(Received for publication, January 23, 1996)
From the
Transcription factor (TF) IIIA, which contains nine zinc finger
motifs, binds to the internal control region of the 5 S RNA gene as the
first step in the assembly of a multifactor complex that promotes
accurate initiation of transcription by RNA polymerase III. We have
monitored the interaction of wild-type and truncated forms of yeast
TFIIIA with the 5 S RNA gene. The DNase I footprints obtained with
full-length TFIIIA and a polypeptide containing the amino-terminal five
zinc fingers (TF5) were indistinguishable, extending from nucleotides
+64 to +99 of the 5 S RNA gene. This suggests that fingers 6
through 9 of yeast TFIIIA are not in tight association with DNA. The
DNase I footprint obtained with a polypeptide containing the
amino-terminal four zinc fingers (TF4) was 14 base pairs shorter than
that of TF5, extending from nucleotides +78 to +99 on the
nontranscribed strand and from nucleotides +79 to +98 on the
transcribed strand of the 5 S RNA gene. Protection provided by a
polypeptide containing the first three zinc fingers (TF3) was similar
to that provided by TF4, with the exception that protection on the
nontranscribed strand ended at nucleotide +80, rather than
nucleotide +78. Methylation protection analysis indicated that
finger 5 makes major groove contacts with guanines +73 and
+74. The amino-terminal four zinc fingers make contacts that span
the internal control region, which extends from nucleotides +81 to
+94 of the 5 S RNA gene, with finger 4 appearing to contact
guanine +82. Measurements of the apparent K values of the TFIIIA
DNA complexes indicated that the
amino-terminal three zinc fingers of TFIIIA have a binding energy that
is similar to that of the full-length protein.
Transcription factor (TF) ()IIIA, a sequence-specific
DNA-binding protein, binds to the 5 S RNA gene as the first step in the
assembly of a multifactor transcription complex. Interaction of TFIIIA
with the internal control region (ICR) of the 5 S RNA gene is necessary
for incorporation of multisubunit TFIIIC into the TFIIIA
DNA
complex. Formation of the TFIIIC
TFIIIA
DNA complex is a
prerequisite for recruitment of TFIIIB; the
TFIIIB
TFIIIC
TFIIIA
DNA complex then recruits RNA
polymerase III (reviewed by Geiduschek and Kassavetis(1992) and
White(1994)). Since the initial purification of TFIIIA from Xenopus (Engelke et al., 1980), the mode of interaction of this
TFIIIA with the Xenopus 5 S RNA gene has been extensively
studied. In contrast, molecular characterization of TFIIIB and TFIIIC
and insights into the architecture of the transcription complex have
been largely derived from studies with Saccharomyces
cerevisiae. TFIIIB and TFIIIC have been purified from this
organism, and the genes encoding several of the subunits of these
multisubunit factors have been cloned (reviewed by Gabrielsen and
Sentenac(1991), Geiduschek and Kassavetis(1992), and White(1994); see
Kassavetis et al. (1995)). The topography of these factors in
the yeast TFIIIB
TFIIIC
TFIIIA
5 S DNA complex,
including the location of various polypeptides of TFIIIC and TFIIIB
along the gene, has been determined by DNase I footprinting and
site-specific DNA-protein cross-linking (Braun et al., 1989,
1992a; Kassavetis et al., 1990).
Xenopus TFIIIA, a
component of the abundant 7 S ribonucleoprotein particle present in
immature oocytes, contains nine consecutive zinc fingers (Miller et
al., 1985; Brown et al., 1985) that are responsible for
the DNA and RNA binding properties of the protein (reviewed by Pieler
and Theunissen (1993)). The 30-amino acid zinc finger motif,
characterized by pairs of cysteine and histidine residues, consists of
an autonomously folding domain in which an anti-parallel -sheet
and an
-helix fold around a zinc ion. The two cysteine and two
histidine residues are coordinated to the zinc, and the structure is
stabilized by a hydrophobic core (Párraga et
al., 1988; Lee et al., 1989; Pavletich and Pabo, 1991). Xenopus TFIIIA is an elongated molecule (Bieker and Roeder,
1984) that protects the entire 50-base pair (bp) ICR of the amphibian 5
S RNA gene from cleavage by DNase I (Engelke et al., 1980).
The protein is oriented with its amino terminus toward the 3`-end of
the ICR and its carboxyl terminus toward the 5`-end of the gene (Miller et al., 1985; Vrana et al., 1988). Early models
describing the disposition of the nine zinc fingers of Xenopus TFIIIA over the 50-bp ICR (Fairall et al., 1986; Vrana et al., 1988; Churchill et al., 1990; Berg, 1990)
have been modified to take into consideration the crystal structure of
the three zinc fingers of Zif268 bound to its 9-bp target site
(Pavletich and Pabo, 1991). In these more recent models, based on
interpretation of DNase I footprinting patterns, hydroxyl radical
footprinting patterns, and missing nucleoside analysis of protein-DNA
complexes, the three amino-terminal and three carboxyl-terminal fingers
of the molecule are proposed to wrap around the major groove of the DNA
helix at each end of the ICR in a manner similar to the Zif268-DNA
interaction. The zinc fingers in the middle of the protein are thought
to lie on one side of the helix, with finger 5 contacting the major
groove and fingers 4 and 6 each crossing the minor groove (Clemens et al., 1992; Hayes and Clemens, 1992; Hayes and Tullius,
1992; Fairall and Rhodes, 1992; Hansen et al., 1993).
The 50-bp ICR of the Xenopus 5 S RNA gene contains three elements that contribute to efficient transcription of the gene: the A-box spanning nucleotides +50 to +64, the intermediate element spanning nucleotides +67 to +72, and the C-box spanning nucleotides +80 to +97 (Pieler et al., 1985, 1987; Bogenhagen, 1985). Various studies have indicated that the amino-terminal fingers of Xenopus TFIIIA interact with the C-box element (Vrana et al., 1988; Christensen et al., 1991; Clemens et al., 1992; Hayes and Clemens, 1992; Liao et al., 1992) with a binding energy that is similar to that of the TFIIIA-DNA interaction (Sakonju et al., 1981; Sakonju and Brown, 1982; Fairall et al., 1986; Vrana et al., 1988; You et al., 1991; Darby and Joho, 1992; Liao et al., 1992; Theunissen et al., 1992; Veldhoen et al., 1994). Indeed, a polypeptide containing only the amino-terminal three zinc fingers of Xenopus TFIIIA was found to have high affinity binding for the 5 S RNA gene, protecting the region from approximately nucleotides +74 to +95 from cleavage by DNase I (Christensen et al., 1991; Liao et al., 1992). The third zinc finger has been implicated as a major contributor to the affinity of Xenopus TFIIIA for DNA (Liao et al., 1992; Darby and Joho, 1992; Theunissen et al., 1992; Del Rio et al., 1993; Zang et al., 1995). Recent studies confirm that various fingers make differing contributions to the binding energy of complex formation and indicate that the energetic contribution made by a finger can be influenced by neighboring fingers or sets of fingers (Del Rio et al., 1993; Clemens et al., 1994; Zang et al., 1995). The linkers between the amino-terminal fingers have also been shown to have a critical role in DNA binding, although it is unclear whether this contribution is through positioning of the zinc fingers or through contacts with DNA (Choo and Klug, 1993; Clemens et al., 1994; Zang et al., 1995).
Comparison of the deduced sequences of
TFIIIA from S. cerevisiae and from Xenopus laevis indicated that the two proteins are structurally similar in that
they both contain nine zinc finger motifs (Ginsberg et al.,
1984; Archambault et al., 1992; Woychik and Young, 1992).
However, the amino acid sequences of the corresponding fingers of yeast
TFIIIA and amphibian TFIIIA as well as the linker sequences between the
fingers differ extensively. A distinctive feature of the yeast protein
is an 81-amino acid domain interrupting the consecutive zinc finger
motifs between fingers 8 and 9. In contrast to the 50-bp DNase I
footprints of the Xenopus TFIIIADNA complex (Engelke et al., 1980) and the human TFIIIA
DNA complex (Seifart et al., 1989; Moorefield and Roeder, 1994), the DNase I
footprint of the yeast TFIIIA
DNA complex is 35 bp, extending from
nucleotides +63 to +97 (Braun et al., 1989).
Site-specific DNA-protein photocross-linking has revealed, however,
that yeast TFIIIA is positioned over a longer region of DNA than that
detected by DNase I footprinting (Braun et al., 1992a). The
DNase I footprint includes the C-box element of the yeast 5 S RNA gene.
This 15-bp sequence, which is positioned at nucleotides +81 to
+94, is the only intragenic element that is essential for
efficient in vitro transcription of the yeast 5 S RNA gene
(Challice and Segall, 1989). An extended ICR has been found to be
required for transcription in vivo (Lee et al.,
1995).
We have previously shown that a truncated polypeptide
containing the amino-terminal three zinc fingers of yeast TFIIIA
retains the ability to bind to the yeast 5 S RNA gene (Milne and
Segall, 1993). In the present study, we have analyzed in more detail
the interaction of full-length and truncated forms of yeast TFIIIA with
the 5 S RNA gene. Our data reveal that fingers 6 through 9 of yeast
TFIIIA are not in tight association with DNA in the TFIIIADNA
complex and suggest that the amino-terminal five zinc fingers make
major groove contacts from nucleotides +73 to +94. A
polypeptide containing the amino-terminal three zinc fingers of TFIIIA
has a binding energy that is similar to that of the full-length
protein.
Figure 1:
Characterization of wild-type and
truncated forms of TFIIIA purified from bacteria. A, schematic
representation of wild-type TFIIIA annotated to show the approximate
positions of the carboxyl-terminal end points of the truncated forms of
TFIIIA containing seven zinc fingers (TF7*), four zinc fingers (TF4*),
and three zinc fingers (TF3*). The numbered boxes denote the
nine zinc fingers. The hatched regions refer to the 48- and
35-amino acid sequences present at the amino and carboxyl termini of
the protein, respectively. The stippled region denotes the
81-amino acid domain present between zinc fingers 8 and 9. B,
SDS-polyacrylamide gel analysis. Aliquots of wild-type (WT)
and truncated forms of yeast TFIIIA, purified from bacteria as
described under ``Experimental Procedures'', were subjected
to electrophoresis on a 12% SDS-polyacrylamide gel. Proteins were
visualized by staining with Coomassie Brilliant Blue. The sizes (in
kilodaltons) of molecular mass markers run in lane M are
indicated on the right. These markers were phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa),
carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and
lysozyme (14.4 kDa). The estimated M values of the
proteins purified from E. coli, obtained by comparison with
the mobilities of these molecular mass markers, are given below
preceded by their predicted molecular weight: wild-type TFIIIA, 49,982,
49,500; TF7*, 31,126, 33,500; TF4*, 20,257, 23,500; and TF3*, 17,912,
22,000. C, binding of wild-type and truncated forms of
bacterially produced TFIIIA to the 5 S RNA gene. A 270-bp radioactively
labeled DNA fragment containing the yeast 5 S RNA gene was incubated
prior to electrophoresis on a nondenaturing polyacrylamide gel with the
bacterially produced proteins indicated above the lanes (see
``Experimental Procedures''). No protein was added to the
reaction in lane 1. The mobilities of free DNA and
TFIIIA
DNA complexes are indicated to the right of the
autoradiogram. D, assessment of transcription factor activity. In vitro transcription reactions (see ``Experimental
Procedures'') contained a yeast tRNA gene (lane 1) or the
yeast 5 S RNA gene (lanes 2-7) as template (denoted as t and 5, respectively, above the lanes); partially
purified yeast TFIIIC, TFIIIB, and RNA polymerase III; and the form of
TFIIIA indicated above the lanes. ppA, TFIIIA partially
purified from yeast. The RNAs synthesized in vitro were
analyzed on a 7 M urea, 10% polyacrylamide gel. The
autoradiogram shows the portion of the gel containing tRNA and 5 S RNA.
The various-sized transcripts represent primary and processed
products.
The first step in construction of a
plasmid for expression of TFIIIA truncated after the fourth zinc finger
motif (TF4*) involved cloning the annealed, self-complementary
oligonucleotide 5`-CTAGCTAGCTAG-3` (kindly provided by J. Ingles),
which provides stop codons in all reading frames, into the SmaI site of pJA453 (Archambault et al., 1992) to
generate pJA453-STOP. pJA453 is a pBluescript SK derivative containing the TFIIIA-coding region from the initiator
ATG codon to the HindIII site at codon 171. The NcoI-BamHI fragment of pJA453-STOP was then inserted
between the corresponding sites of pET-11d for expression of TF4. The
first step in construction of plasmids for expression of TF8, TF7*, and
TF3* involved subcloning the 0.85-kilobase pair KpnI-EcoRV fragment, the 0.80-kilobase pair KpnI-XbaI fragment, and the 0.46-kilobase pair KpnI-Bsp1286I fragment, respectively, of pJA454
between the KpnI and EcoRV sites of pJA453-STOP. For
these constructions, the XbaI end of the KpnI-XbaI fragment and the Bsp1286I end of
the KpnI-Bsp1286I fragment were blunted prior to
ligation. The NcoI-BamHI fragment of each of the
resultant plasmids, containing TFIIIA-coding sequence, was then
subcloned between the corresponding sites of the pET-11d expression
vector. This cloning procedure introduced the non-TFIIIA,
polylinker-encoded amino acids EFLQPLAS, SNSCSP, LISNSCSP, and SNSCSP
at the carboxyl terminus of TF8, TF7*, TF4*, and TF3*, respectively.
For the DNase I and methylation protection footprint experiments, genes encoding proteins with carboxyl-terminal truncations that ended after the linker sequence of finger 6 (TF6), finger 5 (TF5), finger 4 (TF4), and finger 3 (TF3) were generated by polymerase chain reaction. Downstream primers introduced a stop codon at the end of the linker. TF6, TF5, TF4, and TF3 had a stop codon introduced after codons 223, 193, 163, and 133, respectively. To facilitate subcloning, these primers also contained a BamHI site at their 5`-ends. These primers were used in a polymerase chain reaction, with pJA454 as template and the universal primer as the upstream primer, to generate the truncated TFIIIA-coding sequences. The polymerase chain reaction products were cut with NcoI and BamHI and cloned between the corresponding sites of pET-11d.
For the DNase I experiments, pET-11d-based plasmids were also constructed for bacterial expression of amino-terminal truncated forms of TFIIIA. Plasmids JA454-2 and JA454-3 (Milne and Segall, 1993), which encode TFIIIA proteins lacking the sequence for codons 2-43 and codons 2-69, respectively, were digested with KpnI. After the resultant ends had been blunted by treatment with the Klenow form of DNA polymerase, the DNA was digested with BamHI. The pET-11d expression vector was digested with NcoI, and the resultant ends were treated sequentially with S1 nuclease and the Klenow form of DNA polymerase prior to digestion of the DNA with BamHI. The KpnI-BamHI fragments that had been excised from pJA454-2 and pJA454-3 were then subcloned between the NcoI and BamHI sites of pET-11d; this generated plasmids for expression of variant forms of TFIIIA that lack the sequence prior to finger 1 (1FS) and that lack most of the sequence prior to finger 2 (2FS), respectively. The sequence of all polymerase chain reaction-amplified DNA and all subcloning junctions was verified by the chain termination method (Sanger et al., 1977).
In vitro transcription assays were performed as described (Taylor and Segall, 1985) using the yeast 5 S RNA gene (p19-5S) (Challice and Segall, 1989) or a tRNA gene as template (pPm16) (Olson et al., 1981). A 50-µl reaction contained 18 µl of a yeast-derived heparin-agarose fraction (fraction h) (Taylor and Segall, 1985), which contains TFIIIB, TFIIIC, RNA polymerase III, and, as indicated, partially purified yeast TFIIIA or bacterially produced TFIIIA.
We next measured the affinity of
TFIIIA for the 5 S RNA gene by incubating a constant amount of protein
with increasing amounts of a radiolabeled 5 S DNA fragment. Protein-DNA
complexes were separated from free DNA by electrophoresis on a
nondenaturing gel (Fig. 2A). The relative amounts of
free DNA and bound DNA were determined at each input DNA concentration,
and the apparent dissociation constant (K) was
derived by nonlinear regression analysis of the data (Fig. 2A; see ``Experimental Procedures'').
We found that the K
of the protein-DNA complex was
0.11 nM (Table 1). For comparison, the K
values measured for the Xenopus TFIIIA-DNA interaction
range from 0.42 to 2.2 nM (Hanas et al., 1983;
Romaniuk, 1990; Del Rio and Setzer, 1991; Liao et al., 1992;
Del Rio et al., 1993).
Figure 2:
Determination of apparent dissociation
constants. Apparent dissociation constants (K) were determined by gel retardation
analysis of reactions containing a constant concentration of TFIIIA and
variable concentrations of end-labeled 5 S DNA as described under
``Experimental Procedures.'' A, a nonlinear
regression analysis of representative data obtained with wild-type
TFIIIA (WT). The concentration of TFIIIA was
3
nM, and the concentration of 5 S DNA varied from 0.04 to 0.70
nM. B, a nonlinear regression analysis of
representative data obtained with TFIIIA containing seven zinc fingers
(TF7*). The concentration of TF7* was
4 nM, and the
concentration of 5 S DNA varied from 0.04 to 0.70 nM. C, a nonlinear regression analysis of representative data
obtained with TFIIIA containing four zinc fingers (TF4*). The
concentration of TF4* was
20 nM, and the concentration of
5 S DNA varied from 0.04 to 0.70 nM. D, a nonlinear
regression analysis of representative data obtained with TFIIIA
containing three zinc fingers (TF3*). The concentration of TF3* was
15 nM, and the concentration of 5 S DNA varied from 0.07
to 1.85 nM. The insets in A-D show the
gel retardation experiments from which the data were derived for each
plot. The reaction in the first lane in each gel inset
contained no TFIIIA. The K
value obtained
in each experiment is denoted. As determined from the
[P
] values obtained in these examples,
the preparations of wild-type protein, TF7*, TF4*, and TF3* were 11,
12, 2.5, and 13% active, respectively.
We also determined the binding affinities of carboxyl-terminal truncated forms of yeast TFIIIA. Versions of TFIIIA that contained the first three zinc fingers (TF3*), the first four zinc fingers (TF4*), and the first seven zinc fingers (TF7*) were purified from bacteria. These truncated forms of TFIIIA contained up to 25 amino acids after the last intact zinc finger; this additional sequence (denoted by the asterisk) included a portion of the next zinc finger and six to eight amino acids introduced from vector sequence (see ``Experimental Procedures''). We considered that this sequence would be relatively unstructured and would be unlikely to have a significant effect on the affinity measurements. Each form of truncated TFIIIA eluted from Bio-Rex 70 at a unique salt concentration, ran as a single band of the expected molecular mass on an SDS-polyacrylamide gel (Fig. 1B), and was active in binding to the 5 S RNA gene (Fig. 1C). Consistent with the reduction in size of the carboxyl-terminal truncated proteins, the protein-DNA complexes formed with the truncated proteins (Fig. 1C, lanes 3-5) had increased mobilities relative to the complex formed with wild-type protein (lane 2). The protein-DNA interactions visualized in the gel shift assay were shown to be specific by monitoring the effects of addition of competitor DNAs (data not shown). As expected from the observation that the 81-amino acid domain located between fingers 8 and 9 of yeast TFIIIA is essential for the transcriptional activity of the protein (Milne and Segall, 1993), bacterially purified TF7*, TF4*, and TF3* were unable to support in vitro transcription of the 5 S RNA gene (Fig. 1D, lanes 5-7).
Removal of zinc fingers 9 and 8 from yeast TFIIIA did not have a
significant effect on the apparent affinity of the protein-DNA
interaction (Fig. 2B and Table 1). Removal of
zinc fingers 9 through 5 led to an 2-fold increase in the apparent K
(Fig. 2C and Table 1), and
removal of zinc finger 4 led to an additional 3.5-fold increase in the
apparent K
(Fig. 2D and Table 1). Using the measured K
values, we
calculated the
G
values for the protein-DNA
interactions (Table 1). This representation of the data
emphasizes that a polypeptide containing the amino-terminal three zinc
fingers of yeast TFIIIA has a high binding energy; the binding energy
of TF3 was 90% that of the full-length protein. In previous studies
using protein synthesized in vitro, we were unable to detect a
TFIIIA-DNA interaction with a truncated molecule containing only the
amino-terminal two zinc fingers (Milne and Segall, 1993) or with a
truncated molecule containing only fingers 3 through 9. (
)This suggests that if these proteins can bind to the 5 S
RNA gene, they do so with a relatively high K
. It
should be noted that our analysis using truncated proteins would not
necessarily detect the effects that finger-DNA interactions in one
portion of the molecule might exert on the energetics of interactions
occurring elsewhere in the complex (for example, see Del Rio et
al.(1993), Clemens et al.(1994), and Zang et
al.(1995)).
Figure 3:
DNase I protection analysis of
TFIIIADNA complexes. DNA fragments containing the yeast 5 S RNA
gene uniquely end-labeled on the transcribed (A) or
nontranscribed (B) strand were incubated with bacterially
produced wild-type TFIIIA (WT); carboxyl-terminal truncated
forms of TFIIIA containing eight zinc fingers (TF8), seven zinc fingers
(TF7*), six zinc fingers (TF6), five zinc fingers (TF5), four zinc
fingers (TF4), and three zinc fingers (TF3); and amino-terminal
truncated forms of TFIIIA beginning before the first zinc finger (1FS)
or before the second zinc finger (2FS). The DNA fragments produced by
brief treatment of the reactions with DNase I were analyzed on a
sequencing gel (see ``Experimental Procedures''). The
reactions in the third lanes had no added protein. The first and second lanes contain the cleavage products
of A + G and C + T chemical sequencing reactions,
respectively. Nucleotide positions of the 5 S RNA gene are indicated on
the sides of the panels. Also shown is a schematic representation of
the extent of DNase I protection in the various protein-DNA complexes (C). The sequence of the 5 S RNA gene is given from
nucleotides +58 to +112; the ICR is boxed. The
extent of DNase I protection on the nontranscribed strand (NT; hatched rectangles) and transcribed strand (T; solid rectangles) in complexes formed with wild-type TFIIIA,
TF8, TF7*, TF6, TF5, TF4, and TF3 is shown. The shaded portions of the solid rectangles indicate uncertainty in
positioning the boundary of the protected region. The solid
arrowheads denote DNase I-sensitive sites within the region
protected by wild-type TFIIIA, TF8, TF7*, TF6, and
TF5.
TFIIIA containing four intact zinc fingers generated a smaller DNase I footprint on the 5 S RNA gene than did TF5. On the transcribed strand, TF4 appeared to protect a 20-bp region, extending from nucleotides +79 to +98 (Fig. 3A, ninth lane). Comparison of the relative intensities of the bands at nucleotide +76 suggested that the upstream boundary of this footprint might extend to nucleotide +76. On the nontranscribed strand, the protected region extended from nucleotides +78 to +99 (Fig. 3B, ninth lane). Comparison of the footprints obtained with TF4 and TF3 showed that deletion of the fourth zinc finger led to a change in the DNase I footprint only on the nontranscribed strand (Fig. 3, A and B, tenth lanes). Whereas TF4 provided protection from nucleotides +78 to +99, TF3 did not protect residue +78 from DNase I digestion. In summary, our data confirmed, as previously suggested (Milne and Segall, 1993), that yeast TFIIIA, like its amphibian counterpart, binds to the 5 S RNA gene with its carboxyl terminus toward the 5`-end of the gene. Comparison of the DNase I protection patterns obtained with the carboxyl-terminal truncated proteins TF5, TF4, and TF3 indicated that the amino-terminal three zinc fingers span the ICR, which maps approximately from nucleotides +81 to +94 (Challice and Segall, 1989); the presence of finger 4 leads to only a modest change in the protection pattern at the 5`-end of the ICR; and the presence of finger 5 leads to protection of an additional 12 bp upstream of the ICR.
Inspection of the DNase I footprints generated by the two
amino-terminal truncated proteins indicated that TFIIIA lacking the
non-zinc finger amino-terminal extension (1FS) and TFIIIA lacking the
first zinc finger (2FS) did not protect nucleotide +98 on the
transcribed strand (Fig. 3A, eleventh and twelfth lanes). Additionally, protection of nucleotides
+96 and +94 on the transcribed strand (Fig. 3A, twelfth lane) and nucleotide
+97 on the nontranscribed strand (Fig. 3B, twelfth lane) was reduced in the 2FSDNA complex (Fig. 3A, twelfth lane). These data position
finger 1 at the 3`-end of the ICR.
Figure 4:
Determination of guanine residues
protected from methylation in TFIIIADNA complexes. DNA fragments
containing the yeast 5 S RNA gene uniquely end-labeled on the
transcribed (A) or nontranscribed (B) strand were
incubated with wild-type TFIIIA (WT) or a truncated form of
TFIIIA containing six zinc fingers (TF6), five zinc fingers (TF5), four
zinc fingers (TF4), or three zinc fingers (TF3) purified from bacteria.
The reactions in the second lanes had no added protein.
Shortly after the addition of dimethyl sulfate, the reactions were
applied to a nondenaturing gel to resolve protein-DNA complexes from
free DNA. DNA recovered from free DNA (second lanes) or from
protein-DNA complexes (third to seventh lanes) was
cleaved at the modified G residues and analyzed on a sequencing gel
(see ``Experimental Procedures''). The first lanes contain the products of an A + G chemical sequencing reaction
of the DNA probe. Nucleotide positions of the 5 S RNA gene are
indicated on the left-hand sides of the panels; the rectangles on the right-hand sides denote the G residues protected from
methylation on the transcribed (open rectangles) and
nontranscribed (closed rectangle) strands of the wild-type
TFIIIA
DNA complex. In C, the sequence of the 5 S RNA
gene is given from nucleotides +58 to +112. The ICR is boxed, and G residues protected from methylation in the
wild-type TFIIIA
5 S DNA complex are indicated (
, transcribed
strand;
, nontranscribed strand).
We expressed yeast TFIIIA in E. coli and found, as
observed by others (Ottonello et al., 1994), that bacterially
produced yeast TFIIIA supports accurate in vitro transcription
of the 5 S RNA gene. Using wild-type and truncated forms of TFIIIA that
had been purified from bacteria, we studied the interaction of the
protein with the 5 S RNA gene using DNase I and dimethyl sulfate as
probes. We note that DNase I allows only approximate mapping of the
boundaries of a protein-DNA complex. First, not all phosphodiester
bonds are cleaved efficiently by this enzyme in naked DNA. Second, the
boundary of a footprint could be influenced by a weak/transient
interaction of a finger with DNA, displacement of a weakly bound finger
by DNase I, or steric hindrance between the enzyme and DNA-bound
protein. Despite these limitations, inspection of the protection
patterns allowed us to position various portions of yeast TFIIIA along
the 5 S RNA gene. Although yeast TFIIIA and Xenopus TFIIIA
both contain nine zinc fingers, the 40-kDa amphibian protein generates
a 50-bp DNase I footprint (Engelke et al., 1980), whereas the
50-kDa yeast protein generates a 35-bp footprint (Braun et
al., 1989; this study). We found that the smaller footprint of
yeast TFIIIA can be accounted for by the absence of an intimate
interaction of yeast fingers 6-9 with DNA. Studies of the Xenopus TFIIIA-DNA interaction (Clemens et al., 1992;
Fairall and Rhodes, 1992; Hayes and Clemens, 1992; Hayes and Tullius,
1992; Del Rio et al., 1993) suggest that the carboxyl-terminal
three fingers (fingers 7-9) of Xenopus TFIIIA wrap
around the major groove of the DNA helix at the 5`-end of the amphibian
ICR and immediately upstream of this element. The three zinc fingers in
the middle of the protein are thought to lie on one side of the helix,
with finger 5 contacting the major groove and fingers 4 and 6 each
crossing the minor groove. The amino-terminal three zinc fingers are
proposed to wrap around the major groove of the DNA helix, contacting a
13-bp region of the C-box (Christensen et al., 1991; Hayes and
Clemens, 1992; Clemens et al., 1992; Liao et al.,
1992; Bogenhagen, 1993; Hansen et al., 1993; Veldhoen et
al., 1994) with an affinity that is similar to that of the entire
protein (Liao et al., 1992; Choo and Klug, 1993; Zang et
al., 1995). Our methylation protection analysis indicated that
finger 5 of yeast TFIIIA is in close contact with G and
G
, placing it in the major groove, as is finger 5 in the Xenopus TFIIIA
DNA complex. Because the DNase protection
analysis indicated that finger 5 provides protection to nucleotide
+64, the finger 5-DNA interaction presumably extends upstream of
nucleotides +74 and +75. The methylation protection analysis
also indicated that G
was not protected from modification
in the yeast TF3
DNA complex, was partially protected in the
TF4
DNA complex, and was completely protected in the TF5
DNA
complex. This suggests that yeast finger 4 makes a major groove contact
with G
and that this interaction occurs more efficiently
when finger 5 is docked at G
/G
. If this is
the case, it is possible that the 12-bp difference between the DNase I
footprints of TF5 and TF4 represents loss of protection not only by
finger 5, but also by finger 4, i.e. DNase I might partially
displace finger 4 from the TF4
DNA complex. This could explain why
the only difference between the DNase I protection patterns of the
yeast TF4
DNA and TF3
DNA complexes was protection of
nucleotide +78 in the TF4
DNA complex. The interactions
suggested above, which position the major groove contacts made by
fingers 4 and 5 10 bp apart on the same side of the helix, require that
the finger 4-finger 5 linker, which is only five amino acids, cross the
minor groove (e.g. see Kochoyan et al.(1991)).
TFIIIA-induced bending of DNA (Braun et al., 1992b) might
facilitate this proposed spacing. Alternatively, it is possible that
the finger 4-finger 3 linker is responsible for protection of G
and that most of the finger lies over the minor groove, extending
from approximately nucleotides +82 to +75, leaving this
region accessible to DNase I. Finally, it is possible that finger 4
approaches DNA in a novel manner. In the above model, irrespective of
how finger 4 interacts with DNA, fingers 3, 2, and 1 would then be
responsible for the major groove contacts represented by protection of
G residues from positions +85 to +94. Although this differs
from the model proposed for the Xenopus TFIIIA
DNA
complex, in which the amino-terminal three zinc fingers contact 13 bp
of the C-box element (Clemens et al., 1992; Liao et
al., 1992) and finger 4 lies over the minor groove, the fact that
G
is not protected from methylation in the yeast
TF3
DNA complex argues against G
being contacted by
finger 3. If finger 3 is in contact with G
, then our data
indicate that this contact occurs only in the presence of finger 4. In
this case, TF3, which binds to DNA with an affinity that is similar to
that of the full-length protein, would have only two fingers in contact
with DNA. We note that using protein synthesized in vitro, we
did not detect an interaction between the 5 S RNA gene and a
polypeptide containing the first two fingers of yeast TFIIIA (Milne and
Segall, 1993).
Further studies are required to allow detailed
comparisons between the finger-DNA interactions in the yeast and Xenopus TFIIIADNA complexes. Exact positioning of
individual fingers awaits deduction of the three-dimensional structure
of the protein-DNA complexes. As discussed above, however, our
preliminary study suggests that some differences may exist. This is
perhaps not unexpected. Yeast TFIIIA does not contain the conserved
linker motif TGEK found between fingers 1 and 2 and between fingers 2
and 3 of Xenopus TFIIIA. The His-His spacings, which could
affect the structure of the DNA-binding
-helix, also differ
between fingers 1, 3, and 5 of the TFIIIA proteins from the two
organisms (Ginsberg et al., 1984; Archambault et al.,
1992; Woychik and Young, 1992). These differences, in addition to
differences in potential DNA-contacting amino acids, could lead to
differences in the DNA binding properties of the polypeptides.
Deduction of the structures of protein-DNA crystals of Zif268
(Pavletich and Pabo, 1991), GL1 (Pavletich and Pabo, 1993), and
Tramtrack (Fairall et al., 1993) has revealed that zinc
fingers can dock into the major groove of DNA with variations in base
and phosphate contacts and in the spacing of adjacent binding sites.
Linkers have been shown to have a major influence on the binding
affinity of zinc fingers (Choo and Klug, 1993; Clemens et al.,
1994; Zang et al., 1995). Furthermore, it is interesting to
note that the amino-terminal three fingers of yeast TFIIIA serve not
only in DNA binding, but also to recruit TFIIIC to the TFIIIA
DNA
complex (Milne and Segall, 1993). The observations that putative human
TFIIIA (Arakawa et al., 1995; Drew et al., 1995) and Xenopus TFIIIA share more extensive identity than do Xenopus TFIIIA and yeast TFIIIA (Archambault et al.,
1992; Woychik and Young, 1992) and that no similarity has yet been
found between the deduced amino acid sequences of subunits of human
TFIIIC and yeast TFIIIC (L'Etoile et al., 1994; Lagna et al., 1994; Sinn et al., 1995) are consistent with
the notion that a yeast-specific TFIIIA-DNA interaction is established
to direct assembly of yeast TFIIIC into the transcription complex.