From the Department of Biochemistry, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205
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ABSTRACT |
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The influence of transcription factor (TF) IIE on mRNA synthesis in vivo was examined in a temperature-sensitive yeast mutant. A missense mutation in the conserved zinc finger domain severely weakened TFIIE's transcription activity without appreciably affecting its quaternary structure, chromatographic properties, or cellular abundance. The mutation conferred recessive slow-growth and heat-sensitive phenotypes in yeast, but quantitative effects on promoter utilization by RNA polymerase II ranged from strongly negative to somewhat positive. Heat-induced activation of the HSP26, HSP104, and SSA4 genes was attenuated in the mutant, indicating dependence on TFIIE for maximal rates of de novo synthesis. Constitutive HSP expression in mutant cells was elevated, exposing a negative (likely indirect) influence by TFIIE in the absence of heat stress. Our results corroborate and extend recent findings of differential dependence on TFIIE activity for yeast promoters, but reveal an important counterpoint to the notion that dependence is tied to TATA element structure (Sakurai, H., Ohishi, T., and Fukasawa, T. (1997) J. Biol. Chem. 272, 15936-15942). We also provide empirical evidence for conservation of structure-activity relationships in TFIIE's zinc finger domain, and establish a direct link between TFIIE's biochemical activity in reconstituted transcription and its function in cellular mRNA synthesis.
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INTRODUCTION |
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Transcription factor (TF)1 IIE is one of several general transcription factors (the others are TFIIB, TFIID, TFIIF, and TFIIH) that collectively enable RNA polymerase II to carry out promoter-dependent transcription in cell-free systems derived from a wide variety of eukaryotic organisms (Refs. 1-3; reviewed in Ref. 4). TFIIE binds to RNA polymerase II (5, 6), TFIIH (6-9), and Gal11p, a subunit of various RNA polymerase II "holoenzymes" (10-13), and is thought to recruit TFIIH to preinitiation complexes in position to catalyze DNA unwinding and phosphorylation of the carboxyl-terminal repeat domain in the largest subunit of polymerase (4, 5, 7-9).
In mammalian reconstituted systems, TFIIE and TFIIH effect DNA strand separation at the start site prior to initiation (promoter melting) in an ATP-dependent manner (Refs. 14 and 15; and reviewed in Ref. 16). Heteroduplex "bubbles" upstream of and encompassing the start site, where TFIIE closely contacts DNA (17), can bypass the need for both factors (15, 18). In reactions employing topologically relaxed DNA templates and limiting nucleoside triphosphate concentrations, TFIIE and TFIIH (with ATP or dATP as a co-factor) also help nascent transcription complexes overcome a barrier to elongation encountered close downstream of the start site (promoter escape; Refs. 19-21). In reconstituted systems comprising human proteins, the requirement for TFIIE and TFIIH depends on promoter stability and DNA superhelicity (19, 22, 23). In similar systems from yeast and flies, TFIIE seems essential for productive initiation regardless of template topology (3, 24, 25).
TFIIE consists of two dissimilar polypeptide subunits (1-3). Genes encoding the TFIIE subunits in Saccharomyces cerevisiae, designated TFA1 and TFA2, are essential for cell viability (2), and both gene products (Tfa1p and Tfa2p, respectively) are required for TFIIE activity in vitro (25). The Tfa1p subunit has homology to the 56-kDa subunit of human TFIIE (2), most notably in a zinc finger domain (26) that is crucial for human TFIIE activity in vitro (27, 28). Conditional tfa1 mutations, including those that affect this domain, cause recessive growth defects associated with reductions in steady-state levels of total cytoplasmic polyadenylated RNA, consistent with a broad requirement for TFIIE in mRNA synthesis (29, 30).
To help delineate the role of TFIIE in transcription more precisely, we
assessed the effects of a missense mutation in its zinc finger domain
on steady-state levels and de novo induction of specific
mRNAs in vivo. The mutation conferred recessive
phenotypes in yeast including slow growth at 30 °C and lethality at
38-39 °C. At each temperature, however, cellular mRNA levels
were affected in disparate ways that suggested differential dependence
on TFIIE activity. We measured the expression of genes with TATA box
promoter elements conforming to the consensus sequence
5-TATA(A/T)A(A/T)-3
(31), a gene with a non-canonical TATA-like
sequence, and genes lacking a recognizable TATA box. Contrary to a
recent proposal (29), the quantitative effects of the tfa1
mutation argue against a simple correlation between TFIIE dependence
and promoter TATA box content. The TFIIE variant was purified to
apparent homogeneity, and the effects of the zinc finger substitution
on its biochemical activity were examined in a minimal reconstituted
system for promoter-dependent transcription.
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EXPERIMENTAL PROCEDURES |
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Mutagenesis and Allele Replacement--
A double missense
mutation in the TFA1 ORF changing cysteine 124 to serine and
proline 125 to threonine (C124S/P125T) was created in the phagemid
clone pBS/TFA1 (2) by oligonucleotide-directed mutagenesis (32) with
the following primer (mutagenic bases underlined):
5-GGTTACATGTCGACGATTTGTTTGACC-3
. Escherichia
coli strain BMH71-18 mutS (CLONTECH) was
transformed to ampicillin resistance with mutagenesis reaction products
and transformants were screened for plasmids bearing a new
HincII cleavage site created by the mutation. The resulting
allele was designated tfa1-1. This mutation was intended to
disrupt the putative zinc coordination site (26, 27) in the Tfa1p
polypeptide while creating a physical marker in the gene (the
HincII cleavage site) to facilitate its detection in yeast
genomic DNA.
Cellular RNA and Protein Analysis--
Cultures were grown to
mid-log phase at 28 or 30 °C in YPD medium. Half of each culture was
transferred to a prewarmed flask in a 38 °C bath and shaking was
continued. At subsequent times, cells were collected from culture
aliquots by centrifugation for protein analysis (4 ml) or RNA analysis
(10 ml) and immediately frozen in liquid nitrogen and stored at
80 °C. Cells were broken with glass beads to recover soluble
protein (24). Total cellular RNA was recovered by extraction with hot
acidic phenol and ethanol precipitation (36). Protein was quantified
with the Bio-Rad Protein Assay using bovine serum albumin as a
standard. Purified RNA was quantified by absorbance at 260 nm, and
specific mRNA content was analyzed by Northern blot hybridization
with 32P-labeled probes. Radioactivity hybridized to blots
was quantified with a Fuji PhosphorImager and MacBAS software
(Hitachi).
Purification of Bacterially Expressed TFIIE--
The
TFA1 and TFA2 ORFs were cloned in pET vectors
(Novagen) for overexpression in E. coli strain BL21(DE3)
(37). pET21-TFA1 contains the wild-type TFA1 ORF and 189 bp
of 3-flanking sequence (2) cloned into the NdeI site in
pET21a(+). pET21-TFA2 contains the wild-type TFA2 ORF and 40 bp of 3
-flanking sequence (2) cloned between the NcoI and
XhoI sites in pET21d(+). pET21-TFIIE, derived from
pET21-TFA1 and pET21-TFA2, contains both ORFs, each transcribed from
its own T7 promoter (construction details available on request). The
260-bp MunI fragment in pET21-TFIIE was replaced with the
corresponding fragment from ptfa1-1::URA3 to yield the mutant
co-expression plasmid pETIIE-C124S. Co-expression plasmids were
sequenced to ensure the absence of unwanted mutations in the
TFA1 ORFs.
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RESULTS |
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Phenotypes of a Heat-sensitive TFIIE Mutant-- To investigate the phenotypes of a substitution mutation in the TFIIE zinc finger domain, heterozygous yeast bearing the recessive C124S/P125T mutation (TFA1/tfa1-1) were sporulated to obtain haploid progeny. Phenotypically normal or slow-growing haploid offspring were routinely recovered in a 2:2 ratio, indicating allele segregation at a single locus (Fig. 1A). As shown by Southern blot hybridization (Fig. 1B), the C124S/P125T mutation co-segregated with the slow-growth phenotype at 30 °C (Fig. 1A, upper panel) and with inviability at 38-39 °C (Fig. 1A, lower panel). Co-segregation was observed in three independently derived heterozygotes.2 Both of these phenotypes were reversed by transformation of tfa1-1 cells with an integrating plasmid containing the wild-type TFA1 gene (pTFA1::URA3), but not with its allelic derivative, ptfa1-1::URA3.
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Constitutive Transcription in tfa1-1 Yeast-- To test whether the tfa1-1 phenotypes coincided with defects in transcription in vivo, cellular mRNA levels were monitored at 30 and 38 °C. The mRNAs chosen for analysis are short-lived in normal cells and rapidly lost at the restrictive temperature in heat-sensitive mutants affecting TFIIH (41-44) or RNA polymerase II (45). Their steady-state levels are therefore closely tied to rates of synthesis (41-45). The genes contain a canonical TATA box promoter element (ACT1, MET19) or a non-canonical TATA-like sequence (STE2), or lack any TATA element within 300 bp upstream of the initiator codon (CDC7, CDC9, and RAD23).
In mutant cells at 30 °C (Fig. 2A, zero time points), steady-state mRNA levels were either normal (RAD23, ACT1) or reduced to 65-75% (CDC7, CDC9) or 50% (MET19, STE2) of wild-type levels, as shown in plots for quantified Northern blot signals in Fig. 2B (curves for ACT1 and STE2 mRNA content were superimposable with those for RAD23 and MET19, respectively,2 and are omitted for clarity of presentation). Most of these mRNAs dwindled further in tfa1-1 cells after a temperature jump. RAD23 and ACT1 mRNA, which started out at normal levels, fell to 50% of normal within 20 or 30 min, respectively (Fig. 2B, left panel, and data not shown). During the same period, mRNA levels for MET19 (Fig. 2B, left panel) and STE2 (data not shown) fell to 20-30% of normal, respectively, consistent with the faster turnover of these messages (41, 42). However, despite these steep initial declines (consistent with abrupt cessation of synthesis), levels of RAD23, ACT1, MET19 and STE2 mRNA eventually stabilized at 20-30% of wild-type levels, reflecting residual synthesis and/or defective mRNA turnover (Fig. 2B, left panel, and data not shown). Steady-state levels of soluble Tfa1-C124S/P125T protein remained normal in mutant cells at 38 °C, as shown by immunoblotting (Fig. 2A, bottom panel; the immunoblot signal shown corresponded to roughly 1 ng of TFIIE per 10 µg of total soluble extract protein).2 The mutation evidently depressed mRNA levels by changing TFIIE's specific activity (or subcellular localization) rather than its abundance in the cell.
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Transcriptional Activation in tfa1-1 Yeast-- To test whether the TFIIE mutation blocked transcriptional activation, as do mutations affecting TFIIH subunits Rad3p (41) or Ssl2p/Rad25p (42), we monitored mRNA levels for the heat-shock genes HSP26, HSP104, and SSA4 (46-48). In the absence of heat stress (28 °C), the tfa1-1 mutant expressed these genes at 2-3-fold higher levels than did wild-type cells (Fig. 3, zero time points, and data not shown). This could conceivably stem from low constitutive expression of SSA1 and SSA2, whose products negatively regulate the heat-shock transcription factor, HSF (46, 47), and from low levels of a repressor that works through negative cis-acting elements in the HSP26 promoter (48). Derepression of HSP26 could also reflect chronic stress associated with a general transcription defect, such as from nutrient deprivation (see Ref. 48). (The doubling times for logarithmically growing cultures of wild-type and tfa1-1 cells were 1.5 and 2.5 h, respectively (see also Fig. 1A).) In any case, since wild-type TFIIE has never been shown to directly repress gene transcription in vitro, the elevated constitutive expression of HSP genes in mutant cells most likely derives from reduced expression of negative regulatory genes.
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Basal Transcription Activity in Vitro-- To investigate the biochemical basis of these transcription phenotypes, TFIIE heteromers containing wild-type Tfa2p and wild-type or variant Tfa1p were purified from E. coli for analysis. In either case, the two subunits remained associated during ammonium sulfate fractionation and anion and cation exchange chromatography (Fig. 4A). The C124S/P125T substitution did not preclude interaction with the Tfa2p subunit, consistent with findings for similar substitutions in human (27, 28) or yeast TFIIE (30). Each heteromer eluted from a DEAE column in a symmetric peak at 760 mM potassium acetate,2 and from a heparin column at 280 mM potassium acetate (Fig. 4B). Both heteromers had identical subunit stoichiometries (Fig. 4A) and an apparent native molecular mass of 260 kDa, as judged by size exclusion chromatography (Fig. 4C). All of these properties were indistinguishable from those of TFIIE purified from yeast whole cell extract (25), strongly suggesting that the C124S/P125T mutation does not grossly perturb the overall structure of TFIIE.
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DISCUSSION |
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We have examined the effects of a temperature-sensitive mutation in the conserved zinc finger domain of TFIIE on mRNA accumulation in vivo. The effects of the mutation appear to be gene-specific, with different promoters affected to different extents. While it might be imagined that these effects do not truly gauge the dependence on TFIIE because tfa1-1 is a "weak" allele, the underlying premise that TFIIE's contribution to transcription is uniform and obligatory for all promoters is not entirely consistent with biochemical data (19, 22, 23). Moreover, Sakurai et al. (29) recently described a stronger TFIIE loss-of-function mutation (tfa1-21) bearing substitutions that destabilize the heteromer and, at the nonpermissive temperature, cause wholesale degradation of both subunits in vivo. ACT1 mRNA levels decay to zero in tfa1-21 cells after a temperature shift (29), indicating a strict dependence on TFIIE. Yet even in this seemingly null background, mRNA levels for HIS3, GAL4, and GAL80 are affected much less severely (29). The variegated effects on gene expression reported here for tfa1-1 are therefore not peculiar to a weak allele.
Variable dependence on TFIIE is well documented for human promoters
in vitro (22, 23). Our results, together with those of
Sakurai et al. (29), provide evidence for this phenomenon in
living cells. A total of 18 different genes have been examined in
tfa1 mutants (Ref. 29 and this report), with effects ranging from negligible (the +1 start site in the HIS3 transcription
unit; Ref. 29) to modest (GAL80, CDC7) to severe
(MF2, MET19). How might these differences
arise? Differential effects on HIS3 and GAL80
start site utilization in the tfa1-21 mutant led Sakurai et al. (29) to postulate a link between TFIIE dependence and canonical TATA box promoter elements. While TATA elements may somehow
influence the degree of dependence TFIIE for those genes and others
such as ACT1 (29), mRNAs for the TATA-less genes CDC9 and RAD23 suffer equally sharp declines in
the tfa1-1 mutant (Fig. 2), even though this allele is
weaker than the tfa1-21 allele, at least with regard to
effects on cellular TFIIE levels and ACT1 expression (Fig. 2
and Ref. 29). These results clearly indicate that TATA elements are not
necessary for establishing a strong dependence on TFIIE activity
in vivo.
Since ACT1 mRNA synthesis evidently depends on TFIIE (29), its persistence at 38 °C in tfa1-1 cells (Fig. 2) probably reflects the residual activity exhibited by the purified TFIIE variant, TFIIE-C124S/P125T (Fig. 5B). While, in a quantitative sense, the transcription activity of this variant seems more severely defective in vitro than in vivo, interactions with other macromolecules in the cell, like RNA polymerase II (5, 6), TFIIH (6-9), Gal11p (10), and/or molecular chaperones, may ameliorate the mutation's possible effects on the conformational stability of the zinc finger domain (26), effects that could be exacerbated by the relatively harsh conditions of purification. In any case, our results establish a direct (albeit qualitative) connection between TFIIE's activity in reconstituted "basal" transcription systems (1-3, 24, 25) and its function in cellular mRNA synthesis (Refs. 29 and 30, and this work). In addition, since an analogous cysteine-to-alanine substitution in the zinc finger of human TFIIE affects its biochemical properties in similar ways (27), our findings provide empirical evidence for a functionally significant and phyllogenetically conserved structure-activity relationship in the TFIIE zinc finger domain.
Each step in the initiation pathway for RNA polymerase II (reviewed in Ref. 4) may potentially pose a kinetic barrier to transcription (51). Recruitment of polymerase to the promoter can be rate-limiting for transcription in vivo (13). Promoter utilization in such cases should be relatively insensitive to fluctuations in TFIIE activity if polymerase recruitment occurs independent of TFIIE, as shown in vitro (4). If TFIIE's role is limited to promoter melting or promoter escape (14, 15, 19-21), the sensitivities of promoters to changes in levels of TFIIE activity should be proportional to the energetic cost of these isomerizations, which need not be the same for all promoters (51). The degree of dependence on TFIIE in vivo may thus be tied to the helical stability of DNA in the vicinity of the start site, as seen for human TFIIE on "naked" DNA templates (15, 19, 22, 23). This stability may be intrinsic to DNA sequence, but could also be influenced in a conditional way by the topological effects of chromatin structure (13). In Drosophila cells, heat-shock gene activation appears to hinge on the release of transcriptionally engaged polymerases stalled 20-40 bp downstream of the start site (52, 53). If this represents a general case in which promoter escape is rate-limiting, one might predict dependence on TFIIE and TFIIH for maximal rates of HSP transcription, as reported here for TFIIE (Fig. 3B) and elsewhere for TFIIH (41). Further work is needed to fully delineate TFIIE's promoter specificity in vivo, and to try to recapitulate it in vitro so that dynamic effects of promoter structure can be correlated to gene activity in the cell.
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ACKNOWLEDGEMENTS |
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We thank B. Paulus, A. Argyrou, and Y. Jeng for help with mutagenesis and plasmid construction; M. Healy, B. Ahn, P. Tran, and A. Hoffman for transcription proteins; D. Levin, C. Connelly, P. Hieter, S.-J. Lin, V. Culotta, S. Guzder, L. Prakash, F. Nouvet, S. Michaelis, W. Walter, E. Craig, M. Vos, and S. Lindquist for yeast strains and plasmids; and W. Walter and E. Craig for antibody against yeast HSF.
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FOOTNOTES |
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* This work was supported in part by grants from the National Institute of General Medical Sciences and the American Cancer Society (to M. H. S.) and by Training Grant CA09110 from the National Cancer Institute (to P. T.). The Biochemistry DNA core facility was supported by a National Institute of Environmental Health Sciences Grant ES03819.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Junior Faculty Research Award from the American
Cancer Society. To whom correspondence should be addressed. Tel.:
410-614-2709; Fax: 410-955-2926; E-mail:
msayre{at}welchlink.welch.jhu.edu.
1 The abbreviations used are: TF, transcription factor; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; bp, base pair.
2 P. Tijerina and M. H. Sayre, unpublished observations.
3 M. Deng and M. H. Sayre, unpublished observations.
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REFERENCES |
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