in vitro effects of the mutant TFIIF proteins on TFIIS-induced transcript cleavage should prove useful in testing these possibilities.
Structural studies with co-complexes of either TFIIS or TFIIF with yeast RNAPII allow some additional predictions regarding the functional interplay of TFIIF and TFIIS (CHUNG et al. 2003; KETTENBERGER et al. 2003; ASTURIAS 2004). When either binds to RNAPII, significant movement is observed among the mobile regions of RNAPII (CHUNG et al. 2003; KETTENBERGER et al. 2003). A comparison of the co-crystal structure containing RNAPII and the C-terminal half of TFIIS (KETTENBERGER et al. 2003) with a cryo-electron microscope-determined structure of an RNAPII:TFIIF complex (CHUNG et al. 2003) suggests that both TFIIF and the C-terminal half of TFIIS closely approach RNAPII subunit Rpb5p. Physical associations between Rpb5p and TFIIS, Taf14p, and Tfg2p were also observed during large-scale stringent protein affinity purification (KROGAN et al. 2004). Although Rpb5p has been shown to play a role in transcriptional activation (MIYAO and WOYCHIK 1998), it is unclear whether it influences initiation or elongation. It should also be noted that both structures were determined using purified RNAPII, not using an elongation complex, and there are significant structural changes during elongation that might compromise some of these predictions about protein interactions (GNATT et al. 2001). In addition, the TFIIS-containing co-crystal complex lacks domain I. One could speculate that the flexible variable region in domain I allows the conserved four-helix bundle to make contact with Rpb5p, additional RNAPII subunits, and/or TFIIF subunits. Both TFIIS and TFIIF also have physical interactions with the mediator complex, and TFIIF is proposed to be essential in the transition from initiation to elongation that involves a mediator:elongator handoff (OTERO et al. 1999; HSU 2002a,b). Perhaps TFIIS functions during this process as well, and its recent identification in association with the GAL1 promoter in yeast positions it for this type of activity (PRATHER et al. 2005). It might be poised, along with TFIIF, to assist in promoter escape like its functional homolog in bacteria, GreA (HSU et al. 1995). Further genetic and biochemical studies will be needed to elucidate the mechanistic interplay between TFIIF and TFIIS during the various stages of the transcription cycle.
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