Cell, Vol. 71, 21 l-220,

October

16, 1992, Copyright

0 1992 by Cell Press

PCF4 Encodes an RNA Polymerase III Transcription Factor with Homology to TFIIB Alfred0 Lopez-De-Leon, Monett Librizzi, Karen Puglia, and Ian M. Willis Department of Biochemistry Albert Einstein College of Medicine Jack and Pearl Resnick Campus Bronx, New York 10461

Summary A dominant mutation in the PCF4 gene of S. cerevisiae was isolated as a suppressor of a tRNA gene A block promoter mutation. In vitro studies indicate that PCF4 is a stoichiometrically-required RNA polymerase Ill (pol III) transcriptlon initiation factor. We show that the PCFC7 mutation increases the number of transcriptionally competent preinitlation complexes by affecting a limiting activity in yeast cell extracts that is squelched by excess TFIIIC. The PCM gene encodes a TFIIB homolog whose size, biochemical, and genetic properties are consistent with those of the 70 kd subunit of TFIIIB. The TFIIB homology of PCF4 suggests a means for determining the polymerase specificity of a gene. Introduction Transfer RNA (tRNA) genes are representatives of the simplest class of RNA polymerase III (pol Ill) transcription units (Geiduschek and Tocchini-Valentini, 1988). Their transcription is directed by two highly conserved cis-acting elements, the A and B blocks, located downstream of the initiation site. Sequences upstream of the transcription start site also contribute to the transcriptional activity of tRNA genes, but in general these effects have not been attributed to specific recognition elements. Two multisubunit transcription factors (TFs), Ill6 and IIIC, are required in addition to pol Ill to reconstitute this process in vitro on tRNA gene templates (reviewed in Gabrielsen and Sentenac, 1991). TFIIIC is a large multi-subunit complex that binds to the intragenic A and B blocks. The factor has been studied most extensively in yeast, where it is known to have a molecular mass greater than 500 kd and to comprise at least five poiypeptide subunits (55, 60, 95, 135, and 145 kd; Bartholomew et al., 1990; Gabrielsen and Sentenac, 1991). Based on their copurification following chromatography on numerous ion-exchange and affinity resins, these subunits form a highly stable complex. Many experiments have demonstrated the involvement of individual subunits in transcription (for example, see Gabrielsen et al., 1989; Parsons and Weil, 1990). Among these, the UV crosslinking studies of Bartholomew et al. (1990, 1991) are especially informative, since they have revealed the spatial organization of TFIIIC subunits along the DNA. TFIIIB is the principal initiation factor of RNA polymerase Ill. Through interactions with promoter-bound TFIIIC, the factor is specifically recruited to the 5’ flanking region of

tDNAs (Kassavetis et al., 1990). The complexities of this binding reaction are not well understood. However, it is known that the resulting interactions between TFIIIB and DNA confer remarkable stability to the complex; the TFIIIB-DNA complex is resistant to concentrations of salt or heparin that suffice for the quantitative removal of TFIIIC. The differential stability of these factor-DNA interactions has been exploited to show that transcription complexes that have been stripped of TFIIIC are able to direct multiple rounds of initiation by pol Ill (Kassavetis et al., 1990). These important experiments demonstrate the role of TFIIIC (and TFIIIA) as an assembly factor and that of TFIIIB in specific recognition by pol Ill. Despite these achievements, the complete polypeptide composition of TFIIIB has not been established. Earlystudiesshowed that antibodies raised against a 60 kd TFIIIB polypeptide from yeast inhibited pol Ill transcription in yeast and HeLa cell systems (Klekamp and Weil, 1986). Gel-purified and renatured 80 kd protein was also shown to reconstitute low levels of transcription with TFIIIC and pol Ill. More recently, two polypeptides with estimated molecular sizes of 70 and 90 kd have been resolved from TFIIIB preparations (Kassavetis et al., 1991). Both of these components are necessary for transcription and both have been photocrosslinked to DNA in the region bound by TFIIIB (Bartholomew et al., 1991). The relationship between the 70 and 90 kd polypeptides and the previously isolated 60 kd polypeptide is not clear. However, the presence of the 90 kd polypeptide as a contaminant in less pure TFIIIC preparations provides an explanation for the low level of transcription in reactions containing the renatured 60 kd polypeptide. This explanation suggests that the 60 and 70 kd components are one and the same. Recent studies in a number of laboratories involving organisms from yeast to humans have shown that the TATAbinding protein (TBP) is a general transcription factor serving all three classes of eukaryotic RNA polymerase (Comai et al., 1992; White et al., 1992; Schultz et al., 1992; Cormack and Struhl, 1992). With regard to the pol ill system, TBP has been shown to participate in transcription of vertebrate U6 snRNA genes where the promoters are located exclusively upstream of the coding sequence (Lob0 et al., 1991; Simmen et al., 1991) as well as tRNA and 5S genes where the promoters are internal (White et al., 1992; Schultz et al., 1992). As many of these genes lack an identifiable TATA box, their transcription appears analogous to that of TATA-less pol Ii genes. It is now apparent that the widespread role of TBP in transcription escaped attention initially because of the relatively stable association of TBP with many other transcription factors (TATAbinding protein-associated factors or TAFs; Dynlacht et al., 1991; Comai et al., 1992). In yeast cell extracts, an unknown proportion of the TBP copurifies with TFIIIB. Chromatography over more than 5 different columns does not resolve these components (Klekamp and Weil, 1986; Kassavetis et al., 1989). Recently however, fractionation of TBP from TFIIIB subunits was achieved by gel filtration

Figure 1. Genetic Selection the PCF4-7 Mutation PCF4-1.

0

A

sup9-e

A 19

A

supSl+

0

sups

1

6 Mata

Matu PCF4-

1

ura3-52::URA3/sup9-eA trpr-1 met& 7 /euZ-3,112 his3-11,75

19-sups1

PCF4 ura3-52::lJRA3/sup9-eA trpi-1 met&?-l leu2-3,112 arg4-17

under moderately high ionic conditions (Margottin et al., 1991). To study the structure, function, and regulation of pal III transcription factors we have developed a genetic approach in yeast. Previously, we reported a strategy for targeting mutations in components of the pol Ill transcription apparatus of Saccharomyces cerevisiae (Willis et al., 1989). Suppression of amber nonsense mutations in auxotrophic markers was rendered dependent on the expression of a mutated dimeric nonsense suppressor, sup9-e A79-supS7, from Schizosaccharomyces pombe (Figure 1A). In wild-type cells, transcription of this construct is impaired by a promoter mutation (Al 9) in the A block of the sup4e gene. Eight independent dominant mutant strains were isolated that suppressed the sup9-e A79 mutation, allowing expression of the cotranscribed supS7 amber suppressor tRNA. In vitro experiments showed that extracts from a strain bearing the PCF7-7 (polymerase C factor) mutation increased transcription up to 6-fold over wild-type levels (Willis et al., 1989). Current evidence indicates that this gene encodes a component or a regulator of TFIIIB (Willis et al., 1992). In this work, we report the isolation and characterization of PCF4-7. The PCF4-7 gene encodes a homolog of TFIIB yet it activates pol Ill gene transcription. Our results indicate that PCM is the limiting factor for transcription by this polymerase in vivo and in vitro. The studies described here and in the accompanying paper by Buratowski and Zhou support the conclusion that PCF4 encodes the 70 kd subunit of TFIIIB Results Genetic Characterization and Cloning of PCF4-7 PCF4-7 is one of several dominant mutations that was isolated in a selection for extragenic suppressors of a tRNA gene A block promoter mutation (Willis et al., 1989; A. L.-D.-L. and I. M. W., unpublished data). The phenotype conferred by PCF4-7, as with the other PCF mutations,

19-sups

1

and Phenotype

of

(A) Schematic representation of the dimeric Al&sups7 suppressor. The internal A and S block elements of both genes are shown. The A blockof thesup9-egenecontainsaG-A mutation at position 19. The arrows represent the dimeric tRNA precursor whose synthesis is directed by the promoter of the sup9-e gene. The broken arrow reflects the low level of transcription of this construct in wild-type cells and the corresponding suppressor minus phenotype. The solid arrow signifies the increased transription in PCF mutant strains and the expression of supS1 activity. (8) Tetrad analysis of a PCF4-7 heterozygote. The panel shows the level of supS7 amber suppressor activity (growth on Trp- Met- medium) in colonies arising from nine dissected tetrads. The genotypes of the parental strains are also shown. The amber suppressible mutations are trpl-1 and met&l.

sup$e

involves the expression of sups7 activity from a dimeric construct, supQe A79-supS7, which is otherwise not expressed in wild-type cells (Figure 1A). Accordingly, the mutant phenotype can be observed only in the presence of this heterologous gene. No other phenotypes are apparent in PCF4-7 strains. The cells grow well at 30°C and are not heat or cold sensitive. The sups7 suppression phenotype of PCF4-7 is illustrated in Figure 1 B, which shows a tetrad analysis of a diploid strain that is heterozygous for the mutation. Wild-type, nonsuppressing colonies and sups7 expressing colonies segregate 2:2 among the progeny in all 9 tetrads. Thus, the suppression phenotype is conferred by a single nuclear mutation. The PCF4-7 gene was cloned by constructing a library of mutant strain DNA in the centromeric plasmid vector, pRS315. This material was transformed into the wild-type strain, IWDl, which carries a chromosomal copy of the sup9-eA79-supS7 gene and the amber suppressible nonsense mutations frpl-7 and met8-7. Plasmid DNA was extracted from six Trp+ Met+ colonies and transformed into bacteria. Subsequent restriction analysis showed that the same clone had been recovered in each case. Plasmid DNA (pRSM3) from a single Escherichia coli transformant was then reintroduced into the yeast strains IWDl and IWl B6 to test whether sup9-e A 79-supS7-dependent suppression could be restored. These strains differ by the presence and absence, respectively, of the dimeric suppressor gene. Figure 2A shows that both the sup9-e A79sups7 gene and the pRSM3 plasmid must be present to confer nonsense suppressor activity. The dominant phenotype of pRSM3 and its phenotypic dependence on the dimeric suppressor argue that the cloned insert contains the PCF4-7 gene. This was confirmed by showing that a cloned wild-type PCF4+ gene directs integration of a marker gene (LEU2) to the PCF4-7 locus (see Experimental Procedures). The PCF4 gene was physically mapped to the right arm of chromosome VII in the region between pet54 and rad3 by Southern blotting of fractionated yeast chromosomes and by hybridization to an ordered set of

A TFIIB 213

Homolog

Activates

Pol Ill Transcription

A Strain

Plasmid

B Medium

His-

Medium

Containing

the Cloned

37 c

30 c

Figure 2. Phenotypes 1X74-7 Gene

Lys-

of S. cerevisiae

Strains

(A) Strains IWlB6 and IWDl containing either control plasmid (pRS315) or the recombinant clone (pRSM3) bearing the PCF4-7 gene were grown with selection for the plasmid marker (LfU2) and replica plated to medium lacking tryptophan and methionine where amber suppressor activity is required for growth. Three colonies of each type are shown. (B) Transformants of strain FW466 containing plasmids pRS315 (upper row) or pRSM3 (lower row) were grown and replica plated to duplicate lysine or histidine dropout media. Lys- plates were incubated at 25 and 30°C while His plates were incubated at 30 and 37%. These temperatures are semipermissive and permissive, respectively, for the his4-9125 and (~~2-67 mutations in this strain.

yeast clones in k (provided by L. Riles and M. Olsen, Washington University). Given the general role of TBP in transcription by all three nuclear RNA polymerases, we wished to determine whether PCf4-7 might affect general transcription of pol II genes. This question was addressed using strain FW486, which harbors cold-sensitive mutations in two amino acid biosynthetic genes (1~~2-67 and his4-9728). These mutations result from Ty and solo S insertions upstream of the respective coding sequences and disrupt the normal transcription of these genes (see Simchen et al., 1984 and references therein). Acontrol plasmid, pRS315, orplasmid pRSM3 containing the PCF4-1 gene was used to obtain Leu+ transformants of strain FW486. The colonies were then replica plated to Lys- or His- media and incubated at temperatures that were permissive and semipermissive for the two mutations. The results (Figure 28) indicate that the PCf4-7 gene has no effect on the ability of this strain to grow on these media at either temperature. We conclude that the PCF4-1 gene does not function as a general activator of pol II genes. Sequence Analysis of PCF4-1 Reveals Homology to TFIIB Subcloning and unidirectional deletions defined a minimal fragment of about 2.6 kb that conferred the mutant phenotype. Within this fragment, the nucleotide sequence revealed a single large open reading frame of 596 aa (Figure

3). This sequence predicts a Mr of 66,900 and a pl of 6.91 for the PCF4 protein. A search of the Genbank Data Base by the Tfasta program revealed that the N-terminal half of the deduced PCM sequence displays significant similarity to the entire 316 aaof the general pol II transcription factor, TFIIB. The maximal alignment to human TFIIB produced by the program GAP resulted in 46% similarity with 25% identity and 6 gaps. In the representation shown in Figure 3, the number of gaps was reduced to 3 with only a slight drop in similarity (43%). The SUA7 gene of yeast has recently been shown to encode a TFIIB homolog that functions in start site selection by pol II (Pinto et al., 1992). We therefore also compared the deduced amino acid sequence of PCF4 with that of SUA7. Interestingly, PCF4 is no more similar to SUA7 than it is to human TFIIB. The best alignment by GAP indicated 46% similarity with 20% identity and 6 gaps (data not shown). The extent of sequence divergence between these two yeast proteins is surprising but not entirely unexpected. SUA7 shows only 52% similarity to TFIIB from higher eukaryotes whereas the sequences of human, Xenopus, and Drosophila TFIIB are 85% similar. This divergence between SUA7 and higher eukaryotic TFllBs has been suggested to reflect differences in start site selection between these organisms (Pinto et al., 1992; Yamashita et al., 1992). Examination of the alignments between PCF4 and human TFIIB provides an indication of conserved and nonconserved functional domains in these proteins. The putative Zn*+ finger in the N-terminal region of TFIIB is conserved in PCF4 (Figure 3) and also in SUA7 (Pinto et al., 1992). The motif in both yeast proteins is Cys-XP-CysXl 7-Cys-X2-Cys, whereas in human TFIIB the second cysteine is replaced by histidine and the finger length is reduced to 15 aa. Also noteworthy is the spacing of this N-terminal domain relative to the rest of the protein. Both TFIIB and SUA7 contain an additional 20-25 aa (depending on alignment parameters) that are absent in PCF4. TFIIB is known to contain 2 imperfect 76 aa repeats (Ha et al., 1991). The corresponding repeat regions in PCF4 show 25% and 28% identity, respectively, to human TFIIB (Figure 3). It is possible that a common function may be sewed by these sequences. Basic regions have been noted to precede the start and overlap the end of the first imperfect repeat in TFIIB. These sequences are predicted to form amphipathic helices that exhibit a pronounced clustering of basic residues (Ha et al., 1991; Yamashita et al., 1992). Although some basic residues are apparent in the corresponding regions of PCF4 (Figure 3), helical wheel analysis does not predict a similar structure. The basic repeats may therefore participate in TFIIB-specific interactions. The C-terminal 310 aa of PCF4 are not related to any sequences currently in the data base. Other than a higher density of charged residues (37% versus 24% for the TFIIB homology domain), this region does not have any distinguishing features. Multiple Copies of the PCS4 Gene Suppress the sup9-e A79 Promoter Mutation In an accompanying paper in this issue, Buratowski and Zhou report the isolation of TDS4, an allele-specific, multi-

Cdl 214

1 . . . . . . . ..MPVCKNCIiQTE?ERDLSILVCKA~~~S~~NPIVSEV :I . . .: .: . ..,..,..,,:, I.., .,..I 1 l4ASTSP.LDALPRVTCPNEPDAILVEDYRAODNICPECGLWGDRVIDVGS

PCF4

TFIIB PCF4 TFIIB

41 : 50

42 TFGETSAGyWQGSFI~AGQ.........................SIIAA .: . I.: ,. :.I :,.:I 51 EWRTFSNDKATKDPSRVGDSQNPLLSDGDLSTMIGKGTGAASFDEFGNSK

PCF4

67

TFIIB

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SYALRIPEYITDAAFQWYKLALANNF :::I 1.; . .::I .. .. . INLPRNIVDRTNNLFKQVYEQKS

116

. ..QVQMAATHIARKAVELDLVP

246

150

PCF4

Figure 3. Comparison of the Deduced Acid Sequences of PCF4 and Human

Amino TFIIB

Sequence alignment was performed using the GCG program GAP. Amino acids that constitute the putative Zn2+ finger are shown in bold type. The two imperfect amino acid repeats identified in human TFIIB are indicated by arrows. Shadowed residues in human TFIIB represent the basic regions previously predicted to form amphipathic helices. The corresponding basic clusters in PCF4 are also shown in shadow and were subjected to helical wheel analysis (see text).

TFIIB PCF4 TFIIB PCF4

217

GRRPAGIAGACILLACRMNNLRRTHTEIVAVSHVAEETIQQRLNEFKNTK 11.1 :: ;:;.I.:/:. . . :\\:.!I..:. 11: I:./... GRSPISVMAAIYMASQASAEKRTQKBIGDIAGVADVTIRQSYRLIYPRA

TFIIB

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PCF4

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TFIIB

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AAKLSVQKFRENDVEDGEARPPSFVKNRKKE :. :..: :..I:. .. PDLFPTDFKFDTPVDKLPQL..............................

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316 316

317

LNKNPILTQVLGEQELSSKBVLFYLKQFSERRARWERIKATNGIDGENI

366

367

YHEGSENETRKRKLSEVSIQNEHVEGEDKETEGTEEKVKKVKTKTSEEKK

416

417

ENESGHFQDAIDGYSLJXTDPYCPRNLHLLPTTDTYLSKVSDDPDNLKDVD

466

467

DEELNAHLLNEEASKLKERIWIGLNADFLLEQESKRLKQEADIATGNTSV

516

517

KKKRTRRRNNTRSDEPTKTVDAAAAIGLMSDLQDKSGLHAEESGD

566

567

FTTADSVKNMLQKASFSKKINYDAIDGLFR*

596

copy suppressor of a temperature-sensitive TBP mutation. Since both PCF4 and TDS4 mapped to the same region of chromosome VII and the properties of TDS4 suggested a role in pol Ill transcription, we compared their nucleotide sequences. This comparison revealed absolute identity throughout the coding region and the adjacent flanking DNA except for a single point mutation upstream of the gene (data not shown). Since the TDS4 gene was cloned from a wild-type library (S. cerevisiae strain S288C), this result suggests that suppression of the sup%e A79 promoter mutation by PCF4-7 is a consequence of overexpression of the wild-type protein rather than the increased activity of a mutant protein. This wasconfirmed by showing that multiple copies but not a single copy of the TDS4 gene isolated by Buratowski and Zhou can express sups-e A79-sups7 suppressor activity (data not shown). These findings imply that the expression of tRNA genes, and presumably all pol Ill genes in yeast (see below), is limited in vivo by the amount of PCF4 protein. PCF4-1 Is a G&m-al Activator of Pol Ill Transcription In Vitro To define the biochemical function of the PCF4 gene product, a series of in vitro experiments were performed using transcriptionally competent whole-cell extracts derived from PCF4-7 and wild-type strains. Initially, we examined the ability of PCF4-7 cell extracts to transcribe a variety

of pol Ill genes under conditions that permitted multiple rounds of initiation. In agreement with the in vivo prediction that PCF4-7 is an activator of pol Ill genes, the mutant cell extract showed increased transcription over wild type for all the templates tested (Figure 4A). Each gene can be assigned to one of three groups based on the level of activation. A biochemical basis for these three tiers of activation has not been explored, but may reflect either differences in the internal promoter and/or upstream sequences of the genes tested, differences in the spacing of the internal control regions, or a requirement for factors other than Ill6 and IIIC that may be limiting transcription in our extracts (e.g., TFIIIA in the case of the 5s gene). Differences in the level of activation (mutant versus wildtype transcription) have been observed in PCF7-7 cell extracts depending on whether or not preinitiation complexes are preformed (Willis et al., 1992). Additionally, Giardina and Wu (1990) have reported that the presence of nucleoside triphosphates (NTPs) during complex formation can affect transcription levels. Whether these conditions might influence transcription in PCF4-7 cell extracts was assessed by performing multiple round reactions in which preinitiation complexes were preformed in the presence or absence of NTPs. Transcriptional activation under these conditions was relatively constant and did not vary significantly from non-preincubated reactions (compare Figure 46, diagonally hatched bars and Figure 4A,

;,;FllB

Homolog

Activates

Pol Ill Transcription

A cene (Urn.) somin. + w sxtr.ct +1or2

-

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stop

B

14

-

w,

u

-

u

w,

12 10

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a

(A) Schematic of the reaction; complexes were formed by incubating a limiting amount (lim.) of template (supg-e, 40 ng) and carrier DNA with excess extract protein. Transcription was initiated upon addition of the second extract and the full complement of NTPs. (8) Mixing of wild-type and PCF4-1 cell extracts. Each bar of the histogram represents the average amount of transcription (in counts per minute) fmm duplicate reactions (shown below). The order of extract addition is given on the abscissa; W, wild-type; M, mutant. Filled bars indicate single extract additions; diagonally hatched bars indicate homologous extract additions; cross-hatched bars indicate heterologous extract additions.

6 4 2 0

Figure

4. General

Transcriptional

Properties

5. PCF4-1

Affects

the Limiting

Factor

In Vitro

of PC&#-7 Cell Extracts

(A) Transcription activity of different pol Ill genes was examined under multiple round transcription conditions with excess amounts of template (600 ng) and equal amounts of extract protein (75 kg). (B) Effect of extract preincubation in the presence or absence of NTPs on transcriptional activation. The data represented in diagonally hatched columns are from multiple round reactions in which equal amounts of extract protein and excess sup%e DNA as in (A) was preincubated for 60 min. at 25% before initiation. Where indicated, no NTP, ATP (1.2 mM), or CTP (0.6 mM) was included in the preincubation. The horizontally hatched column shows the results of single round (SR) reactions (see Experimental Procedures).

sups-e). A similar level of activation was also obtained when the polymerase was limited to one initiation event per active transcription complex (Figure 46, horizontally hatched bar). Thus, the PCF4-7 mutation affects a step at or prior to initiation in such a way as to increase the number of transcriptionally active complexes. The PCF4-1 Mutation Affects a Limiting Component That Is Required in Stoichiometric Quantities for Initiation In Vitro Extract mixing experiments were performed to examine whether the mutant extract could reproduce the dominant phenotype seen in vivo. One extract was allowed to assemble preinitiation complexes on a limiting amount of template before addition of a second extract and NTPs to initiate transcription (Figure 5A). An important requirement

of these experiments is that the template be sequestered completely into specific complexes after incubation with the first extract. These conditions were established by transcription assays (template and carrier DNA titrations) and by gel-mobility shift experiments (data not shown). The results of mixing wild-type with PCF4-7 extracts are shown in Figure 58. When the second extract added was the same as the first (diagonally hatched bars), a P-fold increase was observed in the amount of transcription over reactions containing a single addition of the same extract (sofid bars). In heterologous mixtures (crosshatched bars), the combination of wild type first and mutant second resulted in a level of transcription very similar to reactions containing a single addition of mutant extract. Similarly, when the order of extract addition was reversed (i.e., mutant followed by wild-type), the amount of transcription was again nearly identical to the single addition of mutant extract. As there is no free template in these reactions, the e-fold increase seen with homologous extracts suggests that the second extract is providing a factor that is limiting for transcription. Moreover, since complexes assembled with wild-type components can be activated to give mutant levels of transcription, the factor affected by the /3X4-1 mutation must be the limiting factor in the extracts and it must be diffusible. The alternative viewpoint, that a wildtype component is limiting the ability of the mutant to func-

Cdl 216

A (lim.) + Extract

Figure 6. PCF4 Is Required Amounts for Initiation

20 25 “C

(Variable) + ATP, GTP, UTP

Heparin + CTP

Extract

Gene

2

1

0 Protein

.2!L+ 25 “C

(Fold

10’ -

stop 25 “C

in Stoichiometric

The relationship between transcription and the amount of extract added after transcription complex assembly on a limiting amount of template was determined under sinole round reactions conditions. After an initial incubation of sups-e DNA (40 ng) with excess PCf4-7 extract protein (73 ug), ternary complexes were formed by the addition of varying amounts of the homologous extract (30-146 pg) and the appropriate NTPs (see Experimental Procedures). Subsequently, nascent transcripts were elongated in the presence of heparin.

3

Increase)

tion, is not the case since addition of wild-type extract to mutant complexes results in a level of transcription equal to, but not double, that seen for a single addition of mutant extract. Whether the limiting factor in PCF4-7 extracts is required in stoichiometric or catalytic amounts for transcription was examined according to the scheme in Figure 6A. As in the previous experiment, preinitiation complexes were allowed to form on a limiting amount of template. Subsequently, varying amounts of the same extract were added together with an incomplete set of NTPs. After a further incubation, the missing NTP was provided with heparin, which limits initiation to a single round. The results show that transcription increased in proportion to the amount of added extract (Figure 6). Linear regression analysis revealedaslopeof approximatelyone(1 .l). Additionally, the number of transcripts synthesized indicated that 500/o100% of the assembled complexes were functional at the highest protein concentrations tested. The high efficiency of transcription in these reactions together with the linearity of the response argues that the PCF4 gene product is stoichiometrically required for initiation. Studies in our own labandbyothers(e.g., Kassavetisetal., 1969) haveshown that the factor limiting transcription in yeast whole-cell extracts is TFIIIB. Thus, the fact that the PCF4-7 mutation affects a stoichiometrically required limiting factor is consistentwith theviewthat PCF4encodesasubunitofTFIIIB. TFIIIC Can Squelch the Mutant Activity in PCF4-1 Cell Extracts Further in vitro support of the hypothesis that PCF4 is a subunit of TFIIIB is provided by the following experiment. A limiting amount of template was quantitatively, specifically, and stably bound during a preincubation with an increasing molar excess (2.5 to 15-fold) of partially puri-

fied TFIIIC. Subsequently, wild-type or PCF4-7 cell extract was added with NTPs to provide the remaining transcription components. In the absence of TFIIIC in the preincubation, the level of transcription in the PCF4-7 extract was 4.6-fold higher than the wild-type extract (Figure 78). However, as the amount of exogenous TFIIIC in the preincubation increased (from O-to 15fold molar excess relative to the template), the difference in transcription between the two extracts declined following a hyperbolic curve that approached the abscissa (Figure 78). This outcome excludes the possibility that F’CF4 encodes a subunit of TFIIIC, since the predicted result in that event would have revealed no increase in transcription over wild type for any of the tested TFIIIClsup4e molar ratios. Why then does total transcription decrease as a function of the amount of preincubated TFIIIC and why is the extent of the decrease different for the two extracts (Figure 7A)? Only one likely explanation has emerged that can account for these observations. The data are consistent with a squelching effect (Ptashne, 1966) of TFIIIC wherein the increasing molar excess of this factor sequesters increasing quantities of the limiting factor, namely TFIIIB, in the extract. Total transcription decreases because less TFIIIB is available to bind the preformed TFIIIC-DNA complexes. Consistent with the view that the PCF4-7 mutation increases the amount of a wild-type protein, the decrease in transcription by added TFIIIC is greater for PCF4-7 extracts (90% reduction) than for wild-type extracts (40% reduction), since more TFIIIB is available to be titrated. Discussion Two genes, PCF7 and PCF4, have been isolated from our collection of tRNA gene A block promoter mutation suppressors. In both cases, the mutant phenotype is con-

A TFIIB 217

Homo@

Activates

Molar

Pol Ill Transcription

Excess

of

Exogenous

of

Exogenous

TFIIIC

I3

00 hlolar

Figure 7. Excess Cell Extracts

2s

53

Escess

TFIIIC Squelches

1co

Transcriptional

150 TFlllC

Activation

by RX-47

The experimental details are given in the text and in Experimental Procedures. (A) Total gene transcription is plotted as a function of the molar excess of exogenous wild-type TFIIIC to supge during a preincubation; wild-type (closed circle) and PCf4-1 (triangle). (B) The data in (A) were used to determine the level of activation by PCf4-7 extracts over wild-type extracts (ordinate axis).

ferred by a dominant mutation. Thus, at least one of the functions of the encoded factors is to influence tRNA gene expression positively. In vitro experiments have shown that the positive effects of the PCFl-1 and PCF4-1 mutations are exerted at the transcriptional level (Willis et al., 1989; the current study). For PCF7-7, increased transcription is likely to have resulted from an increase in the activity of thegeneproduct(Willisetal., 1992) whereasforPCF4-7, the cellular concentration of the factor appears to be affected. These observations indicate that yeast cellshave considerable potential to increase the level of transcription by pol Ill. Moreover, the activities or concentrations of the components in this process appear to have been optimized through evolution rather than maximized. Biochemical Properties of PCF4 Our analysis of transcription in wild-type and PCF4-7 cell extracts has revealed that PCF4 is a general activator of pol Ill transcription (Figure 4A) that is stoichiometrically

required for initiation (Figure 8). Additionally, we have shown that the PCF4-1 mutation increases the number of transcriptionally competent complexes (Figure 48) by affecting a limiting activity in yeast cell extracts (Figure 5) that can be squelched by excess TFIIIC (Figure 7). These properties suggest that PCF4 encodes a subunit of TFIIIB. TFIIIB is the limiting factor in yeast whole-cell extracts (e.g., see Kassavetis et al., 1989) and interacts directly with TFIIIC during preintiation complex formation (Gabrielsen and Sentenac, 1991). This interaction occurs via a component in a TFIIIB subfraction termed TFIIIB’. The TFIIIB’fraction stabilizes the binding of TFIIIC to the gene and affords protection from DNAase 1 cleavage to both DNA strands in the region immediately upstream of the TFIIIC footprint (Kassavetis et al., 1991). Photocrosslinking has shown that this fraction contains a polypeptide whose molecular size by SDS-polyacrylamide gel electrophoresis (70 kd) is very similar to that deduced for PCF4 (86.9 kd). Additionally, the location of the 70 kd polypeptide on DNA has been established by photocrosslinking to be between the transcription start site and position -38. Since the region covered by this subunit overlaps with that for the 135 kd subunit of TFIIIC (Bartholomew et al., 1991), the 70 kd protein is likely to interact with TFIIIC and thereby be squelched by an excess of this factor. These data are all consistent with the view that PCF4 encodes the 70 kd subunit of TFIIIB. Further support for this notion is evident from the deduced sequence of PCF4 and the fact that the same gene was recovered by Buratowski and Zhou (Cell, this issue) as an allele-specific, multi-copy suppressor of a TBP mutation. These genetic data constitute strong evidence that PCF4 (alias TDS4) interacts directly with TBP and are consistent with knowledge that TBP copurifies as a relatively stable complex with TFIIIB (Margottin et al., 1991). A clear explanation for this interaction is provided by the finding that the N-terminal half of PCF4 displays significant homology with the general pol II transcription factor, TFIIB (Figure 3). TFIIB is known to bind to TATA box-containing templates that have prebound TFIID or recombinant TBP. The formation of a TFIIB-containing complex is required for subsequent binding of pol II and the other basal factors (reviewed by Roeder, 1991). Thus, one of the functions of TFIIB that justifies its conservation in PCF4 is to provide interactions with TBP. Determination of Polymerase Specificity The finding of a TFIIB homology domain in PCF4 suggests a means whereby polymerase-specific transcription complexes are assembled. We propose that the unique identity of pol Ill and perhaps pol I transcription complexes is conferred by proteins containing a TBP interaction domain fused to sequences that determine interactions with polymerase-specific factors or subunits. Accordingly, PCF4 constitutes a pol Ill specificity factor, since it apparently mediates interactions between TBP and other components of the pol Ill system. Given the likelihood that PCF4 is the 70 kd subunit of TFIIIB, we expect that the unique C-terminal domain of this protein will interact with at least

Cell 218

gene +

gene/lllC

\, %,,,,,, “,,,,,,,,w,,,,)

+ lllB/TBP

,,llllllllll) -

1118’ (PCF4) IIIB” TBP

A

i gene/lllC/IIIB/TBP Figure 6. A Model for the Binding Ill Transcription Complex

Equilibria

in the Assembly

of a Pol

The direction of the equilibrium for the multi-subunit TFIIIC and TFIIIBTBP complexes is drawn favoring complex formation based on biochemical data that indicate that these entities are relatively stable (Gabrielsen and Sentenac, 1991; Margottin et al., 1991). The apparent equilibrium constant for TFIIIC binding to the promoter of a tANA gene is taken from Baker et al. (1986). Double-headed arrows indicate that the equilibrium constant is unknown.

one TFIIIC subunit (the proximal 135 kd subunit; Bartholomew et al., 1991), other TFIIIB subunits (e.g., the 90 kd polypeptide of TFIIIB”), and possibly subunits of pol Ill. PCF4 Limits Pol Ill Transcription In Vivo and In Vitro Our data indicate that the levels of PCF4 are limiting in vitro and in vivo. The latter conclusion was reached based on the identity of the deduced coding sequence of PCFC1 and its wild-type counterpart (TDS4) and on the fact that a single copy of the PCF4-1 gene activates supQ-e AlQsups7 expression whereas multiple copies of the wild-type gene (TDS4) are required for this phenotype. This observation implies that all of the other pol Ill factors must be present in stoichiometric excess relative to PCF4. It also demonstrates that a diminished interaction between TFIIIC and the A block can be compensated by increasing the cellular concentration of a protein that functions downstream in the pathway of preinitiation complex assembly (Figure 8). The limiting nature of PCF4 in vivo raises the possibility that pol Ill transcription may be controlled on a global scale by regulating the amount or activity of this factor. Accordingly, PCF4 expression may be tightly coupled to cell growth. We note howeverthat regulation at this level does not exclude other pol Ill factors as potential regulatory targets. Binding Equilibria in the Assembly of a Pol Ill Transcription Complex A surprising finding of these studies was that criteria commonly used to establish template-limiting conditions in a transcription assay may overlook important aspects of the reaction. In our experiments, conditions for linear transcription kinetics were determined from experiments in

which the time of incubation, amount of extract, carrier, and template DNA were each varied independently. Our notion that the template was truly limiting under a chosen set of conditions was further supported by the fact that gel-mobility shift experiments showed complete formation of specific complexes. However, under these conditions the addition of either more template or more extract led to a proportional increase in transcription (Figures 5 and 6). This paradox is rationalized in Figure 6, which depicts the assembly of pol Ill transcription complexes as a series of equilibria. The scheme serves to point out that some of the interactions between transcription components are relatively high affinity and proceed, for most practical purposes, to completion. An example of this type of interaction is the binding of TFIIIC to the B block promoter element. The apparent equilibrium constant (Kapp) for this reaction is about log M-’ (Baker et al., 1966). Interactions of other factors (either among themselves or with the DNA) may be much weaker, however, and thus may result in the partitioning of such factors between bound and free states. The properties of TFIIIB or perhaps more correctly, a TFIIIB-TBP complex, demonstrate this point. At template concentrations at or near the K, for transcription, the equilibrium between DNA-bound and freeformsof TFIIIB-TBP can be shifted in a direction favoring transcription by the addition of more template or more complex to the reaction. Thus, the binding of TFIIIB to pol Ill genes that was known previously as the rate-limiting step in transcription complex assembly (Geiduschek and Tocchini-Valentini, 1966) is also thermodynamically limiting under equilibrium conditions. We suggest that this equilibrium is influenced by the PCF4-7 mutation through sequential increases in the amount of the limiting PCF4 protein, the amount of TFIIIBTBP complex, and, finally, the number of complete preintiation complexes (Figure 8). The scheme also illustrates several other points that have been raised by these and other studies including the squelching of TFIIIB and the association of TFIIIB with TBP (Margottin et al., 1991). Continued genetic and biochemical studies of PCF4 and other PCF gene products will no doubt provide additional insights concerning the steps in this process. Experimental

Procedures

Genetic Methods Standard methods for growth, mating, and tetrad analysis of yeast were employed (Sherman et al., 1986). Scoring of nonsense suppression was performed on synthetic minimal media containing the appropriate auxotrophic supplements. Most of the S. cerevisiae strains used in this work are described by Willis et al. (1989). Strain FW486 (MATa his4-9726 /ys2-67 f8u2-701 ade8) was kindly provided by Fred Winston. The original mutant strain containing PCf4-7 (mutant #18-10) was crossed initially to the wild-type strain IWDI to introduce a chromosomal copy of the S. pombe supg-eAlOsupS7 suppressor gene. Two or more outcrosses to IWDI were performed before linkage and tetrad analysis. All strains from thesecrosses had oneof the following general genotypes: MATa ura3-52::URA3(sup9-aA 74supS7)leu2-3,7 72 trpl-7 met8-7 hisl17,15 PCFX or MATa ura3-52::lJRA3(sup&e A79wpS7) leu2-3,7 72 trpl-7 met8-7 arg4-17 PCFY. Plasmid pUNTDS4 was used to direct integration of a LEU2 marker at the PCF4 locus of a dipioid strain heterozygous for the PCF4-7 mutation (see above). As a result of integration at the PCF4-7 locus, all of the haploid Leu’ progeny obtained by random spore analysis expressed sups7 suppressor activity. The sup9-e A79-sups7 suppressor was evicted from strains to be

A TFIIB 219

Homolog

Activates

Pol Ill Transcription

used for extract preparation by growth on synthetic complete medium containing 0.5 mglml 5fluoro-orotic acid (Boeke et al., 1987).

Plasmfd

DNAs

The wild-type PCF4 gene (gifl of Stephen Buratowski) was cloned as an 4.3 kb Xhol-Sphl fragment from YEpTDS4 into the plasmid pUNlOO, which had also been cleaved by these enzymes. The resulting plasmid, pUNTDS4, was cleaved by Bglll to direct integration at the PCF4 locus. Plasmids containing sup9e, sup9e A79, and 5S RNA genes are described by Willis et al. (1989). Plasmids containing the tRNA”ctRNA&P gene (pYM205) and the SUQS gene (pPM16) are described by Willis et al. (1992). The SUP53 tRNALW gene was provided by Chris L. Greer. A plasmid containing the S. cerevisiae U6 snRNA gene that included the downstream B block was obtained from David Brow. Plasmid pGem4 was from Promegaand pRS315 was provided by Mike Snyder.

Cloning and Molecular Analysis of PCF4-1 Chromosomal DNA from a PCF4-7 strain (#18-10; Willis et al., 1989) prepared according to Sherman et al. (1986) was subjected to partial Sau3Al digestion and size fractionated on a lo%-40% sucrose gradient. Fragments with sizes of 6-8 kb were used to construct a total genomic library by cloning into the BamHl site of plasmid pRS315. An estimated 7000 Amp’ transformants were obtained in E. coli strain DH5a.Transformedcellsweregrowninasingle2OOml batchofTerrific broth plus ampicillin for the preparation of plasmid DNA. The amplified library was used to obtain Leu+ transformants of yeast strain IWDl. Colonies were then replica plated onto medium lacking tryptophan and methionine. Trp’ Met+ colonies appeared within 2 days. Plasmid DNA was extracted from cultures of these colonies and recovered as Amp’ transformants in E. coli DH5a. The location of the PCF4-7 gene within the cloned insert was determined initially by subcloning and then by generating a nested set of exonuclease Ill deletion clones (Erase-A-Base, Promega). All clones were tested for PCf4-7 function by transformation of IWDl and selection for suppressor activity. Double stranded DNA sequencing was performed using Sequenase Version 2 (US Biochemical). Computer analysis of the DNA sequence and database searches were carried out using programs of the University of Wisconsin Genetics Computer Group (Devereux et al., 1984). Preparation

of Whole-Call

Extracts

and Purification

of TFIIIC

Yeast cultures (3.6 I) were harvested at an 00, of 4.0, washed once with cold sterile water, and the cell pellets were stored at -70 OC. Whole-cell extracts were prepared by glass bead disruption of 20 g (wet weight) of wild-type or PC&7 cells as described previously (Willis et al., 1989) except for the following modifications. Broken cells were extracted with 0.5 M (NH&SO, prior to the 100,000 x g spin. After the final 70% (NH&SOI precipitation, the resuspended protein (E-10 ml) was fractionated on Sephacryl S200. A fraction equal to one and a half sample volumes containing all of the excluded protein and some partially included material was collected and stored at -70°C in 150 ul aliquots. Protein concentrations in crude extracts were determined spectrophotometrically by the method of Kalb and Bernlohr (1977) and ranged from 5 mglml to 10 mglml. The concentration of purified TFIIIC was determined using the BioRad protein assay and bovine serum albumin as a standard (Bradford method). TFIIIC was partially purified by serial chromatography on BioRex 70 and DEAE-Sephadex A25 as described by Kassavetis et al. (1989). This material was then subjected to gel filtration on Sephacryl S300. TFIIIC activity was monitored and quantified by gel-mobility shift and transcription assays as described by Willis et al. (1992).

Transcription

Assays

Fast protein liquid chromatography-purified nucleoside triphosphates were purchased from Pharmacia and radiolabeled nucleotides were from Amersham. Standard multiple round transcription assays contained excess template DNA (600 ng), limiting amounts of extract (up to 75 ug protein), and were performed according to Willis et al. (1989). All preincubations were at 25OC and actual transcriptions were at 15°C unless otherwise indicated. A number of variations of the standard assay were routinely performed on all extracts to optimize transcription and to ensure linear kinetics. These included extract (O-125 ug), template (O-600 ng, with constant total DNA concentration), and carrier

DNA (O-600 ng) titration experiments. Transcription assay reaction buffer contained 10 mM HEPES-KOH (pH 7.9) 10% glycerol, 1.2 mM ATP, 0.6 mM CTP, 0.6 mM UTP, 130 mM NaCI, 10 mM MgCI,, 2 mM dithiothreitol, and [a-=P]GTP (25 uM, 8 Cilmmol) in a total volume of 50 ul. In extract mixing experiments, the initial preincubation was in a total volume of 50 ul and contained 10 mM HEPES-KOH (pH 7.9) 10% glycerol, 130 mM NaCI, 10 mM MgCI,, 2 mM dithiothreitol, and carrier DNA (pGem4, 560 ng). The second extract (75 ug of protein) and the full complement of NTPs were added subsequently in the same buffer, bringing the final reaction volume to 100 pl. For the experiment in Figure 7, the initial preincubation contained sup9e DNA (6.5 fmol) and increasing amounts of TFIIIC (O-97.5 fmol, determined by gel shift analysis) in 30 ul of 10 mM HEPES-KOH (pH 7.9) 10% glycerol, 130 mM NaCI, 10 mM MgCl*, 2 mM dithiothreitol. After the preincubation, reactions were supplemented with the full complement of NTPs, label, salts, carrier DNA (560 ng), and excess wild-type or PC/+7 extract protein (75 ug, determined by template titration), bringing the final volume to 50 pl. Single round reactions were performed using excess sup4e DNA (600 ng) and extract protein (75 ug) as described by Willis et al. (1992). Ternary complexes were allowed to form at 25OC for 20 min in a 50 ul reaction containing 10 mM HEPES-KOH (pH 7.9) 10% glycerol, 1.2 mM ATP, 0.6 mM UTP and [@P]GTP (25 PM, 16 Cilmmol), 130 mM NaCI, 10 mM MgCl*, and 2 mM dithiothreitol. Nascent transcripts were elongated during a 10 min incubation at 15OC after addition of CTP (0.6 mM) and heparin (300 pg/ml). For the experiment shown in Figure 6,73 ug of PCF4-7 extract protein was used in the initial preincubation. The preincubation buffer was supplemented with carrier DNA (pGEM4, 27 nglug of extract protein). After incubation at 25°C for 20 min, varying amounts of the same extract (30-146 ug) that had been preincubated with carrier DNA (as above for 20 min at 4OC) were added. This was followed immediately by addition of ATP, UTP, and [a-32P]GTP (25 uM, 8 Cilmmol) to the usual final concentrations, and the incubation was continued for 30 min. The reaction volume was now 100 ul. Nascent transcripts were extended in the presence of heparin as before. Linear regression analysis was performed with SigmaPlot from Jandel Scientific.

Acknowledgments We are very grateful to Steve Buratowski for providing the sequence of the 7094 gene prior to publication and for the YEp24 plasmid containing this gene. Thanks also to Jon Warner, LaDonne Schulman, and lndra Sethy for helpful comments on the manuscript. This work was supported by grants to I. M. W. from the National Institutes of Health (GM42728) and the Alexandrine and Alexander Sinsheimer Fund and BRSG funds to the Albert Einstein College of Medicine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

May 21, 1992;

revised

July 31, 1992.

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