Domain Organization and Functional Properties of Yeast
Transcription Factor IIIA Species with Different Zinc
Stoichiometries*
Silvia
Pizzi,
Giorgio
Dieci,
Paolo
Frigeri,
Giovanni
Piccoli
,
Vilberto
Stocchi
, and
Simone
Ottonello§
From the Institute of Biochemical Sciences, University of Parma,
I-43100 Parma and the
Institute of Biological Chemistry,
University of Urbino, I-61029 Urbino, Italy
 |
ABSTRACT |
Transcription factor IIIA (TFIIIA) binds to the
5 S rRNA gene through its zinc finger domain and directs the assembly
of a multiprotein complex that promotes transcription initiation by RNA
polymerase III. Limited proteolysis of TFIIIA forms with different zinc
stoichiometries, in combination with DNA binding and in
vitro transcription analyses, have been used herein to
investigate the domain organization and zinc requirements of
Saccharomyces cerevisiae TFIIIA. Species containing either
nine, six, or three zinc equivalents were produced by reductive
resaturation and controlled metal depletion of recombinant TFIIIA.
Partial digestion of the metal-saturated, 9 Zn2+-liganded
factor yields a stable intermediate comprising the eight N-terminal zinc fingers, and a less stable fragment corresponding to a
C-terminal portion including the ninth finger. Proteolyzed TFIIIA has
the same 5 S DNA binding ability of the intact protein yet no longer
supports in vitro 5 S rRNA synthesis. Both the structural compactness and the 5 S DNA binding ability of the TFIIIA form only
containing 3 zinc ions are severely compromised. In contrast, the 6 Zn2+-liganded species was found to be indistinguishable
from metal-saturated TFIIIA. By demonstrating the existence of three
classes of zinc-binding sites contributing differently to yeast TFIIIA
structure and function, the present study provides new evidence for the
remarkable flexibility built into this complex transcription factor.
 |
INTRODUCTION |
Transcription factor
(TF)1 IIIA, the prototype of
the zinc finger protein superfamily (1), binds to the internal control region (ICR) of the 5 S rRNA gene and directs the assembly of a
multiprotein complex, containing TFIIIB and TFIIIC, that promotes accurate initiation of transcription by RNA polymerase III (2-4). As
the first gene-specific transcription factor identified in eukaryotes
(5), Xenopus laevis TFIIIA, which is both a DNA- and an
RNA-binding protein, is the best characterized with respect to the
interaction with the 5 S DNA ICR and the 5 S rRNA transcript (6). The
study of its role in promoting transcription, however, has been partly
hampered by the difficulties encountered in the purification of the
other components of the amphibian RNA polymerase III transcription
apparatus. The situation is somehow reversed in the case of
Saccharomyces cerevisiae. In yeast, the availability of
highly resolved and purified transcription components (7) has allowed
detailed investigations of the mechanism of 5 S rRNA gene
transcription (8-11), yet the gene for yeast TFIIIA has been cloned
much later than its amphibian homologue (12, 13), and only recently the
yeast protein has been obtained in a recombinant form suitable for
structural and functional studies (14-16).
Binding of 5 S DNA by Xenopus TFIIIA depends on a large
protein domain comprising nine adjacent zinc fingers of the
Cys2-His2 type, in which a zinc ion is
coordinated by invariant cysteine and histidine residues (1, 17).
Studies conducted on isolated finger polypeptides have shown that zinc
coordination is essential for the correct folding of a finger domain
(18-21). A number of studies, mainly based on footprint analysis of
zinc finger deletion mutants, have demonstrated that N-terminal and
C-terminal zinc fingers contact, respectively, the 3'- and 5'-moieties
of the 5 S DNA ICR (6, 22-24). This orientation is thought to
facilitate protein-protein contacts between the small C-terminal extra
finger region of TFIIIA, in which the transcription activation function has been shown to reside (22, 25), and the other components of the
transcription machinery. This modular structure, initially revealed by
limited proteolysis studies conducted on the native protein (1, 22, 26,
27), is reminiscent of the organization of RNA polymerase II
transcriptional activators. In the case of TFIIIA, however, the
DNA-binding domain is much larger than the activation domain, as if the
requirements for a complex mode of binding had to be satisfied, and the
proper functioning of the activation region strongly depends on its
position relative to the DNA-binding domain (25). Indeed, despite a
large interaction surface (a total of 52 base pairs covered by nine
adjacent zinc fingers), the estimated binding affinity of
Xenopus TFIIIA is comparable to that of other DNA-binding
proteins that occupy much smaller regions (28, 29). In the case of
yeast TFIIIA, the native domain organization of the protein has not
been probed yet, and the definition of the boundary between the DNA
binding and the activating region is further complicated by the fact
that the most C-terminal finger is separated from the others by an 81-amino acid-long linker region containing residues that are critical
for transcription activation (30, 31). This unique feature of yeast
TFIIIA, together with its high sequence divergence (20% identity) from
the well characterized amphibian homologue (which is unable to bind the
yeast 5 S DNA ICR and does not reconstitute 5 S rRNA synthesis in a
yeast in vitro transcription system (32)), makes the yeast
protein a particularly interesting candidate for detailed structural
and functional studies.
Among the various strategies that have been employed to study the
functional role of individual TFIIIA fingers and to understand the
reasons for such an extended DNA-binding surface, the analysis of
deletion and so called "broken finger" mutants has been the most
widely used. Whereas the first approach makes use of artificially truncated polypeptides, with the inherent risk of anomalous structural rearrangements, the second approach allows, in principle, the disruption of individual zinc fingers without introducing any major
alteration in the overall structure of the protein. These two
complementary approaches have contributed to elucidate the topography
of individual finger-DNA interactions on the 5 S rRNA gene as well as
the contribution of different fingers to DNA or RNA binding (reviewed
in Refs. 6 and 24). More recently, through the combined use of these
two approaches, it has been inferred that the interaction between
TFIIIA and 5 S DNA is a complex one, in which all fingers can be
equally involved in 5 S DNA binding (without the thermodynamic
dominance of any specific subset of fingers) and that functional
interactions between fingers significantly contribute to the overall
binding energy (33). In keeping with the complex mode of binding
emerging from these studies, the solution structure of the first three
zinc fingers of Xenopus TFIIIA complexed with 5 S DNA also
evidenced an important role of finger-finger interactions in
determining specific DNA binding (34, 35), and the recently solved
crystal structure of the first six zinc fingers of Xenopus
TFIIIA bound to the cognate DNA revealed that different fingers can
contact DNA in a very different manner (36, 37). Given the complexity
of the TFIIIA DNA-binding domain and the functional interdependence of
individual fingers, it would be most informative to address the overall
organization of TFIIIA by changing the zinc coordination state of the
protein without introducing any amino acid sequence alteration. Such a strategy could be based on the stepwise removal of zinc ions from metal-saturated TFIIIA. The existence of distinct classes of
zinc-binding sites with different metal affinities has been documented
previously for Xenopus TFIIIA (38, 39), yet the structural
and functional consequences of partial zinc removal have not been
examined, and nothing is known about the significance of this
heterogeneity in zinc binding affinity.
By taking advantage of the availability of transcriptionally competent,
purified recombinant yeast TFIIIA (15) and of a highly resolved
in vitro transcription system (40), we set out to
investigate, with a non-mutagenic approach, the role of zinc ions in
determining the properties of the yeast transcription factor. We first
determined the native domain organization of the protein by means of
limited proteolysis. TFIIIA forms with different zinc stoichiometries
were then generated and characterized from a structural and functional
point of view.
 |
EXPERIMENTAL PROCEDURES |
Yeast TFIIIA Expression and Purification--
Yeast TFIIIA was
expressed at high levels in Escherichia coli BL21(DE3) cells
co-transformed with plasmid pET-IIIA and a multicopy plasmid carrying
the gene for a minor tRNAArg(AGA/AGG) species (15).
Recombinant TFIIIA purification followed a previously described
procedure (15) with a few modifications. One of these was the
introduction of a nucleic acids removal step at the beginning of the
purification procedure. Accordingly, the pellet obtained from the
initial ammonium sulfate precipitation (50%
(NH4)2SO4 saturation) of the
soluble lysate obtained from 2 liters of bacterial culture was
dissolved in 30 ml of TEGZ-
buffer (50 mM Tris-HCl, pH
7.5, 50 µM EDTA, 10% glycerol, 50 µM ZnSO4, 2 mM
-mercaptoethanol), dialyzed for
4 h against the same buffer containing 0.6 M NaCl
(TEGZ-
/0.6), and centrifuged at 12,000 × g for 15 min. After adjustment to final NaCl and protein concentrations of 0.25 M and 0.75 mg/ml with TEGZ-
buffer, the clarified
supernatant (360 ml) was adsorbed in batch to 50 ml of DEAE-Sephacel
(Amersham Pharmacia Biotech) equilibrated in TEGZ-
/0.25 buffer, and
unbound proteins were then directly subjected to Bio-Rex 70 (Bio-Rad)
chromatography. Other modifications were the elimination of
-mercaptoethanol from all the buffers used after Bio-Rex 70 chromatography and an improved final purification step, carried out on
a heparin Ultrogel A4R (IBF Biotechnics) column (1 ml) equilibrated and
washed with HNG buffer (30 mM Hepes-Na, pH 8.0, 10%
glycerol) containing 0.1 M NaCl (HNG/0.1) and eluted with a
0.3-1 M NaCl gradient. Pooled fractions from heparin
Ultrogel chromatography contained yeast rTFIIIA at greater than 90%
homogeneity and were subsequently concentrated to a final volume of 7 ml, corresponding to a protein concentration of 1.25 mg/ml. During the
initial phases of purification, protein concentrations were determined
with the method of Bradford (41), whereas an
280 value
of 27,100 M
1 cm
1 (15) was used
to determine TFIIIA concentration in highly purified samples.
Limited Proteolysis of TFIIIA--
Samples of TFIIIA (3 µg
each) were digested (37 °C) with trypsin (Sigma, T8642) in a final
volume of 10 µl, at a 1:1000 protease/TFIIIA (w/w) ratio, in
trypsinolysis buffer (30 mM Hepes-Na, pH 8.0, 100 mM NaCl, 8% glycerol). Identical amounts of TFIIIA were
used for S. aureus V8 protease (Fluka, 45712) digestions
(15-60 min, 37 °C), which were conducted in PBS buffer (80 mM Na2HPO4, 25 mM
NaH2PO4, 100 mM NaCl, 8% glycerol)
at a 1:5 protease/TFIIIA ratio. Digestions were blocked by either the
addition of 1 mM AEBSF (Sigma) or by a 2 min boiling in
SDS-PAGE loading buffer. Proteolysis products were resolved by SDS-PAGE
(42), visualized by Coomassie Blue staining, and quantitated by
densitometric analysis carried out on digitized images with the
Multi-Analyst/PC software (Bio-Rad).
N-terminal Sequencing of Protease-resistant
Polypeptides--
Samples of TFIIIA (9 µg each) were digested with
either trypsin or V8 protease under standard limited proteolysis
conditions (30 min at 37 °C). The resulting polypeptides were
resolved by SDS-PAGE on a 12.5% preparative gel and transferred to a
polyvinylidene difluoride membrane (Millipore,
Immobilon-PSQ). The membrane was briefly stained with
Coomassie Blue, and then slices corresponding to the proteolytic
fragments were excised and subjected to N-terminal sequencing using an
automated LF 3000 Protein Sequencer equipped with an on-line Gold high
pressure liquid chromatography system (Beckman) for the detection of
phenylthiohydantoin amino acids. Phenylthiohydantoin-amino acids were
separated on an Ultrasphere ODS column (Beckman).
DNA Binding Assays--
For electrophoretic mobility shift
assays, a 450-base pair DNA fragment, containing the yeast 5 S RNA
gene, was prepared from plasmid pUC9-5S (a kind gift of P. A. Weil
(43)) by BamHI-HindIII digestion and end-labeled
with [
-32P]dCTP and the Klenow fragment of DNA
polymerase I (Amersham Pharmacia Biotech). After labeling and
purification, DNA fragment concentration was determined by ethidium
bromide staining. Binding reaction mixtures contained varying amounts
(from 2.5 to 25 ng) of the different TFIIIA samples, 1 ng of labeled
DNA fragment, and pUC19 plasmid DNA at a 5:1 (w/w) DNA/protein ratio.
The composition of the binding buffer (prepared with double-distilled
water, further purified by passage through Chelex 100 resin, sodium
form (C-7901, Sigma) (44)), was 30 mM Hepes-Na, pH 8.0, 120 mM KCl, 10% glycerol, and 0.5 mg/ml ultrapure bovine serum
albumin (Boehringer Mannheim). Reaction mixtures (20 µl) were
incubated for 30 min at 20 °C and loaded on a non-denaturing 4%
polyacrylamide gel, containing 20 mM Tris-HCl, pH 8.0, and
5% glycerol, that had been pre-run for 2 h at 4 °C.
Electrophoresis was conducted at 4 °C for 2.5 h (200 V), with
two changes of the running buffer (20 mM Tris-HCl, pH 8.0, 5 mM
-mercaptoethanol). Bound and free DNA were
quantified by scintillation counting of excised gel slices as described
previously (45). For the determination of TFIIIA-5 S DNA apparent
dissociation constants, a fixed amount of TFIIIA (10 ng) was incubated
with increasing concentrations of the labeled DNA fragment (0.025-0.5 nM). At each input DNA concentration, the ratio of bound to
free DNA was determined from phosphorimages of dried gels obtained with
the Personal Molecular Imager FX (Bio-Rad), using the Multi-Analyst/PC software (Bio-Rad). Dissociation constants were determined by linear
regression analysis of Scatchard plots using SigmaPlot 4.0 (Jandel
Scientific Software). Standard error for individual Kd determinations varied from 15 to 35%.
In Vitro Transcription Assays--
Template DNA was the yeast
5 S rRNA gene contained in plasmid pUC9-5S. 250 ng of pUC9-5S were
preincubated at 20 °C for 30 min in a 45-µl reaction mixture (30 mM Hepes-KOH, pH 7.9, 100 mM KCl, 5 mM MgCl2, 8% glycerol, 8 units of RNasin
(Ambion), 1 mM DTT) containing an internally balanced set
of complementing fractions as follows: 20 ng of recombinant TBP (46),
60 ng of recombinant TFIIIB70 (46), and 0.5 µg of B" fraction (47), which together reconstitute TFIIIB activity, and 5 µg of a glycerol gradient fraction containing partially purified TFIIIC and RNA polymerase III (45). Following preincubation, RNA synthesis was started
by adding 5 µl of a solution providing ATP, CTP, and GTP to a final
concentration of 500 µM, UTP to a final concentration of
25 µM, and 10 µCi of [
-32P]UTP (800 Ci/mmol, Amersham Pharmacia Biotech). Multiple rounds of transcription
were allowed to proceed for 10 min at 20 °C; reaction products were
then purified, resolved, and quantified as described (15). For single
round transcription experiments, 5 S DNA was first preincubated with
TFIIIC, TFIIIB, RNA polymerase III and limiting concentrations of
TFIIIA for 30 min at 20 °C, in a volume of 33 µl. Stable
elongation complexes paused at nucleotide 10 (48) were then allowed to
form by adding 5 µl of a solution supplying CTP and GTP to a final
concentration of 500 µM, UTP to 25 µM, and
15 µCi of [
-32P]UTP. After 10 min at 20 °C, 2 µl of a solution providing 500 µM ATP and 300 µg/ml
heparin (Sigma, H2149 type) were added to complete the synthesis of
initiated transcripts under conditions of blocked reinitiation. The
number of transcripts synthesized in a single round of transcription
was taken to equal the number of active transcription complexes formed
during preincubation, i.e. the number of active TFIIIA
molecules. 5 S RNA transcripts were quantified from phosphorimages
of dried gels obtained with a Personal Molecular Imager FX (Bio-Rad)
using the Multi-Analyst/PC software (Bio-Rad). The number of
transcripts corresponding to each phosphorimaging signal was determined
by comparison with the signal produced by a known amount of
[
-32P]UTP. All solutions used for in vitro
transcription assays were made with Chelex-treated, double-distilled
water and ultrapure reagents.
Preparation of Yeast TFIIIA Species with Different Zinc
Stoichiometries--
Zinc-saturated TFIIIA was prepared by incubating
the purified protein (1.25 mg/ml) for 1 h at 20 °C in the
presence of 5 mM DTT, followed by a 4-h dialysis against
HNG/0.35 buffer containing 50 µM ZnSO4, and
by the final removal of unbound zinc with a 20-h dialysis against the
same buffer without added zinc. The 6 Zn2+-liganded TFIIIA
species was obtained upon incubation of metal-saturated TFIIIA (1 h,
20 °C) in the presence of 0.5 mM
4-(2-pyridylazo)resorcinol (PAR, Sigma) and removal of excess reagent
by a 20-h dialysis (two changes) against HNG/0.35 buffer. The TFIIIA
species containing 3 zinc eq was similarly prepared by incubation (30 min, 20 °C) with 2 mM EDTA, followed by the removal of
excess EDTA with a 20-h dialysis (two changes) against HNG/0.5 buffer
(an increased salt concentration was required in this case to avoid the
precipitation of the partially unfolded protein). Dialyses were
conducted at 4 °C under nitrogen, and the different TFIIIA samples
were clarified by centrifugation (12,000 × g, 10 min)
prior to protein determination. All dialysis buffers were made with
ultrapure reagents using double-distilled, Chelex-treated water.
Zinc and Sulfhydryl Group Determination--
Samples of the
different TFIIIA species were diluted to a final concentration of 1 µM with HNG/0.35 buffer containing 0.1 mM
PAR, adjusting the spectrophotometer to zero absorbance immediately before protein addition; higher protein (3 µM) and salt
(0.5 M) concentrations were used in the case of
EDTA-treated TFIIIA. In PMPS titration experiments, absorbance changes
at either 500 or 250 nm (in the case of titrations conducted in the
absence of PAR) were measured after each PMPS addition. Zinc release in
the presence of PAR only was similarly analyzed by measuring absorbance changes at 500 nm as a function of time. Zinc release from TFIIIA was
determined indirectly by monitoring the formation of a
(PAR)2·Zn2+ complex (
500 = 66,000 cm
1 M
1), whereas
mercaptide bond formation was directly followed at 250 nm. PMPS and PAR
stock solutions (4 and 5 mM, respectively) were prepared
with Chelex-treated buffers as described previously (44, 49). For the
determination of the zinc content of buffers and reaction mixtures, PAR
was added to a final concentration of 0.1 mM, and the
decrease in absorbance at 500 nm was measured after the addition of 1 mM EDTA. All spectrophotometric measurements were conducted
with a Cary 13E spectrophotometer.
 |
RESULTS |
Limited Proteolysis of Yeast TFIIIA--
We initially probed the
structural organization of yeast TFIIIA by limited proteolysis.
Transcriptionally competent recombinant yeast TFIIIA, purified from an
overproducing E. coli strain, and either trypsin or V8
protease were utilized for this analysis. As shown in Fig.
1A, a 33.5-kDa proteolytic
fragment (p33.5) was already clearly detectable after a 10-min
incubation in the presence of trypsin (lane 2); it became
the main product of trypsinolysis following an additional 20-min
digestion that led to the complete disappearance of full-length TFIIIA
(lane 4) and remained stable even after a 1-h incubation in
the presence of trypsin (data not shown). A smaller and less
represented fragment of about 15 kDa (p15) was generated at the same
time as p33.5 (lane 2) and decreased upon further digestion,
concomitantly with the appearance of an 11.5-kDa fragment (p11.5,
lane 4). Two other minor fragments of 17 kDa (p17) and 30.5 kDa (p30.5) first appeared after 20 min of trypsinolysis (lane
3) and slowly accumulated thereafter.

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Fig. 1.
Limited trypsin digestion of yeast
TFIIIA. A, time course of limited trypsinolysis.
Recombinant yeast TFIIIA (9 µg) was incubated at 37 °C in the
presence of trypsin (9 ng). Aliquots of the reaction mixture, each
corresponding to 3 µg of TFIIIA, were removed at the indicated times,
analyzed by SDS-PAGE on a 12.5% gel, and visualized by Coomassie Blue
staining. Trypsin-resistant polypeptides, whose N-terminal sequences
were subsequently determined, are identified on the right by
their apparent molecular masses (kDa). The TFIIIA sample in lane
1 (A) was incubated for 30 min in proteolysis buffer
with no added trypsin. The migration positions of molecular weight
markers run on the same gel are indicated on the left.
B, zinc dependence of the TFIIIA trypsinolysis pattern.
Reaction mixtures (3 µg of TFIIIA each) containing TFIIIA only
(lane 2) or TFIIIA supplemented with either 50 µM ZnSO4 (lane 3) or 500 µM EDTA (lane 4) were incubated for 10 min at
20 °C prior to trypsin addition. Before trypsinolysis, the sample in
lane 5 was first preincubated for 10 min with 500 µM EDTA, followed by an additional 10-min incubation in
the presence of 500 µM ZnSO4. A control
sample consisting of purified TFIIIA incubated for 30 min at 37 °C
in the absence of trypsin is shown in lane 1. Protease
digestion conditions were the same as in A. The migration
positions of molecular weight markers run on the same gel (15%
polyacrylamide) are indicated on the left; partially
sequenced, trypsin-resistant polypeptides are identified on the
right by their estimated molecular masses (kDa).
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Fig. 1B shows that this trypsin cleavage pattern was not
modified by the addition of excess zinc to the proteolysis mixture (cf. lanes 2 and 3). In contrast, the
addition of a 100-fold molar excess of EDTA (500 µM)
prior to trypsin digestion caused a complete loss of the 33.5-kDa
fragment and of all the other smaller polypeptides (Fig. 1B,
cf. lanes 2 and 4). The residual
undigested protein observed in this latter case likely results from
nonspecific self-aggregation of zinc-depleted TFIIIA. We interpreted
the destabilizing effect of EDTA as the result of a conformational
alteration of TFIIIA induced by zinc removal. Indeed, protease
resistance was fully restored by a 10-min incubation of EDTA-treated
TFIIIA with 500 µM Zn2+ prior to proteolysis
(Fig. 1B, lane 5).
In yeast TFIIIA there are 62 potential trypsin cleavage sites (lysine
and arginine) and 54 additional sites for the V8 protease (glutamate
and aspartate), yet the latter enzyme also generated, as the main
product, a stable, zinc-dependent intermediate of 34.5 kDa
(data not shown), thus confirming the existence in TFIIIA of region(s)
that in the native protein are especially accessible to proteases.
To define the boundaries of the protease-resistant regions of yeast
TFIIIA, we next determined the N-terminal sequence of the polypeptide
fragments generated by either trypsin or V8 protease and inferred the
positions of the corresponding C-terminal cleavage sites by matching
the size of each fragment with the occurrence of downstream cleavage
sites for either enzyme. As summarized by the data reported in Fig.
2, the main products of partial digestion generated by the two proteases essentially correspond to the same polypeptide fragment. The 33.5-kDa, trypsinolysis fragment is generated
by cleavage at Arg39 and ends in a region, centered around
Arg325, containing five contiguous lysine and arginine
residues; a corresponding 34.5-kDa polypeptide fragment, formed by
cleavage at either Glu28 or Glu31 (two
N-terminal sites that are cleaved with equal efficiencies) and ending
in a region centered around Glu329, was generated by V8
protease. The N-terminal sequence of all the smaller-sized fragments
generated by trypsin was also determined. The p15 fragment starts at
Lys328 and thus represents the C-terminal portion of TFIIIA
that is missing from the 33.5-kDa polypeptide. Due to a gel artifact, the molecular mass measured for this fragment is higher than the size
of 12.8 kDa that would be expected for a polypeptide comprised between
Lys328 and the C terminus. The p11.5 polypeptide also
starts at Lys328, indicating that this late appearing
fragment results from further C-terminal cleavage of p15. Similarly,
p30.5 and p17 represent secondary cleavage products of the 33.5-kDa
polypeptide. The p30.5 fragment, which has the same N terminus as
p33.5, is generated from it through further C-terminal digestion,
whereas the p17 fragment, starting at Arg180 within the
fifth finger, is likely the product of a second cleavage event at the N
terminus.

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Fig. 2.
Domain mapping of yeast TFIIIA.
A, schematic representation of yeast TFIIIA showing the nine
zinc finger motifs (numbered boxes), the small N-terminal
and C-terminal extra finger regions, the short linkers joining zinc
fingers 1-8, and the 81-amino acid linker region interposed between
the last two fingers. The positions of trypsin cleavage sites yielding
the p33.5 and the p15 fragments are indicated by arrows.
B, distribution of potential V8 protease (V8
prot.)(upper diagram) and trypsin (Tryps.)
(lower diagram) cleavage sites (indicated by short
bars) within the polypeptide sequence of yeast TFIIIA. The
N-terminal cleavage sites for V8 protease and trypsin (as determined by
sequencing), and the deduced C-terminal ends of the larger fragments
generated by either protease (34.5 and 33.5 kDa, respectively) are
marked with arrows. In the case of trypsin cleavage,
Arg327 also corresponds to the cleavage site that gives the
smaller products of trypsinolysis (p15 and p11.5).
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In its native state, yeast TFIIIA thus appears to be constituted by a
highly structured, protease-resistant domain comprising the first eight
zinc fingers (p33.5 in Fig. 1) and a small, less stable C-terminal
domain comprising the last finger and about half of the 81-amino acid
linker interposed between fingers 8 and 9 (p15 in Fig. 1). These two
domains are connected by a flexible polypeptide region, centered on a
cluster of basic amino acid residues, which is highly susceptible to
proteolytic cleavage.
Functional Characterization of Partially Proteolyzed Yeast
TFIIIA--
Having defined the limited proteolysis pattern of yeast
TFIIIA, we next analyzed the functional competence of the partially proteolyzed protein by gel retardation and in vitro
transcription assays. For these experiments, TFIIIA was first subjected
to limited proteolysis; trypsin was then blocked with the specific
inhibitor AEBSF, and upon verification of the complete conversion into
the 33.5-kDa form by SDS-PAGE analysis, samples of the same reaction mixture were directly used for DNA binding and transcription assays. Data reported in Fig. 3A show
that partially proteolyzed TFIIIA has the same (or even higher) DNA
binding ability of full-length TFIIIA (cf. lane 4 with lanes 2 and 3). Mobility shift competition experiments, conducted with a 5 S rRNA gene-containing plasmid and
with the same plasmid lacking the 5 S DNA as competitors, further
revealed that intact and partially proteolyzed TFIIIA also have an
identical binding specificity for the 5 S rRNA gene (data not shown).
As shown by data previously obtained with deletion mutants of yeast
TFIIIA (30), a minimum of three fingers is required for 5 S DNA
binding. It thus follows that the 33.5-kDa polypeptide, rather than the
less stable (and poorly represented) p15 and p11.5 fragments, is
actually responsible for the specific interaction with the 5 S rRNA
gene. Consistent with its reduced size, the 33.5-kDa polypeptide formed
complexes of distinctively higher electrophoretic mobility. Only at
increasingly high concentrations, proteolyzed TFIIIA gave rise to a
larger complex, likely resulting from the incorporation into the
primary complex of a second molecule of the 33.5-kDa fragment
(cf. lane 4 with lanes 2 and
3).

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Fig. 3.
5 S rRNA gene binding and transcriptional
activation properties of partially proteolyzed TFIIIA.
A, 5 S DNA binding. A 450-bp radioactively labeled DNA
fragment containing the yeast 5 S rRNA gene (1 ng) was incubated,
prior to non-denaturing gel electrophoresis, with the following protein
samples: 10 ng of freshly thawed TFIIIA (A, lane
2); 10 ng of TFIIIA preincubated for 30 min at 37 °C in
proteolysis buffer lacking trypsin (A*, lane 3);
increasing amounts of TFIIIA subjected to a 30-min trypsin digestion at
37 °C (10, 20, and 40 ng in lanes 4-6, respectively).
Trypsin was blocked with 1 mM AEBSF before incubation with
the DNA fragment; no protein was added to the sample in lane
1. The migration positions of the free DNA fragment
(f), the TFIIIA·DNA complex (B), and the
smaller sized p33.5·DNA complex (b) are indicated on the
left. The more slowly migrating complex in lane
6, presumably resulting from DNA binding by a dimeric aggregate of
proteolyzed TFIIIA, is marked by an asterisk. All the lanes
shown come from the same exposure of a single gel. B,
transcription analysis. The transcription capacity of proteolyzed
TFIIIA was tested in an in vitro transcription system
reconstituted from rTBP, rTFIIIB70, B" fraction, and a fraction
supplying both TFIIIC and RNA polymerase III activities, with the yeast
5 S rRNA gene as a template. Increasing amounts of TFIIIA (from 7.5 to
120 ng), incubated for 30 min at 37 °C in the absence (lanes
2-4) or in the presence (lanes 5-9) of trypsin, were
added to transcription reaction mixtures. Transcripts synthesized in
reaction mixtures lacking exogenous TFIIIA, or supplemented with
freshly thawed TFIIIA (15 ng), are shown for comparison in lanes
1 and 12, respectively. Trypsin digestion was stopped
by the addition of 1 mM AEBSF prior to in vitro
transcription. Transcripts in lanes 10 and 11 (T/in) come from control reactions in which AEBSF-blocked trypsin was
added to a reconstituted in vitro transcription system
containing 15 ng of untreated TFIIIA. The position of transcripts (5 S
RNA) after resolution on a polyacrylamide gel is shown.
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In a parallel set of experiments (Fig. 3B), we tested the
ability of partially proteolyzed TFIIIA to support 5 S rRNA synthesis in a yeast in vitro transcription system consisting of
recombinant TBP, recombinant TFIIIB70 plus a partially purified B"
fraction (which together reconstitute TFIIIB activity (47)), and a
glycerol gradient fraction contributing both TFIIIC and RNA polymerase III activities (45). In this system, only background levels of
transcription, due to the presence of trace amounts of contaminating TFIIIA in the TFIIIC/RNA polymerase III fraction, could be detected in
the absence of exogenously added TFIIIA (Fig. 3B, lane
1). The addition of increasing amounts of control TFIIIA
(preincubated for 30 min at 37 °C in proteolysis buffer lacking
trypsin) resulted in a large increase of transcription, and a plateau
was reached upon supplementation of 30 ng of TFIIIA (lanes
2-4). In contrast, the addition of up to 120 ng of partially
proteolyzed TFIIIA failed to raise transcription above the background
level (lanes 5-9). The observed transcriptional impairment
of partially proteolyzed TFIIIA was not due to the accidental
degradation of other transcription components by unblocked trypsin,
because the addition of pre-blocked trypsin to a fully reconstituted
transcription system did not interfere with 5 S rRNA synthesis
(compare lanes 10 and 11 with lane
3).
We conclude from these data that the protease-resistant, eight zinc
finger domain of yeast TFIIIA is fully competent for specific 5 S DNA
binding yet is unable to support 5 S rRNA transcription. In keeping
with the results of previous deletion mutant analyses (30, 31), the
transcription activation function of yeast TFIIIA thus appears to be
associated with a 100-amino acid long C-terminal domain (corresponding
to the p15 polypeptide in Fig. 1) that includes the last finger and
part of the linker region separating fingers 8 and 9.
Different Classes of Zinc-binding Sites in Yeast TFIIIA--
To
gain insight into the contribution of zinc ions to the conformational
stability of yeast TFIIIA, we determined the metal content of
unmodified rTFIIIA and of the EDTA-treated TFIIIA species previously
found to be extremely sensitive to trypsin and V8 proteolysis. Zinc
release from either form of TFIIIA was induced by chemical modification
of cysteine residues with p-(hydroxymercuri)benzenesulfonate (PMPS) and was monitored in the presence of the metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR) (44, 49). The release of
protein-bound zinc ions was determined by measuring absorbance changes
at 500 nm (the absorption maximum of the
(PAR)2·Zn2+complex) resulting from the
titration of TFIIIA with PMPS (Fig. 4A), whereas mercaptide bond
formation between PMPS and TFIIIA cysteine residues was monitored at
250 nm in parallel titrations conducted with PMPS only (Fig.
4B). As shown in Fig. 4A, the titration of both
forms of TFIIIA with PMPS resulted in a linear increase of absorbance,
up to plateau values corresponding to the release of 7 and 3 zinc eq,
respectively, from the untreated and the EDTA-treated species. Taken
together with limited proteolysis data (Fig. 1), these results indicate
that the presence of 7 zinc ions per protein molecule is sufficient to
confer to yeast TFIIIA its distinctive protease resistance properties,
whereas the removal of 4 additional zinc ions leads to a reversible
destabilization of protein structure. Also apparent in Fig. 4
(cf. A and B) is the good correlation between A500 and A250
curves in the case of the untreated form, confirming that zinc release
from TFIIIA was indeed due to cysteine modification. More specifically,
the fact that break points in both curves occur at nearly identical
PMPS equivalent values, corresponding to the release of 7 zinc ions and
the titration of 14 reactive sulfhydryl groups, implies that, like in
Xenopus TFIIIA (50), the release of 1 zinc ion from a yeast
TFIIIA finger also requires the chemical modification of 2 cysteine
residues. In fact, no such correlation between
A500 and A250 curves was observed in the case of the EDTA-treated species (Fig. 4, cf. A and B), an observation that is best explained by the
presence in this TFIIIA form of PMPS-reactive yet metal-uncoordinated
cysteine residues.

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Fig. 4.
Determination of the zinc and sulfhydryl
group content of untreated and EDTA-treated TFIIIA. A,
zinc determination. Increasing amounts of PMPS were added to untreated
TFIIIA (1 µM; open circles) or EDTA-treated
TFIIIA (3 µM; open triangles) in
Chelex-treated buffer containing 0.1 mM PAR. The
spectrophotometer was adjusted to zero absorbance (500 nm) immediately
before TFIIIA addition. Absorbance changes at 500 nm were monitored
after each PMPS addition, and A500 values
(normalized with respect to TFIIIA concentration) were plotted against
the number of PMPS equivalents added. The number of released zinc
equivalents was determined using an 500 value of 66,000 M 1 cm 1 for the
(PAR)2·Zn2+ complex (see "Experimental
Procedures" for details). B, sulfhydryl group
determination. A PMPS titration of untreated and EDTA-treated TFIIIA
(both at a concentration of 1 µM) was conducted as in
A but in the absence of PAR. Absorbance changes at 250 nm,
recorded after each PMPS addition, were plotted against the number of
PMPS equivalents added.
|
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At variance with the nine predicted zinc finger motifs that are
recognizable in the polypeptide sequence of yeast TFIIIA, the above
titration experiments only revealed 7 zinc ions/protein molecule, a
stoichiometry that did not change following direct zinc supplementation
(data not shown). Similarly, a total of 14 cysteine residues, instead
of the expected 20, was found to be reactive with PMPS. We thus
reasoned that both discrepancies may originate from the oxidation of a
few cysteine residues during TFIIIA expression or purification
(e.g. through mixed disulfide formation with exogenously
supplied
-mercaptoethanol). Accordingly, a resaturation procedure
was worked out, in which disulfide reduction by DTT is followed by the
elimination of the reducing agent in the presence of excess zinc and
the final removal of unbound zinc through anaerobic dialysis against
Chelex-treated buffer. Data reported in Fig.
5 show that 9 zinc eq could indeed be
recovered upon reductive resaturation with the above procedure and that this variation in the number of protein-bound zinc ions was accompanied by a corresponding increase of the number of PMPS-reactive cysteines (from 14 to 18 Cys residues). Interestingly, despite this variation in
the number of protein-bound zinc ions, the trypsin sensitivity of
metal-saturated TFIIIA remained the same as that previously determined
for the 7 Zn2+-liganded TFIIIA species (shown in Fig. 7;
cf. lanes 2 and 3).

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Fig. 5.
Zinc and sulfhydryl group content of
metal-resaturated TFIIIA. Resaturated TFIIIA (1 µM)
was titrated with PMPS in the presence (A) or in the absence
(B) of PAR. Titration conditions were the same as in Fig. 4;
A500 and A250
values were similarly plotted against the number of PMPS equivalents
added.
|
|
Another notable feature of the titration curve reported in Fig.
5A is the disproportionate release of 2 zinc eq (instead of the expected 0.5) upon the addition of the first PMPS equivalent. This
may indicate that some zinc ions can be released from metal-saturated TFIIIA without any prior cysteine modification. Indeed, as shown in
Fig. 6, approximately 2 Zn2+
eq were very rapidly released from metal-saturated TFIIIA in the
presence of PAR only, and a third one was more slowly released upon
further incubation (30 min) in the presence of PAR. Under identical
experimental conditions, only 1 zinc eq was slowly released from
non-resaturated, 7 Zn2+-liganded TFIIIA, implying that the
2 zinc ions missing from this TFIIIA species likely correspond to those
that are rapidly released from metal-saturated TFIIIA. In keeping with
this view, no zinc ion was "spontaneously" released from the
EDTA-treated form of TFIIIA (Fig. 6).

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Fig. 6.
Zinc release from TFIIIA species with
different metal stoichiometries in the absence of cysteine
modification. Untreated TFIIIA (1 µM, open
circles), metal-saturated TFIIIA (1 µM, filled
circles), or EDTA-treated TFIIIA (3 µM, open
triangles) were incubated in Chelex-treated buffer containing 0.1 mM PAR, and the time course of zinc release to PAR was
spectrophotometrically monitored at 500 nm. Upon normalization with
respect to TFIIIA concentration, A500 values
were used to determine the number of spontaneously released zinc
equivalents.
|
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Three zinc ions in metal-saturated yeast TFIIIA are thus associated to
weak metal-binding sites. They can be removed without any prior
cysteine modification, thereby generating a TFIIIA species carrying 6 zinc eq, which still exhibits the characteristic trypsin sensitivity of
metal-saturated and 7 Zn2+-liganded TFIIIA (Fig.
7, lanes 2-4). The apparent
zinc dissociation constant of these three weak binding sites, estimated
on the basis of the Kd of the
(PAR)2·Zn2+ complex (44), is higher than
10
7 M, whereas the zinc ions bound to the
three sites of highest affinity, which are not released even in the
presence of 2 mM EDTA, appear to be kinetically or
thermodynamically trapped.

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Fig. 7.
Limited proteolysis of TFIIIA species with
different zinc stoichiometries. TFIIIA species (3 µg each)
containing 9 (lane 2), 7 (lane 3), 6 (lane
4), or 3 (lane 5) zinc eq were incubated for 30 min at
37 °C in the presence of trypsin under standard conditions (see
"Experimental Procedures" for details). A control sample of TFIIIA
was incubated for 30 min in proteolysis buffer with no added trypsin
(lane 1; A). Trypsin-resistant polypeptides are
identified on the right by their apparent molecular masses
(kDa); the migration positions of molecular weight markers run on the
same gel are indicated on the left.
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Functional Properties of TFIIIA Species with Different Zinc
Stoichiometries--
Having established conditions for the controlled
metal depletion and resaturation of yeast TFIIIA, the three species
thus generated, containing, respectively, 3, 6, and 9 zinc eq, were examined for their DNA binding and transcription activation capacities. The gel retardation assays reported in Fig.
8A show that the PAR-treated form, carrying 6 zinc eq, has a 5 S DNA binding ability
indistinguishable from that of metal-saturated TFIIIA (cf.
lanes 2-5 with lanes 6-9). Apparent
dissociation constants for the TFIIIA·5 S DNA complex were next
determined by incubating constant amounts of TFIIIA with varying
concentrations of the 5 S DNA fragment (see "Experimental Procedures"). As shown in Table I,
apparent Kd values for metal-saturated and 6 Zn2+-liganded TFIIIA differ by no more than 12% and are
similar to the dissociation constant previously reported for the yeast
TFIIIA-5 S DNA interaction (14). In contrast, 3 Zn2+-liganded TFIIIA is severely impaired in its ability to
bind the 5 S rRNA gene (cf. lanes 2-5 with
lanes 10-13). No protein-DNA complex could be detected with
this TFIIIA species under conditions in which metal-saturated TFIIIA
shifted about half of the 5 S DNA fragment (cf. lanes 4 and
12 in Fig. 8A). A similar pattern was observed
when testing the ability of these three TFIIIA forms to support
in vitro transcription of the 5 S rRNA gene (Fig.
8B). The 6 Zn2+-liganded species exhibited the
same transcription activity of metal-saturated TFIIIA (cf.
lanes 2-6 with lanes 8-12). In the case of the
3-zinc form, instead, low levels of 5 S rRNA synthesis could only be
detected at the highest factor concentration (lanes 13-18),
as if the large structural disorganization caused by the removal of 6 Zn2+ ions did not completely inactivate the protein. In
doing these experiments, care was taken to avoid zinc rebinding to
metal-undersaturated TFIIIA. To this end, all the buffers used for DNA
binding and in vitro transcription assays were prepared with
ultrapure reagents and Chelex-treated, double-distilled water, and the
zinc content of 5× concentrated mock reaction mixtures (not containing
TFIIIA) was evaluated with the same PAR procedure utilized for TFIIIA analysis. The amount of zinc thus determined corresponds to a concentration <6 nM in standard (1×) reaction mixtures.
Based on a zinc dissociation constant higher than 10
7
M for the three weak binding sites and assuming that all
this zinc is available to TFIIIA, it can be estimated that such zinc concentration would allow for the resaturation of less than 10% of the
6 Zn2+-liganded TFIIIA molecules used for in
vitro assays. We cannot exclude, on the other hand, that some
contaminating zinc ions may be bound by the intermediate affinity sites
present in 3 Zn2+-liganded TFIIIA. The low residual
activity exhibited by this particular TFIIIA species may thus be due to
either incomplete inactivation or limited zinc rebinding.

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Fig. 8.
5 S DNA binding and transcriptional activity
of TFIIIA species with different zinc stoichiometries.
A, 5 S DNA binding. A 450-bp radioactively labeled DNA
fragment (1 ng) containing the yeast 5 S rRNA gene was incubated with
increasing amounts (2.5, 5, 12.5, and 25 ng) of TFIIIA species
containing 9 (lanes 2-5), 6 (lanes 6-9), or 3 (lanes 10-13) zinc eq. The DNA fragment with no added
TFIIIA was run in lane 1. DNA-protein complexes were
resolved on a 4% non-denaturing polyacrylamide gel and visualized by
autoradiography; the positions of free (f) and bound
(B) fragments are indicated. B, transcription
analysis. Increasing amounts (2, 5, 8, 12, and 16 ng) of zinc-saturated
TFIIIA (lanes 2-6), 6 Zn2+-liganded TFIIIA
(lanes 8-12), and 3 Zn2+-liganded TFIIIA
(lanes 14-18) were added to a partially reconstituted
transcription system (rTBP, rTFIIIB70, fraction B", and a TFIIIC/RNA
polymerase III fraction) programmed with the 5 S rRNA gene. Control
reactions were run in the absence of exogenously added TFIIIA
(lanes 1, 7, and 13). The position of transcripts
(5 S RNA) after resolution on a polyacrylamide gel is shown; all the
lanes shown come from the same exposure of a single gel.
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Table I
Dissociation constants for zinc-saturated and 6 Zn2+-liganded
TFIIIA·5 S DNA complexes
The average apparent Kd ± S.D. values were
determined from N independent experiments, with n
individual data points.
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|
Another potential artifact we considered is that loosely bound zinc
ions, released from 9 Zn2+-liganded TFIIIA in the presence
of PAR, could derive from partially denatured, inactive molecules
present in our TFIIIA preparations. According to this hypothesis, zinc
release upon PAR treatment would all take place at the expense of these
inactive molecules, leaving unchanged the zinc content as well as the
trypsin resistance, DNA binding, and in vitro transcription
properties of active TFIIIA molecules. To test this possibility, we
determined the fraction of active molecules in different TFIIIA
preparations by quantitative single round transcription assays (48)
(see "Experimental Procedures" for details). We found that the
fraction of transcriptionally active molecules ranged from 56 to 95%
in four different preparations yielding TFIIIA species with 6.1 ± 0.3 zinc ions/molecule upon PAR treatment. This lack of correlation
between the fractional activity and the zinc release capacity of
different TFIIIA preparations argues that 3 loosely bound zinc ions are
uniformly released by all TFIIIA molecules in the presence of PAR.
Further support to this interpretation was provided by the
densitometric analysis of limited proteolysis experiments as the one
reported in Fig. 7. Based on the complete loss of detectable
polypeptide fragments resulting from trypsin digestion of 3 Zn2+-liganded TFIIIA (Fig. 7, lane 5), one would
in fact predict a reduced recovery of the trypsin-resistant fragment
(33.5 kDa) in 6 Zn2+-liganded as compared with
zinc-saturated TFIIIA samples. The extent of such decrease, which would
result from degradation of zinc-depleted, inactive TFIIIA molecules,
should vary with fractional activity, being most prominent for TFIIIA
samples with the highest proportion of inactive molecules (such as the
56% active preparation utilized for the experiment reported in Fig.
7). We found, instead, that regardless of fractional activity, equal
amounts of the 33.5-kDa fragment were recovered upon digestion of the 9 Zn2+- and the 6 Zn2+-liganded species.
Importantly, the total densitometric signal (obtained by summing the
p33.5, p30.5, p17, p15, and p11.5 signals) recovered in either case was
always the same as the input TFIIIA signal measured in a control
undigested sample that was run in parallel (lanes 1, 2, and
4 in Fig. 7, and data not shown). It thus appears that
loosely bound zinc ions are not selectively removed from inactive
molecules and that a metal stoichiometry of 6 zinc ions/protein
molecule is not only sufficient to maintain the structural integrity of
TFIIIA but also to preserve its specific 5 S DNA binding and in
vitro transcription activities. The loss of 3 additional zinc
ions, however, results in the formation of a TFIIIA species that is
both structurally and functionally impaired.
 |
DISCUSSION |
Dealing with TFIIIA from the yeast S. cerevisiae, this
study reports new and, we believe, generally relevant information on the role of zinc ions in determining the structural organization and
functional properties of this transcription factor.
Despite the high sequence divergence between yeast TFIIIA and the
homologous transcription factor from Xenopus, our data
indicate that both proteins share a common modular organization. We
find, in fact, that the yeast protein is also organized in a two-domain structure consisting of an N-terminal protease-resistant domain of
about 34 kDa, with autonomous DNA binding ability, and a smaller C-terminal domain, with a lesser protease resistance, that is responsible for transcription activation. At variance with
Xenopus TFIIIA, whose DNA-binding domain comprises all the
nine zinc fingers, the corresponding domain of the yeast factor
includes only eight of the nine fingers, while the ninth finger is
located in the activation domain, together with the C-terminal half of
the linker region between finger 8 and 9 and the short region
C-terminal to the ninth finger. As revealed by limited proteolysis
analysis of the yeast transcription factor, these two domains are
connected by a region that is highly sensitive to protease cleavage and thus presumably flexible and poorly structured. Interestingly, both
trypsin and V8 protease cleave within, or very close to, a cluster of
basic residues (RKRRK) that resembles the basic region (KRKLK)
interposed between the DNA binding and the activation domains of
Xenopus TFIIIA (25). In the Xenopus factor,
however, this basic region lies C-terminal to the ninth finger, a few
residues upstream of the activation domain. In yeast TFIIIA, the
boundary between the two domains (centered around amino acid residues
325-328) lies about 20 residues upstream with respect to a stretch of
hydrophobic amino acids that has recently been shown to play a key role
in the assembly of a productive transcription complex (31). The linker
region between the eighth and the ninth finger, which represents the
site of highest sequence divergence between the yeast and the amphibian
transcription factors, thus appears to be a critical determinant of not
only the functional but also the structural integrity of yeast TFIIIA.
In fact, in vitro transcription activity was not
reconstituted by partially proteolyzed TFIIIA samples, even if they
contained both the C-terminal activation domain (p15/p11.5) and the
DNA-binding domain (p33.5).
The domain organization of the yeast transcription factor is not stably
maintained in an EDTA-treated form of TFIIIA only containing three
zinc-coordinated fingers. Interestingly, despite the profound
structural alterations exhibited by this 3 Zn2+-liganded
TFIIIA species, including a strong tendency toward aggregation, a
native domain organization was recovered following zinc
re-supplementation (Fig. 1). Therefore, zinc coordination is not only
required to maintain a correct structural organization, but it also
promotes the de novo acquisition of such structure starting
from a largely disorganized polypeptide chain.
Based on the successful reconstitution and controlled metal depletion
of 9 Zn2+-liganded TFIIIA, the remaining part of our work
focused on the comparative characterization of TFIIIA forms with
different zinc stoichiometries. This analysis revealed the existence in
the yeast protein of at least three previously unidentified classes of
zinc-binding sites. The most tightly associated are the 3 zinc ions
that remain bound after EDTA treatment, which, as noted above, are not
sufficient to maintain TFIIIA integrity. The 3 zinc ions that can be
extracted by EDTA, but are not released in the presence of PAR only,
identify metal-binding sites of intermediate affinity. The third class of metal coordination sites (estimated Kd higher
than 10
7 M) accounts for the 3 zinc ions that
are readily released just upon incubation with PAR. The presence of
three weak metal-binding sites is consistent with the "spontaneous"
loss of 2 zinc eq during protein purification (with the concomitant
oxidation of 4 cysteine residues) and with the fact that the resulting
7 Zn2+-liganded species slowly released to PAR only 1 additional zinc ion. This indicates that the 2 zinc eq lost in the
course of protein purification correspond to the 2 zinc ions that are
rapidly released from metal-saturated TFIIIA in the presence of PAR.
This observation, along with the strongly biphasic kinetics of loosely
bound zinc release, points to the existence in the yeast protein of an
additional level of heterogeneity among weak metal-binding sites.
The release of approximately 5 zinc eq to PAR and the existence of
distinct classes of zinc-binding sites have been documented previously
for Xenopus TFIIIA (38), yet the functional competence and
structural integrity of TFIIIA species containing discrete subsets of
zinc-coordinated fingers have not been analyzed. In the case of yeast
TFIIIA, the rather unexpected finding is that the species lacking all 3 loosely bound zinc ions has the same affinity for 5 S DNA as the
metal-saturated protein and behaves identically to it in in
vitro 5 S rRNA synthesis assays, the latter conducted under
transcription conditions that require not only the proper assembly of a
preinitiation complex but also a TFIIIA-DNA interaction that is stable
enough to allow efficient reinitiation of transcription (51). Although
the present analysis does not identify the specific fingers that are
metal-depleted in TFIIIA preparations carrying only 6 zinc eq, it may
be useful to confront our data with those produced by broken finger
mutagenesis experiments showing that the simultaneous disruption of
fingers 8 and 9 destroys the ability of TFIIIA to support cell
viability, whereas TFIIIA with a disruption of only finger 8 or finger
9, as well as TFIIIA with a disruption of only finger 5, retains the
ability to support cell viability
(31).2 This comparison leads
to the prediction that fingers 5, 8, and 9 can individually lose their
zinc ion, whereas either finger 8 or 9 should remain zinc-liganded
following PAR treatment. On the same note, finger 1, whose disruption
destroys the ability of TFIIIA to support in vitro
transcription and cell viability,2 should not correspond to
any of the weak zinc-binding fingers identified by our analysis. The
results of broken finger mutagenesis studies conducted on
Xenopus TFIIIA (52, 53) also point to the existence of some
degree of structural and functional nonequivalence among the nine
fingers. What may complicate the comparison between zinc depletion and
finger disruption data, however, is that the defects exhibited by at
least some of the broken finger mutants may result not only from zinc
depletion and finger unfolding, but also from secondary effects
deriving from amino acid substitution. Indeed, at variance with the
situation reported for Xenopus TFIIIA broken finger mutants,
where the disruption of each of eight fingers gave rise to localized
alterations of the limited proteolysis pattern (52), we did not observe
any differential proteolytic fragment distinguishing 9 Zn2+- from 6 Zn2+-liganded TFIIIA. It is thus
difficult to imagine that, at least in our case, the immediate
consequence of zinc removal is finger disruption. Instead, our data
indicate that the loss of 3 loosely bound zinc ions is well tolerated
in the context of an otherwise unmodified protein (i.e.
devoid of any primary sequence alteration).
A far more extreme case of partial metal independence has been reported
previously for Xenopus TFIIIA derived from EDTA-treated 7 S
particles, which only contains 2 zinc eq. This species, albeit never
directly compared with metal-saturated TFIIIA, was found to be
functionally competent despite its severely reduced zinc stoichiometry
(50, 54). In addition, based on the results of circular dichroism
studies conducted on this same species as well as on fully
metal-depleted TFIIIA, it has been proposed that zinc ions may be
primarily involved in the acquisition of TFIIIA folding, rather than in
the maintenance of a correctly organized structure (55). This
interpretation clearly cannot be applied to our data, which show that a
metal stoichiometry exceeding 3 zinc ions/protein molecule is
absolutely required to maintain the native structure of TFIIIA. In
turn, the observed structural and functional integrity of 6 Zn2+-liganded TFIIIA can rely on finger-finger and other
kinds of intramolecular interactions that, by determining the global
three-dimensional organization of the protein, may effectively
compensate for the release of 3 nonessential zinc ions from
metal-saturated TFIIIA. The existence of functionally important
finger-finger interactions in Xenopus TFIIIA is indeed
strongly implied by the results of a recent study showing that the
simultaneous binding of 5 S DNA by all nine fingers involves a
substantial energetic cost (33). The apparent redundancy of 3 zinc ions
in yeast TFIIIA, as revealed by the present analysis of an unmodified,
full-length protein, is thus probably best explained by the remarkably
high degree of structural and functional flexibility built into this
complex transcription factor. A similar case of structural and
functional heterogeneity among zinc fingers has recently been reported
for human metal-response element-binding transcription factor-1, where a subset of loosely bound zinc ions can be removed without effects on
the structure and DNA binding ability (56).
Finally, it may be useful to consider the possible physiological
implications of the existence in yeast TFIIIA of three classes of
zinc-binding sites with very different metal affinities. The presence
of 3 nonessential zinc ions considerably limits, at least in yeast, the
effectiveness of previously proposed regulatory mechanisms based on the
zinc-dependent modulation of TFIIIA activity in response to
changes in zinc availability (57). Far from being key regulators of
transcription factor activity, the three loose metal-binding fingers of
yeast TFIIIA may instead be viewed as an intramolecular zinc reservoir
that can allow the synthesis of an essential component of the
translation machinery, the 5 S rRNA, even under temporary conditions
of severe zinc shortage.
 |
ACKNOWLEDGEMENTS |
We are grateful to Gian Luigi Rossi for
helpful discussions and support, to Tony Weil for plasmid pUC9-5 S,
and to Owen Rowland and Jacqueline Segall for communicating results
prior to publication. We also thank Alessio Peracchi and Riccardo
Percudani for their critical reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported by grants from the National Research
Council of Italy (CNR), Target Project on "Biotechnology," and from
the Ministry of University and of Scientific and Technological Research
(MURST, Rome, Italy).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.
§
To whom correspondence should be addressed: Tel.: 39-521-905646;
Fax: 39-521-905151; E-mail: simone{at}irisbioc.bio.unipr.it.
The abbreviations used are:
TF, transcription
factor; ICR, internal control region; TBP, TATA box-binding protein; r, recombinant; PAR, 4-(2-pyridylazo)resorcinol; PMPS, p-hydroxymercuriphenylsulfonate; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.
2
O. Rowland and J. Segall, personal communication.
 |
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