Download Internal deletion mutants of Xenopus transcription factor IIIA

volume 17 Number 23 1989
Nucleic A c i d s Research
Internal deletion mutants of Xenopus transcription factor IIIA
Jay S.Hanas*, Roanna M.Littell, Chris J.Gaskins and Robert Zebrowski
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center,
Oklahoma City, OK 73190, USA
Received May 17, 1989; Revised and Accepted October 20, 1989
ABSTRACT
Xenopus transcription factor HIA (IFIilA) or TFLLLA mutants with internal deletions were expressed
in E.coli utilizing a bacteriophage T7 RNA polymerase system. TFULA or deletion mutant TFULAS,
isolated from E.coli cell extracts, were identified by SDS PAGE and unmunoblotting with rabbit
antiserum against native TFULA purified from Xenopus immature oocytes. Specific DNA binding
of intact or internally deleted TFLUA was compared by analyzing their abilities to protect the internal
control gene (ICR) of the Xenopus 5S RNA gene from DNase I digestion. Intact protein, synthesized
from a full-length TFLTIA cDNA, bound specifically to the entire ICR (+96 to +43) and promoted
5S RNA gene transcription in vitro. One TFLLTA deletion mutant, expressed from cDNA lacking
the coding sequence for the putative fourth zinc finger (designated from the N-terminus, amino acids
103-132) protected the ICR from DNase I digestion from nucleotide positions +96 to +78. A
second TFLTIA mutant resulting from fusion of putative zinc fingers 7 and 8 (deletion of amino acids
200-224) protected the 5S gene ICR from positions +96 to +63. The DNase I protection patterns
of these mutant proteins are consistent with the formation of strong ICR contacts by those regions
of the protein on the N- terminal side of the mutation but not by those regions on the C-terminal
side of the mutation. The regions of the protein comprising the N-terminal 3 fingers and N-terminal
six fingers appear to be in contact with approximately 18 and 33 bp of DNA respectively on the
3' side of the 5S gene ICR . These internal deletion mutants promoted 5S RNA synthesis in vitro
and DNA renaturation.
INTRODUCTION
Xenopus transcription factor UIA binds specifically to the ICR of the 5S RNA gene,
protecting from DNase I digestion nucleotides +45 to +96 and promotes 5S RNA synthesis
by RNA polymerase III (1,2,3). A large cluster of very strong contacts between TFTHA
and the 5S RNA gene is located between nucleotides +80 to +90 (4). TFIHA requires
zinc for specific binding to the 5S RNA gene and for promotion of transcription (5). Analysis
of the amino acid sequence of ThlLLA (6) revealed 9—11 repeat units of about 30 amino
acids, each having the potential to coordinate zinc via two cysteines and two histidines
(7,8). The TM11A gene appears to encode nine contiguous zinc binding domains (1 through
9 from N-terminal) encompassing approximately three fourths of the protein (9). Potential
zinc binding domains have been found in the coding sequences of many eukaryotic gene
regulatory proteins (10). TFHIA also has the ability to promote renaturation of
complementary strands of DNA, a property dependent upon the structural integrity of the
C-terminal half of the protein (11,12).
Previous studies utilizing deletion analyses from either the N-terminal or C-terminal
ends of TFIHA suggested the N-terminal fingers of the protein have a role in forming
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specific contacts with the 3' region of the 5S gene ICR (13,14) whereas the C-terminal
tail is required for transcription promotion (13). In the present study, internal deletions
in TF1I1A are analyzed in order to better understand the roles of internal domains of TH11A
in the structure and function of this protein. Specific points examined include 1) the variation
in DNase I protection of the 5S gene upon deletion of internal segments of TFlllA and
2) the effect of these internal deletions on the transcription promotion and DNA renaturation
abilities of the mutated TFTUAs.
METHODS AND MATERIALS
Cbning and expression of TFlllA and deletion mutant TFlllA cDNA
A system to express foreign genes in E.coli (15) was kindly provided by Dr. Stanley Tabor.
One plasmid (pGPl-2) contains the bacteriophage T7 RNA polymerase gene under the
control of the lambda P L promoter, the cI857 repressor gene, and a gene for kanamycin
resistance. The other plasmid in this system (pT7-7) contains a T7 RNA polymerase
promoter followed by a ribosome binding site, an ATG initiation codon, a polylinker with
an open reading frame for the synthesis of fusion proteins, and the /3-lactamase gene. A
pT7-7 vector capable of expressing Th'lilA in E. coli was constructed by ligating a 1.5
kb EcoRI insert containing the complete TF11LA cDNA (kindly provided by Dr. Robert
Roeder) into the EcoRI site of pT7-7. For expression of a TFUIA mutant lacking the fourth
zinc binding domain (Fig. 1A), vector pT7IHA4 was constructed in which a filled in Bgl
II site in TFIIIA cDNA (He 102) was ligated to the Pvu II site (Leu 130). This construct
should express a TFIIIA mutant lacking the fourth zinc domain and juxtaposing the third
and fifth domains. For expression of a TFIIIA mutant in which zinc domains 7 and 8
are fused (Fig. IB), vector pT7mA78 was constructed by digesting the 1.5 kb EcoRI
TFIIIA cDNA with TaqI and religating the two large EcoRI-TaqI fragments into EcoRI
digested pT7-7. This construct (deletion of amino acids 200—224) results in the fusion
of zinc fingers 7 and 8 with two cysteines from finger 7 and two histidines from finger
8. Internal deletion constructs were confirmed by restriction enzyme analysis and DNA
sequencing of the mutant junctions in the pT7-7 plasmids.
Expression and isolation of TFlllA from E. coli
E.coli strain K38 (16) harboring pGPl-2 was transformed with plasmid pT7-7 containing
either the 1.5 kb EcoRI TFIIIA cDNA or the deletion inserts. When intact or deletion
TFEUA cDNA inserted into pT7-7 is expressed, a fusion protein is most likely synthesized
containing the first three amino acids from pT7-7 (Met-Ala-Arg), the next 13 amino acids
from the normally untranslated 5' leader of the TFIIIA cDNA (De-Pro-Glu-Ala-Glu-GlyCys-Ser-Val-Ala-Glu-Gly-Glu, ref. 6), followed by TFIIIA amino acids. K38 cells,
containing pGPl-2 and pT7- 7 (with either the intact or deletion inserts) were grown at
37 °C in Luria broth (containing 50 /tg/ml ampicillin and kanamycin) to an A550 of
0.8— 1.0. Cells were harvested by low speed centrifugation and the cell pellets were stored
at —70°C. Typically 1 g of a cell paste was resuspended in 3 ml of sonication buffer
(20 mM Tris-HCl, pH 7.5, 320 mM KC1, 1.5 raM MgCl2, 0.4 mM DTT, and 10 /iM
ZnCy in a 13 ml polystyrene tube. The cell suspension was sonicated for 6 min with
a microtip utilizing an Ultrasonics Cell Disrupter at 50% maximum power output and on
a 50% on-off pulsed cycle. The sonicate was spun at 2000 rpm for 2 min to sediment
cell debris and the supernatant was then spun 6000 rpm for 10 min. This supernatant was
subjected to chromatography on a 0.5 ml BIO-REX 70 cation exchange column equilibrated
with the extraction buffer. The column was eluted sequentially with 0.50, 0.75, and 1.0
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M KC1 (in the sonication buffer). The KCL concentrations in the 0.75 and 1.0 M buffer
fractions were reduced to 0.32 M by addition of appropriate amounts of 0.05M KC1
sonication buffer and centrifugation through an Amicon Centricon-30 concentrator.
Lowering the salt concentration of these fractions yields maximal activity of certain TFULA
mutants. An N-terminal deletion mutant of TFHIA (14) was found to partially inhibit binding
if the mutant protein was treated in this manner (in agreement with a previous report on
N-terminal deletions, ref. 13).
Immunoblot of intact TFIIIA and deletion mutant TFIIIA
0.75 or 1.0 M KC1 chromatographic fractions from E.coli sonicates, containing either intact
TFIIIA, deletion mutant TFIIIA, or no expressed TFIILA, were subjected to SDS PAGE
as described previously (14). Electrophoresed gels were equilibrated in transfer buffer (80
mM Tris-Glycine, pH 8.3, 20% methanol) and the proteins were transferred onto
nitrocellulose (17). The nitrocellulose blot was soaked in 10X PBS plus 2% nonfat milk
solution or 1% BSA followed by washing in l x PBS and 0.05% Tween 20. The blot
was then incubated with rabbit antiserum against Xenopus TFIIIA (at a 500:1 dilution)
in 1% BSA, 10 X PBS, and 0.05% Tween 20. The immunoblot was adsorbed with an
alkaline phosphatase-conjugated, anti-rabbit antibody followed by incubation in a /S-naphthyl
acid phosphate stain (18). TFIIIA in the 0.75 and 1.0 M column fractions was quantitated
by comparison of immunoblots with known amounts of transcription factor.
DNase I protection and DNA renaturation assays
A 303 bp EcoRI-BamHI fragment containing the Xenopus borealis somatic 5S RNA gene
was end-labeled on the coding strand (EcoRI site) with [or32?] dATP and reverse
transcriptase (12). DNase I protection with native TFIIIA or intact/deletion mutant ihlilAs
isolated from E.coli was performed as described previously (14). TFUIA-dependent DNA
renaturation was assayed as described previously (12).
Transcription of the Xenopus 5S RNA gene in an unfertilized egg extract
Unfertilized eggs were collected overnight from mature, Xenopus laevis females after
injection with human chorionic gonadotropin (19). The egg mass was dejellied by washing
in 2% cysteine-HCl (pH 7.8) and then rinsed 4 times with modified Barth's solution (15
mM HEPES, pH 7.6, 88 mM NaCl, 2mM KC1, 1 mM CaCl2, 1 mM MgCl2, 1 mM
MgSO4, 0.5 mM Na2HPO4, and 2 mM NaHCG,) followed by 2 rinses in deionized water.
The rinsed eggs were homogenized in a precooled, teflon-glass homogenizer and the extract
was subjected to high-speed centrifugation (19). Transcription reactions (50 /d, 25 of which
is the egg extract) contained 10 mm HEPES, pH 7.5, 70 mM KCL, 5 mM MgCl2, 0.5
mM DTT, 0.2 mM ATP, GTP, CTP, 0.02 mM UTP, 2 ^Ci [a^P] UTP, 2 pg pSP65-17
plasmid DNA (12), and amounts of native, intact, or deletion mutant TFIILA indicated
in the Figure Legends. Reactions were incubated for 2 hrs at 23°C, stopped by addition
of 3 fi\ 10% SDS, incubated with 1 ng proteinase K for 30 min, followed by phenol
extraction and ethanol precipitation. Gel electrophoresis and autoradiography were
performed as described previously (20).
RESULTS
Isolation and identification of intact TFIIIA and deletion mutant TFIIIA
Intact or deletion mutant TFLHAs were isolated from E. coli sonicates by chromatography
on BIO-REX 70, a cation exchange resin previously used to purify native TFIIIA from
immature oocytes (1,21). Fig. 2A is a coomassie-stained, SDS polyacrylamide gel of 0.75
M KC1 (lane 1) and 1.0 M KC1 (lane 2) fractions obtained from BIO-REX 70
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MTEfVUL CONTROL REGION OF S3 RNA GENE
L M I
T
LTITIf
«|V
tA I
P|
It
'lOUUli
I
t.1 I • 1 S T *
»
I
°K L T I 0
COCH
s
n
Fig. 1. Internal Deletion Mutants of TFIIIA. The juxtaposition of the TFUIA amino acid sequence along the
5S RNA gene ICR is according to the proposed model of Tso et al., 1986 (ref. 9). Amino acid positions in
the boxed areas correspond to those DNA sequences removed from the TFIIIA cDNA. A, finger 4 deletion
mutant; B, finger 7 - 8 fusion mutant.
chromatography of a TFIIIA sonicate. Intact TFTJIA is observed as a coomassie-staining
band (arrow, Fig. 2A) which comigrates with TFIIIA immunoblotted with an alkaline
phosphatase conjugate against rabbit, anti-TFIHA antibody (Fig. 2B, lane 1). The TFIIIA
purity in the 0.75 M KC1 fraction (Fig. 2A, lane 1) was less than in the 1.0 M fraction
Gane 2, estimated to be 10-15%). The purity of TFIIIA in the 1.0 M fraction is dependent
upon the amounts of low molecular weight proteins present and is variable from preparation
to preparation. Lane 2 in Fig. 2B exhibits the immunoblotting of the zinc finger 4 deletion
mutant (Fig. 1A). The mobility of this mutant protein during SDS PAGE is slightly greater
than that of intact protein (lane 1, Fig. 2B) and is consistent with the removal of 30 amino
acids. Panel C in Fig. 2 illustrates immunoblotting of a fraction from E.coli cells not
expressing TFIIIA (lane 1), TFIIIA derived from native 7S particles purified from frog
ovarian tissue (lane 2), and the finger 7 — 8 mutant (lane 3). The fraction from E.coli cells
not expressing TFIIIA did not react with the anti-TFIHA antibody (lane l); this material
also yielded negative results in the assays for TFIIIA function. The slightly increased
mobility of the finger 7 - 8 mutant (lane 3) relative to the native TFIIIA (lane 2) is consistent
with the 9 amino acid difference in mass.
Intact/deletion mutant TFIHA-dependent DNase I protection of the 5S RNA gene
DNase I protection experiments were performed to compare the abilities of TFIIIA and
internal deletion mutant TFHJAs isolated from E.coli to specifically bind the 5S RNA gene.
Fig. 3 is an autoradiogram of the DNase I protection patterns of native TFIIIA (isolated
from oocytes) and intact/ deletion mutant TFIIIA (isolated from E. coli) on the 5S RNA
gene (end-labeled on the coding strand). Native TFIIIA protects nucleotide positions +43
to +96 of the coding strand from DNase I digestion (panel A, lane 2). TFIIIA expressed
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A
1
B
1
TFIIIAfinger 4
deletion
Fig. 2. SDS PAGE and Immunoblotting of intact TFIIIA and deletion mutant TFIIIA. Chromatography of E. coli
sonicates, SDS PAGE, and immunoblotting were performed as described in MATERIALS AND METHODS.
Panel A, Coomassie stained SDS polyacrylamide gel of intact TFIIIA (lane 1, 50 pi 0.75 M fraction, lane 2,
50 pi 1.0 M fraction). Panel B, immunoblot of intact TFHIA (lane 1, 25 pi 1.0 M fraction) and finger 4 deletion
mutant (lane 2, 25 pi 1.0 M fraction). Panel C, immunoblot of chromatography fraction from E.coli cells not
expressing TFIIIA (lane 1, 25 pi 1.0 M fraction), TFIIIA from 7S particles isolated from frog ovarian tissue
(lane 2, 1 pg), and finger 7 - 8 fusion mutant (lane 3, 25 pi 1.0 M fraction).
in and isolated from E. coli also protects from DNase I digestion the coding strand of the
5S RNA gene to the same extent (lanes 3 and 4, 0.75 M fraction, lanes 5 and 6, 1.0 M
fraction). Although TFIIIA purity is significantly less in the 0.75 M KC1 fraction than
the 1.0 M KC1 fraction (cf. lane 1 with lane 2, Fig. 1A), both preparations footprint equally
well as evidenced by similar protection in lanes 3 , 5 , and 6 (note: material in lane 4 was
lost possibly during ethanol precipitation/washing steps). Panels B and C illustrate the DNase
I protection of the 5S RNA gene afforded by the finger 4 deletion mutant (lanes 3 and
4, panel B) and the finger 7—8 fusion mutant (lanes 1 —3, panel C). For the finger 4 mutant,
protection is observed from +96 to +78 on the coding strand with slight DNase I
hypersensitivity observed around +77. Previous work has shown TFIIIA contacts on the
coding and non-coding strands parallel each other (13). We examined DNase I protection
by the finger 4 mutant on the non-coding strand and found similar extent of protection
as observed with the coding strand (not shown). The finger 7 - 8 fusion mutant protects
from DNase I digestion the coding strand of the 5S RNA gene from nucleotides +96 to
+63 (lanes 2 and 3, panel C) with slight hypersensitivity seen at +63 (most apparent
in lane 1). The extent of protection by this mutant is similar to that observed for the 20
kDa tryptic digestion product of TFIIIA (21,11). In addition, this protection pattern is
identical to that observed previously with a TFIIIA mutant lacking the C-terminal 185
amino acids (13).
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B
1 2
3
4
5
6
1 2
3
4
1
2
3
^^^^^^^^^^V * ^^^^^^1
MI
••
+96
— +63
Fig. 3. DNase I protection of the 5S RNA gene by intact and deletion mutant TFIHA. TFmA-containing 7S
particles were purified from Xerwpus immature ovaries (17). Intact and internal deletion mutant TFMAs were
expressed in and isolated from E.coh as described in METHODS AND MATERIALS. DNase I protection assays,
denaturing acrylamide gel electrophoresis, and autoradiography were performed as described in METHODS AND
MATERIALS. The nucleotide positions on the coding strand were derived from ref. 22. Panel A: lane 1, no
7S particle; lane 2, 1 x 1CT8 M 7S particle preincubated 30 min at 23°C with 10 fig/ml RNase A; lanes 3 and
4, 2 a n d 3 x l 0 ~ 8 M intact TFUIA (0.75 M KCL fraction); lanes 5 and 6, 2 a n d 3 x l O " 8 M intact TFIIIA (1.0
M KCI fraction). Panel B: lanes 1 and 2 are the same as in panel A; lanes 3 and 4, 2 and 3 x 10~8 M finger
4 deletion mutant TFIIIA. Panel C, lanes 1-3, 1, 2, and 3 x l O " 8 M finger 7 - 8 fusion mutant TFIIIA. All
reactions contained 1 x 10~9 M end-labeled 5S RNA gene.
Promotion of 5S RNA synthesis by intact and internal deletion mutant TFIIIA
In order to determine if the finger 4 and finger 7 - 8 fusion mutants can promote transcription
of the Xenopus 5S RNA gene, these proteins were assayed in an unfertilized egg extract
which is highly dependent upon addition of both the 5S RNA gene and TFIIIA for promotion
of 5S RNA synthesis. Fig. 4 is an autoradiogram of a denaturing acrylamide gel depicting
the synthesis of steady state levels of 5S RNA in such an extract. In absence of exogenously
added TFUIA, little 5S RNA synthesis is observed from the 5S RNA gene-containing
plasmid flane i). The addition of native TFIIIA (in the 7S particle) isolated from Xenopus
immature ovaries stimulates 5S RNA synthesis as evidenced by the enhanced incorporation
of [a32P] UTP into 5S RNA (lanes 2 and 3). Likewise, the addition of TFUIA expressed
and isolated from E.coli (lanes 4 and 5) or the finger 4 deletion mutant TFIIIA (lanes
6 and 7) stimulate the synthesis of comparable levels of 5S RNA. Fig. 5 is a gel
autoradiogram depicting steady state levels of [32P] 5S RNA synthesized in the presence
of the finger 7 - 8 fusion mutant (lanes 2 and 3) or native TFTHA (lane 4). Although the
finger 7 - 8 fusion protein is able to promote 5S RNA synthesis, the steady state levels
produced are less than observed with the native protein. A control reaction containing
the 5S RNA gene plasmid but no exogenous TFIIIA was electrophoresed in lane 1.
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- 5 S RNA
Fig. 4. Stimulation of 5S RNA synthesis by intact andfinger4 deletion mutant TFIIIA. 7S particles and intact/deletion
mutant TFHIAs were isolated as described in METHODS AND MATERIALS. Transcription assays utilizing
a Xenopus laevis unfertilized egg extract, gel electrophoresis, and autoradiography were performed as described
in METHODS AND MATERIALS. Lane 1, no TFIHA in reaction; lanes 2 and 3, 0.5 and 2.5 xlO~ 7 M 7S
particle in reaction; lanes 4 and 5, 5 and 7 x 10" 8 M intact TFIHA (0.75 M KC1 fraction); lanes 6 and 7, 5
and 7x 10~8 M finger 4 deletion mutant TFIIIA (0.75 M KC1 fraction) in transcription reaction. The concentration
of Xenopus borealis 5S RNA gene-containing plasmid was 1.8xlO~8M in all reactions.
DNA renaturation promoted by intact and deletion mutant TFIIIA
TFIIIA has the ability to promote the renaturation of complementary strands of DNA
(11,12). This ability is lost upon trypsin digestion of the C-terminal half of the protein
(11). As stated in the description of Fig. 3, the TFIIIA finger 7 —8 fusion mutant protects
the 5S RNA gene from DNase I digestion in similar fashion to that described previously
for this N-terminal fragment of TFIIIA generated by trypsin digestion (21,11). We asked
whether this finger 7 - 8 fusion mutant (as well as the finger 4 deletion mutant) could
promote DNA renaturation. Fig. 6 is an autoradiogram of an agarose gel (electrophoresed
in Tris-phosphate buffer) examining the DNA renaturation abilities of intact and deletion
mutant THilAs. In this gel system, double-stranded DNA (end-labeled 5S RNA gene
fragment, lane 1) migrates faster than single-stranded DNA (heat denatured 5S RNA gene
fragment, lane 2). Lanes 3—5, 6—8, and 9—11, contain increasing concentrations of THIllA
expressed and isolated from E.coli, finger 4 deltion mutant, and the finger 7 - 8 fusion
mutant respectively. All three proteins promote the renaturation of complementary DNA
strands as evidenced by the conversion of the single-stranded DNA (upper band) to doublestranded DNA Qower band).
DISCUSSION
According to the zinc finger model for Xenopus transcription factor DIA, the protein contains
9 zinc binding domains encompassing amino acids 11-280. This linear array of fingers
is proposed to bind along the 5S RNA gene ICR in about 5 bp/finger increments (23).
Partial sequence homology among the 5 bp increments led to the conclusion that all 9
1 2
3
4
Wf «•» f P -5S RNA
m.
Fig. 5. Stimulation of 5S RNA synthesis by the finger 7 - 8 fusion mutant of TFIIIA. 7S particles and deletion
mutant TFIHA were isolated as described in METHODS AND MATERIALS. Transcription assays, gel
electrophoresis, and autoradiography were performed as described in the Fig. 4 legend. Lane 1, no TFIIIA in
reaction; lanes 2 and 3, 7 and 14X 10~8M finger 7 - 8 fusion mutant (0.75 M KC1 fraction) in transcription
reaction; lane 4, 5 x 10~7M 7S particles in reaction. All reactions contained 1.8xl0~ 8 M 5S RNA gene plasmid.
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1 2 3 4 5 6 7 8 9 1 0 1 1
ss DNAds DNA-
Fig. 6. Promotion of DNA renaturatkm by intact and deletion mutant TFHIA. Isolation of intact and mutant
TH11A proteins, DNA renaturation reactions, SDS reaction quenching, agarose gel electrophoresis, and
autoradiography were performed as described in METHODS AND MATERIALS. Lane 1, 303 bp fragment
containing the Xenopus 5S RNA gene; lane 2, heat denatured (single-stranded) fragment, no TFIUA added; lanes
3 - 5 , 0.8, 1.6, 2.4X 10~8 M intact TFIIIA added to renaturauon reaction; lanes 6 - 8 , 0.8, 1.6, 2 4xlO~ 8 M
of finger 4 deletion mutant; lanes 9 - 1 1 , 0.8, 1.6, 2.4x 10~8 M finger 7—8 fusion mutant. Protein samples
were from the 0.75 M KC1 fractions. The concentration of 5S RNA gene containing fragment (double-stranded)
in all reactions was 1 x 10~9 M.
fingers were acting as DNA binding entities to a common sequence motif (24). Previous
in vitro mutagenesis studies on TFIIIA suggested the N-terminal fingers were involved
in the initial formation of specific DNA contacts (as deduced by DNase I protection, refs.
13,14) whereas the C-terminal tail of the protein was necessary for transcription promotion
(13). This DNA binding behavior suggested that binding by fingers in die middle and in
the C-terminal region of TFIIIA is dependent upon the initial interaction of the fingers
in the N-terminal region of the protein.
In order to examine certain predictions of the zinc finger model as well as better
understand the function of internal structural elements in '1'hlllA, we constructed internal
deletion mutants of TFIIIA cDNA. The first mutant (Fig. 1A) contains a deletion in mat
region of the cDNA coding for the fourth putative zinc finger (joining De 102 of finger
3 and Leu 133 of finger 5). The second mutant (Fig. IB) contains a deletion in the cDNA
resulting in die potential fusion of die putative zinc binding site of fingers 7 and 8 (joining
Arg 199 of finger 7 and Arg 225 of finger 8). DNA sequence analysis confirmed the
presence of these reading frames in the mutated cDNAs and immunoblot analyses
demonstrated the expression and isolation of proteins slighdy smaller in molecular weight
than intact TFUIA.
In die finger 4 mutant as well as the finger 7 — 8 fusion mutant die registry of fingers
has been shifted 1 finger toward the N-terminus. One DNA binding possiblity for these
mutants is that dieir protection of the 5S RNA gene from DNase I digestion could also
be shifted 5 bp toward the 3' side of the control region because of the sequence similarity
of the contiguous 5 bp binding sites. However, DNase I protection afforded by the finger
4 deletion mutant extended only from nucleotides +96 to about +78 on the 5S RNA gene
(Fig. 3B). This region of protection is consistent widi the TFIIIA zinc finger model in
which die region of protein comprising die first 3 fingers interacts from +96 to +80 (see
Fig. 1). The behavior of this finger 4 deletion mutant is different from a previously described
TFIIIA mutation in which die C-terminal 212 amino acids were deleted (leaving the Nterminal 4 fingers intact, ref. 13). This particular mutant was unable to protect die 5S
gene ICR from DNase I digestion (13). This result coupled with our findings with die
finger 4 deletion mutant suggests that more C-terminal sequences may play a role in the
binding of die finger 1 —3 region to die 5S gene ICR. DNase I protection by the finger
7—8 fusion mutant extended from +96 to about +63 which is consistent with tight binding
by the region of TFIUA comprising the first six fingers (Fig. 1). The footprint boundries
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exhibited by the finger 7 — 8 mutant are very similar to that observed previously for TF1ILA
variants with C-terminal deletions up to and including fingers 9,8,7, or 6 (13). These results
suggest that, although the N-terminal fingers may be interacting with the 5S gene ICR
as predicted by the TFIQA zinc finger model (Fig. 1), the more C-terminal fingers are
not interacting as predicted by the model.
In both the finger 4 deletion mutant and the finger 7 - 8 fusion mutant, the fingers
downstream of the structural change are apparently unable to afford DNase I protection
of the remaining 5' portion of the ICR. One explanation for these findings is the DNA
binding sites (5 bp increments) for the TKUlA fingers are not identical and juxtaposition
of the finger 5—9 cluster next to finger 3 in the finger 4 mutant has garbled the specificity
of the remaining amino acid-nucleotide base interactions. An alternative explanation is
these internal structural changes in TH11A have altered the conformation/folding of protein
domains downstream of the change but not upstream. If such a structural change has taken
place in the finger 4 and finger 7 — 8 fusion mutants, we believe it is not overly disruptive
because both proteins are still capable of promoting DNA renaturation (Fig. 6), a property
dependent in part on the structural integrity of the C-terminal half of the protein (11).
Also in support of the notion that these internal mutations have not drastically altered the
conformation/folding of the C-terminal halves of the mutant proteins is the observation
that both mutants are capable of promoting 5S RNA gene transcription to a certain degree
(Figs. 4, 5). The C-terminal tail of ThILLA is required for transcription promotion and
its 3-dimensional structure most likely remains intact in these mutants. These transcription
results also suggest that strong contacts (as assayed by DNase I protection) between the
C-terminal region of the protein and the 5' portion of the ICR are not absolutely required
for promotion of 5S RNA synthesis.
ACKNOWLEDGEMENTS
This work was supported by a grant from the National Institute of General Medical Sciences.
The costs of publication were defrayed in part by the payment of page charges. Tlas article
must therefore be marked 'advertisement' in accordance with 18 U.S.C. Section 1734 only
to indicate this fact. The authors thank R. Lloyd for assistance with graphics.
•To whom correspondence should be addressed
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