Genetic Interactions of Spt4-Spt5 and TFIIS With the RNA Polymerase II CTD and CTD Modifying Enzymes in Saccharomyces cerevisiae

Corresponding author: Grant A. Hartzog, Department of MCD Biology, 349 Sinsheimer Labs, University of California, Santa Cruz, CA 95064., hartzog{at}biology.ucsc.edu (E-mail)

Communicating editor: M. JOHNSON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genetic and biochemical studies have identified many factors thought to be important for transcription elongation. We investigated relationships between three classes of these factors: (1) transcription elongation factors Spt4-Spt5, TFIIS, and Spt16; (2) the C-terminal heptapeptide repeat domain (CTD) of RNA polymerase II; and (3) protein kinases that phosphorylate the CTD and a phosphatase that dephosphorylates it. We observe that spt4 and spt5 mutations cause strong synthetic phenotypes in combination with mutations that shorten or alter the composition of the CTD; affect the Kin28, Bur1, or Ctk1 CTD kinases; and affect the CTD phosphatase Fcp1. We show that Spt5 co-immunoprecipitates with RNA polymerase II that has either a hyper- or a hypophosphorylated CTD. Furthermore, mutation of the CTD or of CTD modifying enzymes does not affect the ability of Spt5 to bind RNA polymerase II. We find a similar set of genetic interactions between the CTD, CTD modifying enzymes, and TFIIS. In contrast, an spt16 mutation did not show these interactions. These results suggest that the CTD plays a key role in modulating elongation in vivo and that at least a subset of elongation factors are dependent upon the CTD for their normal function.

THE carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II) consists of 26 (in yeast) to 52 (in humans) repeats of the heptapeptide YSPTSPS (DAHMUS 1996 ). The CTD is unphosphorylated prior to transcription initiation and becomes highly phosphorylated soon thereafter. Although CTD phosphorylation is a marker of elongating Pol II, the exact role of the CTD in elongation is not clear (DAHMUS 1996 ). The phosphorylation state or even the presence of the CTD has no effect on the intrinsic elongation activity of Pol II (KIM and DAHMUS 1988 ). However, the observations that CTD kinases and a CTD phosphatase regulate elongation and that truncation of the CTD perturbs elongation suggest that elongation is regulated by CTD phosphorylation in vivo (AKHTAR et al. 1996 ; CHO et al. 1999 ; KOBOR et al. 1999 ; PRICE 2000 ).

Enzymes that modify the CTD's phosphorylation state have been identified in many organisms. Fcp1, a conserved CTD phosphatase, is required for transcription of most yeast genes and may play both positive and negative roles in elongation (CHO et al. 1999 ; KOBOR et al. 1999 ). Several conserved cyclin-dependent kinases are CTD kinases. Srb10/Cdk8 represses transcription by phosphorylating free Pol II and thereby preventing assembly of preinitiation complexes (HENGARTNER et al. 1998 ; SUN et al. 1998 ; GU et al. 1999 ). Kin28/Cdk7, a subunit of general transcription initiation factor TFIIH, is not required for initiation but plays an essential role after initiation, either in promoter escape or early elongation (LEE and YOUNG 2000 ). P-TEFb, a CTD kinase studied in humans and Drosophila, is required for efficient elongation but not initiation in vitro (MARSHALL and PRICE 1995 ; MANCEBO et al. 1997 ; ZHU et al. 1997 ). In yeast, Ctk1 and Bur1/Sgv1 are 42% identical to Cdk9, the catalytic subunit of P-TEFb (ZHU et al. 1997 ). Both Ctk1 and Bur1 can phosphorylate the CTD in vitro and are implicated in the regulation of elongation (LEE and GREENLEAF 1991 , LEE and GREENLEAF 1997 ; ZHU et al. 1997 ; MURRAY et al. 2001 ).

In vitro transcription studies have shown that when P-TEFb is absent or inhibited, transcription elongation, but not initiation, is inhibited (MARSHALL et al. 1996 ; WADA et al. 1998B ). Inhibition of elongation in the absence of P-TEFb function is dependent upon two protein complexes, DSIF and NELF (WADA et al. 1998A ; YAMAGUCHI et al. 1999 ). Interestingly, DSIF can also promote elongation in vitro when nucleotide concentrations are limiting, and both DSIF and P-TEFb are required for Tat-mediated stimulation of HIV transcription in vitro (WADA et al. 1998A ; WU-BAER et al. 1998 ; IVANOV et al. 2000 ; PRICE 2000 ). Elucidating the mechanisms of action and interplay between the CTD, P-TEFb, and DSIF are of clear importance to our understanding of transcription elongation and the regulation of HIV transcription.

DSIF has two subunits, Supt4H and Supt5H, which are conserved across eukaryotes (CHIANG et al. 1996A , CHIANG et al. 1996B ; HARTZOG et al. 1996 ; WADA et al. 1998A ; ANDRULIS et al. 2000 ; GUO et al. 2000 ; KAPLAN et al. 2000 ). The yeast homologs of these proteins, Spt4 and Spt5, also form a protein complex that we refer to as Spt4-Spt5 (HARTZOG et al. 1998 ). Early studies of Spt4-Spt5 led to the model that it affects transcription through interactions with chromatin (SWANSON and WINSTON 1992 ). More recent observations in yeast, including genetic interactions with Pol II subunits and transcription elongation factor TFIIS, implicate Spt4-Spt5 in transcription elongation (HARTZOG et al. 1998 ). Furthermore, like DSIF, Spt4-Spt5 co-immunoprecipitates with Pol II (HARTZOG et al. 1998 ; WADA et al. 1998A ). These observations led us to propose that Spt4-Spt5 regulates transcription elongation by mediating interactions between Pol II and nucleosomes (HARTZOG et al. 1998 ).

In this study, we used genetic analysis in Saccharomyces cerevisiae to investigate the in vivo dependence of Spt4-Spt5 on the Pol II CTD and on regulators of CTD phosphorylation. We find that Spt4-Spt5 function depends on the length of the CTD, the presence of particular phosphoacceptors within the CTD, the function of at least three CTD kinases, and a CTD phosphatase. Interestingly, we find that Spt5 binds to both hypophosphorylated Pol II (Pol II_A) and hyperphosphorylated Pol II (Pol II_O) and that partial truncation of the CTD, or mutations in CTD kinases or FCP1, do not affect Spt5-Pol II binding. Furthermore, we show that transcription elongation factor TFIIS is similarly dependent upon the CTD and CTD kinases.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Media and genetic methods:
Strain construction and other genetic manipulations were carried out by standard methods (ROSE et al. 1990 ). Yeast media, including rich (YPD), minimal (SD), synthetic complete (SC), and 5-fluoroorotic acid (5-FOA) media were made as described previously (ROSE et al. 1990 ). All GHY and FY S. cerevisiae strains used in this study (Table 1) are isogenic to S288C (WINSTON et al. 1995 ). The ctk1::HIS3 mutation was created by cutting plasmid pSZH4 (STERNER et al. 1995 ) with SnaBI and VspI and transforming the digested DNA into a diploid FY strain. This mutation was confirmed by PCR and genetic analysis. The fcp1-110 allele is derived from PCY335.

The dilution spotting growth assay was carried out by growing cells to saturation in liquid media. Cells were counted, pelleted, washed in sterile water, and diluted to 1 x 10⁷ cells/ml. Serial fivefold dilutions of these cells were pipetted onto 5-FOA (10-µl aliquots) or YPD plates (5-µl aliquots) and incubated at 30° for the time indicated. The results of these assays were consistent with those obtained from replica-plating assays using the same strains.

In the plasmid shuffle experiments, multiple transformants were scored for mutant phenotypes.

Plasmids:
All yeast plasmids used in this study were CEN plasmids. RPB1 (26 CTD repeats) is carried on plasmids pRP114 (LEU2) and pRP112 (URA3), rpb1-110 (13 CTD repeats) on pV5, rpb1-101 (11 CTD repeats) on pC1, and rpb1-103 (10 CTD repeats) on pC3 (NONET et al. 1987 ). Plasmids pY1WT(14), pY1WT(7)A5(7), and pY1WT(9)A2(6) have been described previously (WEST and CORDEN 1995 ). Plasmid pGH215, a LEU2 plasmid carrying kin28-16, was created by subcloning a 1.25-kb BamHI/XhoI fragment from YCpLac22-kin28-16 (a gift of Steve Buratowski) into pRS315. Plasmid pGP465, a LEU2 CEN plasmid carrying KIN28, was a gift from Greg Prelich. Plasmids pRS316-CTK1 and pSRB10 were gifts of Arno Greenleaf and Greg Prelich.

Immunoprecipitation and immunoblotting:
The anti-Spt5 antibody and the anti-CTD antibodies 8WG16, B3, H5, and H14 have been previously described (THOMPSON et al. 1989 ; WARREN et al. 1992 ; MORTILLARO et al. 1996 ; HARTZOG et al. 1998 ; PATTURAJAN et al. 1998 ). H5 and H14 were purchased from Covance (Richmond, CA). RPB1(yN-18) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-coupled secondary antibodies were purchased from Bio-Rad (Hercules, CA) and Santa Cruz Biotechnology.

Cells were grown to midlog phase in YPD unless otherwise stated, harvested, and quickly frozen in liquid nitrogen. The frozen cell pellets were ground to a powder with a mortar and pestle under liquid nitrogen and thawed in extract buffer (30 mM HEPES pH 7.4, 200 mM potassium acetate, 1 mM EGTA, 1 mM magnesium acetate, 10% glycerol, 0.05% Tween-20). All extract and elution buffers included the following protease inhibitors: 2 µm pepstatin A, 0.6 µm leupeptin, 2 µg/ml chymostatin, 2 mM benzamidine HCl, 1 mM phenylmethylsulfonyl fluoride. Crude extracts were clarified by centrifugation at 89,000 x g for 30 min. For immunoprecipitations, 5 mg of clarified extract was mixed with 20 µl anti-Flag (M2) antibody-conjugated beads (Sigma, St. Louis) and incubated for 2 hr at 4°. The beads were pelleted and washed four times with 25 volumes of extract buffer. Bound proteins were competitively eluted by incubating the beads with 500 µg/ml of the Flag peptide (Research Genetics, Huntsville, AL), Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, for 15 min. This elution step was repeated twice and eluates were pooled.

Immunoblots were carried out by standard procedures, using the Pierce (Rockford, IL) SuperSignal West Pico reagent to identify HRP-labeled secondary antibodies. To separate Pol II_A and Pol II_O, samples were electrophoresed on 20-cm-long 5% SDS-PAGE gels.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Interactions of SPT4 and SPT5 with rpb1-CTD truncation mutations:
To determine if yeast Spt4-Spt5 function depends on an intact CTD, we constructed spt rpb1 double mutants and used the plasmid shuffle assay to ask if they displayed synthetic mutant phenotypes, a hallmark behavior of genes involved in a common function (GUARENTE 1993 ). We focused on three recessive spt mutations: spt5-194 and spt4, which cause strong Spt^- phenotypes, suppress snf/swi mutations, and cause moderate (spt5-194) to strong (spt4) sensitivity to 6-azauracil, and spt5-242, which confers cold sensitivity and a weak Spt^- phenotype (SWANSON et al. 1991 ; SWANSON and WINSTON 1992 ; HARTZOG et al. 1996 , HARTZOG et al. 1998 ).

The spt rpb1 strains were transformed with a series of LEU2 plasmids carrying rpb1-CTD alleles. Leu⁺ Ura^- transformants, which had lost the wild-type RPB1 URA3 plasmid, were identified by growth on 5-FOA media and scored for mutant phenotypes (Table 2). The spt4 and spt5-194 strains were viable but slow growing when the CTD was truncated to 10 repeats (Table 2). In contrast, a strain carrying the spt5-242 mutation was barely viable when the Rpb1 CTD was truncated to 11 repeats and was inviable when the CTD was truncated to 10 repeats. Thus, the dependence on Spt4-Spt5 function for normal growth is determined in part by the length of the CTD.

Genetic interactions between CTD kinases and SPT4 and SPT5:
We used double mutant analysis to determine if Spt4-Spt5 function depends on one or more CTD kinases in vivo. Complete deletions of the nonessential CTK1 and SRB10 genes were used. In contrast, because BUR1 and KIN28 are essential for life, we used partial loss-of-function alleles, bur1-2 and kin28-16, for these genes. We found that spt4 ctk1 and spt4 kin28-16 double mutants are inviable (Fig 1). Synthetic slow growth defects were observed in spt5-194 ctk1 double mutants, and spt5-194 kin28-16 double mutants were inviable. Furthermore, we found that spt5-194 bur1-2 double mutants display moderate growth defects (data not shown) and we have previously reported that bur1-2 is synthetically lethal with spt4 and the spt5-4 mutation (MURRAY et al. 2001 ). In contrast, neither spt4 nor spt5-194 displayed new mutant phenotypes in combination with srb10.

View larger version (101K): In this window In a new window Download PPT slide   Figure 1. SPT4 and SPT5 display genetic interactions with SRB10, BUR1, CTK1, and KIN28. (A) Strains with the indicated genotypes were assayed by serial dilution onto YPD plates followed by incubation at 30° for 2 days. The indicated strains are as follows: WT, FY602; spt4, GHY166; srb10, GY785; spt4 srb10, GHY813; ctk1, GHY705; spt4 ctk1, GHY690; spt5-194, GHY13; spt5-194 srb10, GHY811; spt5-194 ctk1, GHY715; spt5-242, FY1635; and spt5-242 srb10, GHY845. (B) spt kin28 mutants carrying a URA3 KIN28 plasmid were transformed with a LEU2 kin28-16 plasmid (pGH215) or a LEU2 KIN28 plasmid (pGP465). Transformants were grown in SC-Leu media and serial dilutions were spotted to 5-FOA plates to select for cells that had lost the URA3 KIN28 plasmid. Plates were incubated 5 days at 30° and photographed. The indicated strains are as follows: SPT+, GHY848; spt4, GHY909; spt5-194, GHY881; and spt5-242, GHY938. (C) Summary of the observed growth phenotypes. Strains that grew to form clearly visible large colonies after 2 days were scored as +++; strains that gave poor growth with small colonies after 2 days were scored as ++; strains that grew poorly and did not give colonies clearly visible to the eye until day 3 or later were scored +; strains that were inviable were scored as -.

spt5-242 showed a distinct set of interactions with the CTD kinases. Like spt5-194, spt5-242 caused inviability when combined with kin28-16 (Fig 1B). In contrast, spt5-242 partially suppressed the slow growth phenotype of ctk1 at 30° but not at 15°, the nonpermissive temperature for both mutations (Fig 2 and data not shown). Also, spt5-242 srb10 cells displayed marked synthetic growth defects (Fig 1A). These data are summarized in Fig 1C. The extensive and allele-specific genetic interactions between SPT4, SPT5, and the genes encoding several different CTD kinases suggest that the CTD's phosphorylation state may influence Spt4-Spt5 function.

View larger version (53K): In this window In a new window Download PPT slide   Figure 2. The growth defect of ctk1 strains is suppressed by spt5-242. Strains were struck out to a YPD plate and incubated for 2 days at 30°. Note that the spt5-242 ctk1 double mutant gives larger colonies than the ctk1 single mutant. The indicated strains are as follows: WT, FY120; spt5-242, FY1634; ctk1, GHY630; spt5-242 ctk1, GHY726.

BUR1 and CTK1 interact genetically with KIN28 and the CTD:
It is possible that Kin28, Srb10, Bur1, and Ctk1 collaborate with one another or with other factors to regulate Spt4-Spt5 function. Consistent with this idea, Srb10 and Ctk1 have been reported to perform partially overlapping functions in vivo (KUCHIN and CARLSON 1998 ) and bur1-2 ctk1 double mutants are inviable (MURRAY et al. 2001 ). To extend this analysis, srb10, bur1-2, and ctk1 mutants were crossed to a kin28-16 strain and the double mutant progeny of these crosses were analyzed for new mutant phenotypes. The srb10 kin28-16 double mutants did not display any striking new mutant phenotypes (data not shown). In contrast, bur1-2 kin28-16 and ctk1 kin28-16 double mutants were barely viable, giving visible colonies only after prolonged incubation (Fig 3A and data not shown). We previously reported that a different kin28 mutation, kin28-ts4, does not cause synthetic phenotypes when combined with bur1-2 (MURRAY et al. 2001 ). Thus, our present observations may indicate an allele-specific BUR1 KIN28 interaction or may be due to differences in strain background between the two experiments.

View larger version (51K): In this window In a new window Download PPT slide   Figure 3. Normal Ctk1 function depends on Kin28 and the length and composition of the CTD. (A) Analysis of kin28 ctk1 double mutants. A kin28 strain (GHY848) and a kin28 ctk1 strain (GHY955), both carrying a URA3 KIN28 plasmid, were transformed with a LEU2 KIN28 plasmid or a LEU2 kin28-16 plasmid. Transformants were grown in SC-Leu media, spotted to 5-FOA plates, grown for 3 days at 30°, and photographed. (B) Analysis of ctk1 CTD truncation double mutants. A rpb1 (GHY498) strain and a ctk1 rpb1 strain (GHY665), each carrying a URA3 RPB1 plasmid, were transformed with the indicated LEU2rpb1 plasmids and grown to saturation in SC-LEU media. Transformants were spotted to 5-FOA plates and grown at 30° for 3 days. (C) Analysis of the effect of substituting alanine for serine 2 or 5 of the CTD in ctk1 mutants. The ctk1 rpb1 strain used in B was transformed with LEU2 CEN plasmids containing either the wild-type WT(26), WT(14), WT (9)A2(6), or WT(7)A5(7) alleles of rpb1. Transformants were struck out to a SC-Leu plate, grown for 3.5 days at 30°, and photographed. This plate was replica plated to a SC-Leu plate and a 5-FOA plate, each of which was grown for 2 days at 30° and photographed. The growth of CTK1+ strains with these rpb1 alleles is shown in Fig 4.

CTD truncation mutations are suppressed by srb10 mutations and enhanced by kin28 and bur1 mutations (NONET and YOUNG 1989 ; RODRIGUEZ et al. 2000 ; MURRAY et al. 2001 ). To determine if CTK1 also shows genetic interactions with the CTD, we constructed ctk1 rpb1 double mutants as described above for spt rpb1 double mutants. We found that truncating the CTD to <13 repeats caused ctk1 cells to be inviable (Fig 3B). Combined with the observation of synthetic phenotypes in ctk1 kin28-16 double mutants, these observations are consistent with the model that Bur1, Ctk1, and Kin28 are partially functionally redundant in vivo.

The predominant phosphorylation targets in Rpb1 of yeast are the serine 2 and 5 positions of the CTD repeat (Y₁S₂P₃T₄S₅P₆S₇; BENSAUDE et al. 1999 ). Substitution of serine 2 or 5 with alanine in every repeat of the CTD results in inviability, but rpb1 mutants in which only a subset of these serines are altered are viable (WEST and CORDEN 1995 ). To ask if the ability to phosphorylate position 2 or 5 of the CTD repeat is important for Ctk1 function in vivo, we performed plasmid shuffles on ctk1 rpb1 double mutants. The wild-type URA3 RPB1 plasmid was replaced with one of three LEU2 rpb1 plasmids: pY1AWT(14), in which the CTD is composed of 14 wild-type repeats; pY1WT(7)A5(7), in which the CTD contains 7 wild-type repeats followed by 7 repeats with a serine-to-alanine substitution at position 5; and pY1WT(9)A2(6), with 9 wild-type repeats followed by 6 repeats in which alanine is substituted for serine 2. Transformation of ctk1 rpb1 cells with these plasmids caused mild to strong dominant negative growth defects, and when replica plated to 5-FOA plates, neither the WT(9)A2(6) nor the WT(7)A5(7) plasmid could support viability (Fig 3C). Thus, in the absence of Ctk1, both the length and composition of the CTD take on an added importance in the cell.

Spt5 function depends upon the serine 2 and serine 5 positions of the CTD repeat:
The experiments described above demonstrate a link between Spt4-Spt5 and the Rpb1-CTD and CTD kinases. However, they do not prove that Spt4-Spt5 function depends on particular phosphorylation states of the CTD. Spt4-Spt5 may be directly phosphorylated and thereby regulated by one or more of the CTD kinases. To begin to determine if the in vivo function of Spt4-Spt5 depends on CTD phosphorylation states, we performed plasmid shuffles on spt rpb1 double mutants with the serine-to-alanine CTD substitution plasmids. In the presence of the WT(7)A5(7) rpb1 allele, the spt5-242 rpb1 strain was inviable, and the spt4 and spt5-194 strains displayed severe synthetic growth defects, giving rise to visible colonies only after prolonged incubation (Fig 4 and data not shown). In the presence of the WT(9)A2(6) allele, the spt5-194 strain showed no growth defects, the spt4 strain displayed a moderate growth defect, and the spt5-242 strain displayed severe growth defects (Fig 4). Taken together, these results suggest a requirement for the serine 2 and 5 residues in the CTD repeat for proper Spt4-Spt5 function.

View larger version (69K): In this window In a new window Download PPT slide   Figure 4. Alteration of the serine 2 and serine 5 positions of the CTD repeat causes synthetic phenotypes when combined with spt4 and spt5 mutations. Strains with the indicated SPT genotype, an rpb1 mutation, and a URA3 RPB1 CEN plasmid were transformed with a LEU2 plasmid carrying either the WT(14), WT(9)A2(6), or WT(7)A5(7) rpb1 alleles. Transformants were grown in SC-Leu media and serial dilutions were spotted to 5-FOA plates, which were incubated at 30° for 2 days and then photographed. Note that although the spt5-242 WT(7)A5(7) double mutant was inviable, small colonies were observed for the spt4 WT(7)A5(7) and the spt5-194 WT(7)A5(7) double mutants after 3 or more days of incubation. The indicated strains are as follows: SPT+, GHY498; spt4, GHY628; spt5-194, GHY950; spt5-242, GHY339.

Interactions of SPT4 and SPT5 with FCP1:
Following transcription termination, the CTD must be dephosphorylated before Pol II can be recycled into new preinitiation complexes (LEE and YOUNG 2000 ). To address the role of CTD dephosphorylation in Spt4-Spt5 function we performed double mutant analysis with FCP1 (KOBOR et al. 1999 ). We found that the recessive fcp1-110 mutation (COSTA and ARNDT 2000 ) causes moderate growth defects when combined with spt4 or spt5-242 and a severe growth defect in combination with spt5-194 (Fig 5). These observations are consistent with the idea that either hyper- or inappropriate phosphorylation of the CTD is deleterious in a spt mutant.

View larger version (79K): In this window In a new window Download PPT slide   Figure 5. Synthetic growth defects of fcp1-110 with spt4 and spt5 mutations. The indicated mutants were spotted onto YPD plates and grown for 2 days at 30°. The indicated strains are as follows: WT, GHY417; fcp1-110, OY143; spt4, GHY368; spt4 fcp1-110, OY155; spt5-194, GHY379; spt5-194 fcp1-110, OY152; spt5-242, GHY367; spt5-242 fcp1-110, OY157.

Spt5 associates with different Rpb1 phosphoisoforms:
To determine if Spt5's association with Pol II is dependent on a particular CTD phosphorylation state, we immunoprecipitated a Flag-epitope-tagged derivative of Spt5 (Spt5-Flag) from yeast extracts and analyzed the precipitates by immunoblotting for Rpb1, the largest subunit of Pol II. Using antibodies that recognize either hyper- or hypophosphorylated Pol II to probe the immunoblots, we observed that Spt5 immunoprecipitated both phosphorylated and unphosphorylated forms of Pol II (Fig 6 and Fig 7B). This co-immunoprecipitation depended upon the Flag epitope (Fig 6 and Fig 7) and was still observed in 0.3 M KCl and 0.1% NP-40 (data not shown). Also, using a Spt5-Flag strain with a HA1 epitope-tagged allele of RPB1, we were able to simultaneously detect both forms of polymerase in anti-Spt5-Flag immunoprecipitates, using an anti-HA1 antibody (data not shown).

View larger version (21K): In this window In a new window Download PPT slide   Figure 6. Co-immunoprecipitation of Pol II phosphoisoforms with Spt5-Flag. Anti-Flag immunoprecipitations were performed on whole cell lysates made from wild-type (GHY611) or Spt5-Flag (GHY617) strains. Immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose, and blotted with B3 (-Pol IIO) or 8WG16 (-Pol IIA) antibodies as indicated at bottom. Mock, immunoprecipitate from wild-type extract; WCE, whole cell lysate from Spt5-Flag strain; IP, immunoprecipitate from Spt5-Flag extract.

View larger version (41K): In this window In a new window Download PPT slide   Figure 7. Spt5-Rpb1 co-immunoprecipitations from CTD kinase mutants. Anti-Flag immunoprecipitations were performed on whole cell lysates made from yeast strains with the indicated mutations (Mock, GHY611; WT, GHY617; ctk1, GHY708; kin28-16, GHY1043; srb10, GHY815; rpb1 CTD, GHY681[pC3]; bur1-2, GHY745). For the kin28-16 strain, lysates were prepared from cells grown to log phase at 30° and then shifted to fresh media at 30° or 37° for 40 min (see MATERIALS AND METHODS). Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and blotted with antibodies as indicated. (A) Western blots of whole cell lysates. Each lane contains 50 µg total protein. (B) Western blots of immunoprecipitates. -Ser5-PO4, monoclonal antibody H14; -Rpb1-N, anti-N-terminal antibody RPB1 (yN-18); -Spt5, anti-Spt5 polyclonal.

In a similar set of experiments, DSIF was reported to preferentially associate with Pol II_A (WADA et al. 1998B ). We therefore considered the possibility that Spt5 was co-immunoprecipitating with partially dephosphorylated polymerase rather than Pol II_O. Two observations suggest that this hypothesis is incorrect. First, phosphorylated Rpb1 in the immunoprecipitates migrated at the same reduced mobility observed for Pol II_O in crude extracts (Fig 6). Second, Pol II_A did not preferentially immunoprecipitate with Spt5 (Fig 6). Thus, in yeast, Spt5 associates with both Pol II_A and Pol II_O.

Spt5-Rpb1 association is not dependent on any single CTD kinase:
Having established that Spt5 associates with both Pol II_A and Pol II_O, we next asked if any of the CTD kinases are required for the Spt5-Rpb1 co-immunoprecipitation. Extracts were prepared from Spt5-Flag strains carrying each of the CTD kinase mutants described above and a Spt5-Flag Rpb1-CTD strain in which the CTD was truncated to 10 repeats. These extracts were examined by immunoblotting with an anti-Spt5 antibody and with an antibody directed against the N terminus of Rpb1. Spt5 and total Rpb1 levels were unchanged in these mutants (Fig 7A). The blots were also probed with the antibodies specific for the serine-5-phosphorylated and serine-2-phosphorylated forms of the CTD repeat. Consistent with published results, we observed increased levels of serine-5-phosphorylated Rpb1 in extracts of ctk1 cells (Fig 7; PATTURAJAN et al. 1999 ). In yeast, significant levels of phosphorylation of the serine-2 position of the CTD are observed only around the time of the diauxic shift (PATTURAJAN et al. 1998 ). Consistent with this finding, we did not observe phosphorylation of the serine-2 position of the CTD in our extracts, which were prepared from cells growing in log phase (data not shown).

Anti-Flag immunoprecipitates were examined by immunoblotting (Fig 7B). Rpb1 co-immunoprecipitated specifically with Spt5-Flag from each mutant extract tested, indicating that the physical interaction between Spt5 and Rpb1 is not dramatically affected by the length of the CTD or the activity of any single CTD kinase. Shifting the kin28-16 strain to 37°, its restrictive temperature, for 45 min prior to extract preparation resulted in a significant decrease in the amount of phosphorylated Rpb1 associated with Spt5 (Fig 7B, -Ser5-PO₄) but had no effect on the amount of total Rpb1 associated with Spt5. Thus, Spt5's association with Pol II does not depend upon the CTD's phosphorylation state nor upon any single CTD kinase.

TFIIS-deficient cells display an increased dependence upon the CTD and CTD kinases for normal growth:
Biochemical studies have shown that when Pol II arrests transcription in vitro, it is unable to resume elongation until stimulated to do so by TFIIS (UPTAIN et al. 1997 ). To determine if TFIIS is dependent upon an intact CTD in vivo, we used the plasmid shuffle strategy to test the phenotype of CTD mutations when combined with a deletion of DST1, which encodes TFIIS. dst1 cells grew very poorly when the CTD was truncated to 11 repeats and were not viable when the CTD was truncated to 10 repeats (Table 3). Combining dst1 with either of the WT(9)A2(6) or WT(7)A5(7) rpb1 alleles or the bur1-2, ctk1, or kin28-16 mutations also caused cells to grow poorly (Table 3 and MURRAY et al. 2001 ). In contrast, a dst1 srb10 double mutant displayed no new mutant phenotypes (Table 3). Finally, fcp1-110 was previously shown to cause a strong synthetic growth defect when combined with dst1 (COSTA and ARNDT 2000 ). Thus, DST1 shares a similar set of genetic relationships with the CTD and CTD modifying enzymes as do SPT4 and SPT5.

SPT16 displays genetic interactions with the CTD and CTD modifying enzymes that are distinct from those observed for SPT4, SPT5, and DST1:
To address further the specificity of the interactions we observed between CTD kinases and transcription elongation factors, we expanded our analysis to include SPT16/CDC68. Like Spt4 and Spt5, Spt16 has been implicated in the regulation of transcription through effects on chromatin (MALONE et al. 1991 ; ROWLEY et al. 1991 ). Human Spt16 is a subunit of FACT (facilitates chromatin transcription), which facilitates Pol II transcription through nucleosomes in a defined in vitro system, and a similar complex has been observed in yeast (WITTMEYER and FORMOSA 1997 ; EVANS et al. 1998 ; LEROY et al. 1998 ; ORPHANIDES et al. 1998 , ORPHANIDES et al. 1999 ). The mutation we chose for our analysis, spt16-197, causes phenotypes similar to those caused by the spt4 and spt5-194 mutations but does not show strong genetic interactions with DST1, SPT5, or SPT6, although it has been reported to suppress the 6AU^s phenotype of spt4 mutations (MALONE et al. 1991 ; ORPHANIDES et al. 1999 ). As shown in Table 3, we observed only mild synthetic growth phenotypes when we analyzed rpb1-CTD mutations in a spt16-197 background and no synthetic growth phenotypes when spt16-197 was combined with CTD kinase mutants. This suggests that the observed synthetic phenotypes between mutations in the CTD, CTD kinases, and SPT4 and SPT5 are the result of specific interactions between these genes and are not a general feature of transcription elongation factors.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Spt4-Spt5 and TFIIS functions depend on the CTD and CTD modifying enzymes:
The data presented here demonstrate that the functions of Spt4-Spt5 and TFIIS are interrelated with those of the Pol II CTD and the enzymes that modify its phosphorylation state. Cells that are defective for Spt4-Spt5 or TFIIS function show an increased dependence upon the length and composition of the CTD, Kin28, Bur1, Ctk1, and Fcp1. In contrast to these genetic interactions, the srb10 mutation caused new phenotypes only when combined with spt5-242. Thus, the interdependence of TFIIS and Spt4-Spt5 on the Srb10 CTD kinase, which is implicated in negative regulation of preinitiation complex assembly (HENGARTNER et al. 1998 ; SUN et al. 1998 ), is less clear.

How does the CTD affect Spt4-Spt5 and TFIIS function?
Several models have been proposed to explain the dependence of DSIF on P-TEFb. In one, phosphorylation of the CTD by P-TEFb is proposed to prevent DSIF from binding Pol II and inhibiting elongation (WADA et al. 1998B ). This cannot be true in yeast, since we observe that (1) Spt5 co-immunoprecipitates with both Pol II_A and Pol II_O, (2) unphosphorylated polymerase does not preferentially co-immunoprecipitate with Spt5, (3) truncation of the CTD to 10 repeats does not alter the quantity of Rpb1 that co-immunoprecipitates with Spt5, and (4) mutation of CTD kinases does not appreciably alter Spt5-Rpb1 co-immunoprecipitation (Fig 6 and Fig 7). Our observations are consistent with recent studies demonstrating that Spt5 co-localizes with Pol II_O on Drosophila polytene chromosomes and with in vitro and in vivo studies showing that Spt5 is recruited to transcribed regions of genes (ANDRULIS et al. 2000 ; KAPLAN et al. 2000 ; PING and RANA 2000 ).

Recently, the phosphorylation state of the CTD was found to change as polymerase transcribes across a gene (KOMARNITSKY et al. 2000 ). Initially in elongation, Pol II is heavily phosphorylated on the Ser5 position of the CTD only, wherease late in elongation Ser5 is dephosphorylated and Ser2 phosphorylation predominates. Thus our observations of strong synthetic growth defects in the spt WT(7)A5(7) strains compared to spt WT(9)A2(6) strains may indicate that Spt4-Spt5 functions primarily early in elongation. We initiated chromatin-immunoprecipitation experiments to test this model directly.

P-TEFb has also been shown to directly phosphorylate Spt5 and to prevent its inhibitory function (IVANOV et al. 2000 ; KIM and SHARP 2001 ). Our data do not address this model, and it is possible that Kin28, Ctk1, Bur1, and Fcp1 may all directly regulate phosphorylation of Spt4-Spt5 in addition to the CTD. In this respect, it is interesting to note that bur1 mutants share many phenotypes with spt4 and spt5 mutants (PRELICH and WINSTON 1993 ). Thus, Bur1 may directly regulate Spt4-Spt5 function.

In the absence of a direct Spt4-Spt5-CTD binding interaction, we can propose a speculative model to explain the interdependence of Spt4-Spt5 and the CTD. In this model, perturbation of CTD phosphorylation leads to formation of elongation complexes with altered processivity and an altered dependence on Spt4-Spt5. In this context, our observations that TFIIS and Spt4-Spt5 show a similar dependency on the CTD and CTD kinases are intriguing. The only known biochemical function of TFIIS is to rescue polymerase from arrest (UPTAIN et al. 1997 ). In otherwise wild-type cells, TFIIS is not essential, and dst1 mutations display no mutant phenotypes other than sensitivity to 6-azauracil and mycophenolic acid (UPTAIN et al. 1997 ). This suggests that transcription arrest is normally a rare event, which cells may survive in the absence of TFIIS. On the basis of this and our observation of genetic interactions between DST1, RPB1, and the genes encoding CTD modifying enzymes, we propose that perturbation of CTD function leads to an increased frequency of transcription arrest in vivo and hence an increased dependence upon TFIIS.

Not all elongation factors depend on the CTD:
We found no clear evidence that Spt16 function depends upon the CTD or CTD modifying enzymes. Only a single spt16 allele was tested in this study and it is possible that other alleles of spt16 would have behaved differently. Nevertheless, the results presented here are likely significant, as spt16-197 causes mutant phenotypes that are nearly identical to those caused by spt4 and spt5-194 (MALONE et al. 1991 ). Thus, Spt16 likely functions by a mechanism that is distinct from that of Spt4-Spt5. This conclusion is consistent with that of a recent biochemical study of P-TEFb, DSIF, and FACT (WADA et al. 2000 ).

The interdependence of many other transcription elongation factors and the CTD remains to be determined. Of particular interest is SPT6, which has mutant phenotypes identical to those of spt4 and spt5 mutants and displays unlinked noncomplementation and synthetic lethality when combined with spt4 or spt5 mutations (WINSTON et al. 1984 ; SWANSON and WINSTON 1992 ). In Drosophila, Spt6 shows a distribution on polytene chromosomes similar to that of Spt5, and in human cells it has been implicated in Tat-dependent HIV transcription (WU-BAER et al. 1998 ; ANDRULIS et al. 2000 ; KAPLAN et al. 2000 ). Thus, studies addressing the dependence of Spt6 and other elongation factors on Spt4-Spt5 and the CTD may provide important insights into their roles in transcription.

	ACKNOWLEDGMENTS

We thank Karen Arndt, Steve Buratowski, Jeff Corden, Arno Greenleaf, Greg Prelich, Fred Winston, and Rick Young for gifts of antibodies, plasmids, and strains; Steve Buratowski and Doug Kellogg for advice on experiments; and Greg Prelich and Fred Winston for critical comments on the manuscript. G.H. thanks Fred Winston for support during the early phase of this work. This work was supported by grant GM60479 from the National Institutes of Health to G.H.

Manuscript received May 11, 2001; Accepted for publication July 18, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

AKHTAR, A., G. FAYE, and D. L. BENTLEY, 1996  Distinct activated and non-activated RNA polymerase II complexes in yeast. EMBO J. 15:4654-4664[Medline].

ANDRULIS, E. D., E. GUZMAN, P. DORING, J. WERNER, and J. T. LIS, 2000  High-resolution localization of Drosophila Spt5 and Spt6 at heat shock genes in vivo: roles in promoter proximal pausing and transcription elongation. Genes Dev. 14:2635-2649[Abstract/Free Full Text].

BENSAUDE, O., F. BONNET, C. CASSE, M. F. DUBOIS, and V. T. NGUYEN et al., 1999  Regulated phosphorylation of the RNA polymerase II C-terminal domain (CTD). Biochem. Cell Biol. 77:249-255[Medline].

CHIANG, P. W., E. FOGEL, C. L. JACKSON, K. LIEUALLEN, and G. LENNON et al., 1996a  Isolation, sequencing, and mapping of the human homologue of the yeast transcription factor, SPT5. Genomics 38:421-424[Medline].

CHIANG, P. W., S. Q. WANG, P. SMITHIVAS, W. J. SONG, and E. CROMBEZ et al., 1996b  Isolation and characterization of the human and mouse homologues (SUPT4H and Supt4h) of the yeast SPT4 gene. Genomics 34:368-375[Medline].

CHO, H., T. K. KIM, H. MANCEBO, W. S. LANE, and O. FLORES et al., 1999  A protein phosphatase functions to recycle RNA polymerase II. Genes Dev. 13:1540-1552[Abstract/Free Full Text].

COSTA, P. J. and K. M. ARNDT, 2000  Synthetic lethal interactions suggest a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation. Genetics 156:535-547[Abstract/Free Full Text].

DAHMUS, M. E., 1996  Reversible phosphorylation of the C-terminal domain of RNA polymerase II. J. Biol. Chem. 271:19009-19012[Free Full Text].

EVANS, D. R., N. K. BREWSTER, Q. XU, A. ROWLEY, and B. A. ALTHEIM et al., 1998  The yeast protein complex containing cdc68 and pob3 mediates core-promoter repression through the cdc68 N-terminal domain. Genetics 150:1393-1405[Abstract/Free Full Text].

GU, W., S. MALIK, M. ITO, C. X. YUAN, and J. D. FONDELL et al., 1999  A novel human SRB/MED-containing cofactor complex, SMCC, involved in transcription regulation. Mol. Cell 3:97-108[Medline].

GUARENTE, L., 1993  Synthetic enhancement in gene interaction: a genetic tool come of age. Trends Genet. 9:362-366[Medline].

GUO, S., Y. YAMAGUCHI, S. SCHILBACH, T. WADA, and J. LEE et al., 2000  A regulator of transcriptional elongation controls vertebrate neuronal development. Nature 408:366-369[Medline].

HARTZOG, G. A., M. A. BASRAI, S. L. RICUPERO-HOVASSE, P. HIETER, and F. WINSTON, 1996  Identification and analysis of a functional human homolog of the SPT4 gene of Saccharomyces cerevisiae. Mol. Cell. Biol. 16:2848-2856[Abstract].

HARTZOG, G. A., T. WADA, H. HANDA, and F. WINSTON, 1998  Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae. Genes Dev. 12:357-369[Abstract/Free Full Text].

HENGARTNER, C. J., V. E. MYER, S. M. LIAO, C. J. WILSON, and S. S. KOH et al., 1998  Temporal regulation of RNA polymerase II by Srb10 and Kin28 cyclin-dependent kinases. Mol. Cell 2:43-53[Medline].

IVANOV, D., Y. T. KWAK, J. GUO, and R. B. GAYNOR, 2000  Domains in the SPT5 protein that modulate its transcriptional regulatory properties. Mol. Cell. Biol. 20:2970-2983[Abstract/Free Full Text].

KAPLAN, C. D., J. R. MORRIS, C. WU, and F. WINSTON, 2000  Spt5 and spt6 are associated with active transcription and have characteristics of general elongation factors in D. melanogaster. Genes Dev. 14:2623-2634[Abstract/Free Full Text].

KIM, J. B. and P. A. SHARP, 2001  P-TEFb phosphorylates hSPT5 and RNA polymerase II CTD independently of CAK. J. Biol. Chem. 5:5.

KIM, W. Y. and M. E. DAHMUS, 1988  Purification of RNA polymerase IIO from calf thymus. J. Biol. Chem. 263:18880-18885[Abstract/Free Full Text].

KOBOR, M. S., J. ARCHAMBAULT, W. LESTER, F. C. HOLSTEGE, and O. GILEADI et al., 1999  An unusual eukaryotic protein phosphatase required for transcription by RNA polymerase II and CTD dephosphorylation in S. cerevisiae. Mol. Cell 4:55-62[Medline].

KOMARNITSKY, P., E. J. CHO, and S. BURATOWSKI, 2000  Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Dev. 14:2452-2460[Abstract/Free Full Text].

KUCHIN, S. and M. CARLSON, 1998  Functional relationships of Srb10-Srb11 kinase, carboxy-terminal domain kinase CTDK-I, and transcriptional corepressor Ssn6-Tup1. Mol. Cell. Biol. 18:1163-1171[Abstract/Free Full Text].

LEE, J. M. and A. L. GREENLEAF, 1991  CTD kinase large subunit is encoded by CTK1, a gene required for normal growth of Saccharomyces cerevisiae. Gene Exp. 1:149-167.

LEE, J. M. and A. L. GREENLEAF, 1997  Modulation of RNA polymerase II elongation efficiency by C-terminal heptapeptide repeat domain kinase I. J. Biol. Chem. 272:10990-10993[Abstract/Free Full Text].

LEE, T. I. and R. A. YOUNG, 2000  Transcription of eukaryotic protein-coding genes. Annu. Rev. Genet. 34:77-137[Medline].

LEROY, G., G. ORPHANIDES, W. S. LANE, and D. REINBERG, 1998  Requirement of RSF and FACT for transcription of chromatin templates in vitro. Science 282:1900-1904[Abstract/Free Full Text].

MALONE, E. A., C. D. CLARK, A. CHIANG, and F. WINSTON, 1991  Mutations in SPT16/CDC68 suppress cis- and trans-acting mutations that affect promoter function in Saccharomyces cerevisiae. Mol. Cell. Biol. 11:5710-5717[Abstract/Free Full Text].

MANCEBO, H. S., G. LEE, J. FLYGARE, J. TOMASSINI, and P. LUU et al., 1997  P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro. Genes Dev. 11:2633-2644[Abstract/Free Full Text].

MARSHALL, N. F. and D. H. PRICE, 1995  Purification of P-TEFb, a transcription factor required for the transition into productive elongation. J. Biol. Chem. 270:12335-12338[Abstract/Free Full Text].

MARSHALL, N. F., J. PENG, Z. XIE, and D. H. PRICE, 1996  Control of RNA polymerase II elongation potential by a novel carboxyl-terminal domain kinase. J. Biol. Chem. 271:27176-27183[Abstract/Free Full Text].

MORTILLARO, M. J., B. J. BLENCOWE, X. WEI, H. NAKAYASU, and L. DU et al., 1996  A hyperphosphorylated form of the large subunit of RNA polymerase II is associated with splicing complexes and the nuclear matrix. Proc. Natl. Acad. Sci. USA 93:8253-8257[Abstract/Free Full Text].

MURRAY, S., R. UDUPA, S. YAO, G. HARTZOG, and G. PRELICH, 2001  Phosphorylation of the RNA polymerase II Carboxy-terminal domain by the Bur1 cyclin-dependent kinase. Mol. Cell. Biol. 21:4089-4096[Abstract/Free Full Text].

NONET, M. L. and R. A. YOUNG, 1989  Intragenic and extragenic suppressors of mutations in the heptapeptide repeat domain of Saccharomyces cerevisiae RNA polymerase II. Genetics 123:715-724[Abstract/Free Full Text].

NONET, M., D. SWEETSER, and R. A. YOUNG, 1987  Functional redundancy and structural polymorphism in the large subunit of RNA polymerase II. Cell 50:909-915[Medline].

ORPHANIDES, G., G. LEROY, C. H. CHANG, D. S. LUSE, and D. REINBERG, 1998  FACT, a factor that facilitates transcript elongation through nucleosomes. Cell 92:105-116[Medline].

ORPHANIDES, G., W. H. WU, W. S. LANE, M. HAMPSEY, and D. REINBERG, 1999  The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins. Nature 400:284-288[Medline].

PATTURAJAN, M., R. J. SCHULTE, B. M. SEFTON, R. BEREZNEY, and M. VINCENT et al., 1998  Growth-related changes in phosphorylation of yeast RNA polymerase II. J. Biol. Chem. 273:4689-4694[Abstract/Free Full Text].

PATTURAJAN, M., N. K. CONRAD, D. B. BREGMAN, and J. L. CORDEN, 1999  Yeast carboxyl-terminal domain kinase I positively and negatively regulates RNA polymerase II carboxyl-terminal domain phosphorylation. J. Biol. Chem. 274:27823-27828[Abstract/Free Full Text].

PING, Y. H. and T. M. RANA, 2000  DSIF and NELF interact with RNA Polymerase II elongation complex and HIV-1 Tat stimulates P-TEFb-mediated phosphorylation of RNA polymerase II and DSIF during transcription elongation. J. Biol. Chem. 8:8.

PRELICH, G. and F. WINSTON, 1993  Mutations that suppress the deletion of an upstream activating sequence in yeast: involvement of a protein kinase and histone H3 in repressing transcription in vivo. Genetics 135:665-676[Abstract].

PRICE, D. H., 2000  P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol. Cell. Biol. 20:2629-2634[Free Full Text].

RODRIGUEZ, C. R., E. J. CHO, M. C. KEOGH, C. L. MOORE, and A. L. GREENLEAF et al., 2000  Kin28, the TFIIH-associated carboxy-terminal domain kinase, facilitates the recruitment of mRNA processing machinery to RNA polymerase II. Mol. Cell. Biol. 20:104-112[Abstract/Free Full Text].

ROSE, M. D., F. WINSTON and P. HIETER, 1990 Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

ROWLEY, A., R. A. SINGER, and G. C. JOHNSTON, 1991  CDC68, a yeast gene that affects regulation of cell proliferation and transcription, encodes a protein with a highly acidic carboxyl terminus. Mol. Cell. Biol. 11:5718-5726[Abstract/Free Full Text].

STERNER, D. E., J. M. LEE, S. E. HARDIN, and A. L. GREENLEAF, 1995  The yeast carboxyl-terminal repeat domain kinase CTDK-I is a divergent cyclin-cyclin-dependent kinase complex. Mol. Cell. Biol. 15:5716-5724[Abstract].

SUN, X., Y. ZHANG, H. CHO, P. RICKERT, and E. LEES et al., 1998  NAT, a human complex containing Srb polypeptides that functions as a negative regulator of activated transcription. Mol. Cell 2:213-222[Medline].

SWANSON, M. S. and F. WINSTON, 1992  SPT4, SPT5 and SPT6 interactions: effects on transcription and viability in Saccharomyces cerevisiae. Genetics 132:325-336[Abstract].

SWANSON, M. S., E. A. MALONE, and F. WINSTON, 1991  SPT5, an essential gene important for normal transcription in Saccharomyces cerevisiae, encodes an acidic nuclear protein with a carboxy-terminal repeat. Mol. Cell. Biol. 11:3009-3019[Abstract/Free Full Text].

THOMPSON, N., T. STEINBERG, D. ARONSON, and R. BURGESS, 1989  Inhibition of in vivo and in vitro transcription by monoclonal antibodies prepared against wheat germ RNA polymerase II that react with the heptapeptide repeat of eukaryotic RNA polymerase II. J. Biol. Chem. 264:11511-11520[Abstract/Free Full Text].

UPTAIN, S. M., C. M. KANE, and M. J. CHAMBERLIN, 1997  Basic mechanisms of transcript elongation and its regulation. Annu. Rev. Biochem. 66:117-172[Medline].

WADA, T., T. TAKAGI, Y. YAMAGUCHI, A. FERDOUS, and T. IMAI et al., 1998a  DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. Genes Dev. 12:343-356[Abstract/Free Full Text].

WADA, T., T. TAKAGI, Y. YAMAGUCHI, D. WATANABE, and H. HANDA, 1998b  Evidence that P-TEFb alleviates the negative effect of DSIF on RNA polymerase II-dependent transcription in vitro. EMBO J. 17:7395-7403[Medline].

WADA, T., G. ORPHANIDES, J. HASEGAWA, D. K. KIM, and D. SHIMA et al., 2000  FACT relieves DSIF/NELF-mediated inhibition of transcriptional elongation and reveals functional differences between P-TEFb and TFIIH. Mol. Cell 5:1067-1072[Medline].

WARREN, S. L., A. S. LANDOLFI, C. CURTIS, and J. S. MORROW, 1992  Cytostellin: a novel, highly conserved protein that undergoes continuous redistribution during the cell cycle. J. Cell Sci. 103:381-388[Abstract].

WEST, M. L. and J. L. CORDEN, 1995  Construction and analysis of yeast RNA polymerase II CTD deletion and substitution mutations. Genetics 140:1223-1233[Abstract].

WINSTON, F., D. T. CHALEFF, B. VALENT, and G. R. FINK, 1984  Mutations affecting Ty-mediated expression of the HIS4 gene of Saccharomyces cerevisiae. Genetics 107:179-197[Abstract/Free Full Text].

WINSTON, F., C. DOLLARD, and S. L. RICUPERO-HOVASSE, 1995  Construction of a set of convenient S. cerevisiae strains that are isogenic to S288C. Yeast 11:53-55[Medline].

WITTMEYER, J. and T. FORMOSA, 1997  The Saccharomyces cerevisiae DNA polymerase alpha catalytic subunit interacts with Cdc68/Spt16 and with Pob3, a protein similar to an HMG1-like protein. Mol. Cell. Biol. 17:4178-4190[Abstract].

WU-BAER, F., W. S. LANE, and R. B. GAYNOR, 1998  Role of the human homolog of the yeast transcription factor SPT5 in HIV-1 Tat-activation. J. Mol. Biol. 277:179-197[Medline].

YAMAGUCHI, Y., T. TAKAGI, T. WADA, K. YANO, and A. FURUYA et al., 1999  NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation. Cell 97:41-51[Medline].

ZHU, Y., T. PE'ERY, J. PENG, Y. RAMANATHAN, and N. MARSHALL et al., 1997  Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro. Genes Dev. 11:2622-2632[Abstract/Free Full Text].

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