(Received for publication, January 31, 1997, and in revised form, March 5, 1997)
From the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Zinc fingers in transcription factor IIIA
(TFIIIA) contribute differentially to RNA and DNA binding affinity. We
investigated whether the same putative -helix amino acids in TFIIIA
zinc fingers are essential for both RNA and DNA binding. In published
structures, zinc fingers make DNA base contacts through amino acids
1, +2, +3, and +6 of the recognition helix. Alanine substitution at
these four positions were made in TFIIIA RNA binding zinc fingers,
tz4-7 and DNA binding zinc fingers, tz1-3. Substitution in zinc
fingers 4 or 6 of tz4-7 reduced RNA affinity 77- and 38-fold,
respectively, whereas substitution in zinc fingers 5 or 7 had little
effect. DNA binding affinity of tz1-3 was eliminated by alanine
substitution in any one zinc finger. We determined which amino acids
supported RNA binding by phage display of a library of zinc finger 4 mutants. Lysine at helix position
1 of zinc finger 4 was conserved in all selected tz4-7 fusions. Point mutation of Lys
1
to alanine in zinc finger 4 reduced tz4-7 RNA affinity 30-fold. We
propose that RNA binding by TFIIIA shows similarity to DNA binding in
the use of the recognition helix. Helix positions
1 and +2 may have
particular significance for RNA binding.
In Xenopus oocytes, transcription factor IIIA (TFIIIA)1 has dual functions as a positive transcription factor that binds to the 5 S rRNA gene internal control region and as a storage protein in a 7 S ribonucleoprotein complex with 5 S rRNA (1, 2). Nine tandemly repeated C2H2 zinc fingers at the amino terminus function in binding both 5 S rRNA and the internal control region of the 5 S rRNA gene with high affinity and specificity. Site-specific zinc fingers 4-7, and zinc fingers 1-3 bind independently and specifically to 5 S rRNA and DNA, respectively (3-6). With the exception of the Wilm's tumor protein WT1 (7), other RNA binding C2H2 zinc fingers do not yet have well defined RNA binding sites (8, 9). The other major class of zinc fingers that bind to RNA, the C2HC retroviral nucleocapsid protein, are structurally distinct from TFIIIA (10).
Three structures of C2H2 zinc fingers, from
Zif268 (11), Tramtrack (12), and GLI (13), complexed with their cognate DNA have been solved. In each structure, -helices lie in the major
groove and make base-specific contacts. Zinc fingers 1-3 from TFIIIA
bind the 5 S rRNA gene promoter with high affinity and produce a
compact footprint similar to Zif268 (14-16). A recent mutagenesis
study of TFIIIA has shown that some of the potential amino acid
contacts with DNA are at positions predicted to be
-helical by
comparison with the Zif268 crystal structure (17). The significance of
these putative
-helix amino acids for RNA binding is less clear.
We have investigated whether amino acids in the putative -helices of
zinc fingers are important for RNA and DNA binding affinity.
Site-directed mutagenesis was done using a PCR based procedure. cDNA fragments encoding zinc fingers were amplified from pSPTF15 (18) using specific primers and subcloned into pET28b(+) (Novagen). Mutations were introduced by overlapping PCR using primer pairs encoding the desired amino acid changes.
Protein Expression and PurificationZinc finger
polypeptides derived from TFIIIA were expressed from pET28b as
polyhistidine-tagged molecules in Escherichia coli strain
BL21(DE3). Cells were grown to A600 0.6 in 100 ml of LB, supplemented with 100 µM ZnSO4, 40 mM glucose, and 30 µg/ml kanamycin and induced with 1 mM isopropyl-1-thio--D-galactopyranoside for 3-4 h. Inclusion bodies containing the fusion protein were collected by centrifugation and extracted by sonication in 5 ml of His-binding buffer (5 mM imidazole, 500 mM NaCl, 50 µM ZnSO4, 100 µM
phenylmethylsulfonyl fluoride, 1 mM benzamidine, 20 mM Tris, pH 8.0) containing 6 M urea. The
extract was clarified by centrifugation and passed over a 1-2-ml
nickel-agarose column (HIS-bind resin, Novagen) equilibrated with
His-binding buffer containing 6 M urea. The column was
washed with 10 column volumes of His-binding buffer containing 6 M urea and 6 volumes of column buffer (6 M
urea, 500 mM NaCl, 20 mM Tris, pH 8.0)
containing 40 mM imidazole. Proteins were eluted with 1 M imidazole in column buffer. Protein-containing fractions
were pooled, dialyzed twice against 500 ml of column buffer over a 12-h
period and once against 500 ml of 2 M urea, 150 mM NaCl, 1 mM DTT, 2 mM
CaCl2, 20 mM Tris, pH 7.9, for 4 h.
Derivatives of tz4-7 were digested for 2-4 h with 1-2 units of
thrombin (Novagen)/100 µg of protein at 25 °C. All proteins were
denatured by dialysis against 6 M urea in 500 mM NaCl, 50 µM ZnCl2, 1 mM DTT, 20 mM MES, pH 6.5, and renatured by
stepwise dialysis in the same buffer containing progressively lower
concentrations of urea. The final dialysis buffer was 100 mM NaCl, 50 µM ZnCl2, 1 mM DTT, 20 mM MES, pH 6.5, 20% glycerol.
A 66-base pair AvaI
fragment of the Xenopus borealis oocyte 5 S rRNA gene
internal control region was end-labeled with
-[32P]dCTP using the Klenow fragment of DNA polymerase
I (5). Xenopus laevis oocyte-type 5 S rRNA was made by
in vitro runoff transcription in the presence of
-[32P]CTP (T7 Megascript kit, Ambion). Protein binding
to 32P-labeled RNA and DNA was measured by electrophoretic
mobility shift (5). For Scatchard analysis, fixed concentrations of protein that produce between 20 and 80% shifted nucleic acid were incubated with DNA (0.4-50 nM) or RNA (0.4-20
nM) in 10 µl of binding buffer (0.05% Nonidet P-40, 90 mM KCl, 10 µM ZnCl2, 5 mM DTT, 8% glycerol, 1 mM MgCl2,
20 mM Tris, pH 7.5) for 30 min at room temperature
(21 °C). Nonspecific competitors were also included at 20 µg/ml
poly[d(I-C)] for DNA reactions and 20 µg/ml poly(I-C) for RNA
binding reactions. KD values (Table I) are the mean
of at least three independent determinations.
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Four codons (1, +2, +3, and
+6 of the
-helix) in zinc finger 4 were randomized by constructing
zinc finger 4 from two oligonucleotides, which were annealed and filled
in with the Klenow fragment of DNA polymerase I. Randomized zinc finger
4 DNA was digested with BsiWI, ligated to DNA coding for
fingers 5-7 synthesized by PCR and inserted into
ApaI-SpeI cleaved fd.tet.7000 (19).
Phage preparation and selection were done according to Rebar and Pabo (19) with minor differences. Phage were grown anaerobically and prepared by precipitating twice with polyethylene glycol 8000. Phage (108-109 colony forming units) were incubated in 50 µl of binding buffer with 20 µg/ml poly(I-C) and 5-10 pmol of biotin-labeled 5 S rRNA for 1 h at 21 °C with gentle agitation. Binding buffer (190 µl) was added, and the mixture was split evenly between six microtiter wells coated with streptavidin and blocked with acetylated bovine serum albumin. Reactions were incubated in wells at 21 °C with gentle agitation for 1 h and washed 10 times over 30 min with 150 µl of wash buffer (100 mM KCl, 1 mM MgCl2, 10 µM ZnCl2, 5% glycerol, 5 mM DTT, 10 mM Tris, pH 7.5). Phage were eluted by incubation of each well with 40 µl of 4 M NaCl, 5 mM MgCl2, 10 µM ZnCl2, 5% glycerol, and 15 mM HEPES, pH 7.8. Eluted phage were titered and used to infect K-91 cells, which were grown anaerobically to produce phage for the next round of selection against 5 S rRNA.
The positions of zinc-liganding amino acids in TFIIIA zinc fingers
are similar to those of Zif268, as are the positions of conserved
hydrophobic residues required for zinc finger structure (20). By
analogy to the Zif268 structure, we assumed position 1 of the zinc
finger -helix to be six amino acids amino-terminal to the
zinc-liganded histidine. Nucleic acid binding was determined by
Scatchard plot. The assay conditions we used (20-80% shifted RNA),
provide an accurate measurement of the fraction of shifted RNA, but
absolute values for KD could have significant errors
if the data are not truly linear in a bound/free versus bound plot, despite correlation coefficients greater than 0.8 (Figs.
1 and 2). To estimate potential errors,
we also determined the KD by an alternative method
using protein titration and curve-fitting (3). The values differ by
60% (0.1 nM by Scatchard versus 0.16 nM), but the error is probably less, since the
determination by protein titration assumes fully active protein. We
used scatchard analysis since the fraction of active protein need not
be known and we place greater emphasis on relative affinities rather
than absolute values for KD.
RNA Binding by Zinc Fingers 4-7
Sequence-specific RNA
binding by TFIIIA is mediated through central zinc fingers (3, 6).
Mobility shift assays with tz4-7 show a single shifted species at
protein-RNA ratios that generate multiple shifted bands for tz1-3
(compare Figs. 1A and 2A). When individual zinc
fingers in tz4-7 were substituted at four positions predicted to be
within the finger -helix, substitution in zinc fingers 4 and 6 showed the most profound reduction in RNA affinity. The estimated
dissociation constant for zinc finger 4 substitution (tz4-7A4) was 7.7 nM, a 77-fold reduction from the native tz4-7
KD of 0.1 nM. Alanine substitution in zinc finger 6 (tz4-7A6) reduced RNA affinity 30-fold (Table
I). In contrast, alanine substitution in zinc fingers 5 or 7 (tz4-7A5 and tz4-7A7) produced no significant difference in RNA
affinity compared with native tz4-7. The absence of large effects for
alanine substitution in zinc fingers 5 and 7 suggests that alanine
substitutions per se do not grossly disrupt the structure of
the finger 4-7 fragment.
Clemens et al. (3) previously showed that tz5-7 RNA
affinity was 20-fold lower than tz4-7 (using our nomenclature),
suggesting an important contribution for zinc finger 4. Our data
suggest that substitution of one or more of amino acids
Lys1, Asn+2, Glu+3, and
Val+6 is responsible for this effect. In the context of
tz1-7, a Glu+3
Arg substitution in finger 4 results in
a 4-fold reduction in affinity, which indicates that one or more of the
other alanine-substituted positions,
1, +2, +6, make more significant
contributions to RNA binding affinity. Alanine substitutions in zinc
finger 6 reduce 5 S rRNA affinity to approximately the same degree as a
previously reported Thr
1
Ile substitution (3),
suggesting that Thr
1 may be the most important residue
within zinc finger 6 for 5 S rRNA affinity. Individual zinc finger
"knockout" mutations in TFIIIA that disrupt the zinc liganding
capacity of zinc fingers are in agreement with our findings.
Substitution of histidine with asparagine in central zinc fingers 4, 5, or 6 reduced RNA affinity 2.6-4.1-fold (21). Alanine substitution in
zinc fingers 5 and 7 produced no change in RNA affinity. Yet
qualitative assays showing reduced 5 S rRNA affinity of fingers 5-9
compared with 6-9 and quantitative assays showing a 60-fold reduction
in affinity for fingers 1-6 compared with 1-7 suggest that specific
RNA contacts are made by both finger 5 and finger 7 (3, 5). Such
contacts presumably lie outside of the zinc finger amino acid positions that we mutated.
Alanine substitution
at four putative -helical amino acids in zinc fingers 1 or 2, within
tz1-3, abolished DNA binding (Fig. 2). A discrete band shift was
detectable only at a 1500:1 molar ratio of protein to DNA, and DNA
titration with a fixed protein concentration was not attempted. Alanine
substitution in zinc finger 3 appeared to be less severe, since a
discrete band shift appeared at a high molar ratio of protein to DNA
(300:1). In contrast, alanine substitutions in zinc finger 1 or 2 (tz1-3A1 and tz1-3A2) reduced RNA binding only 3-4-fold.
Substitution in finger 3 (tz1-3A3) has no significant effect on RNA
binding by these zinc fingers. These amino-terminal zinc fingers
contribute to overall RNA affinity of TFIIIA with lower sequence
specificity than tz4-7 and can likely achieve this through two zinc
fingers, which is the minimal RNA binding fragment from the amino
terminus of TFIIIA (5). Consistent with this notion, when any two zinc
fingers of tz1-3 are substituted, a clear band shift was not observed,
suggesting that some of the
-helical amino acids substituted are
involved in the RNA contacts made by tz1-3 (Fig. 2C).
These experiments suggest amino acids that make significant contributions to TFIIIA DNA affinity are in part the same as Zif268. The effect of alanine substitution on the three-finger amino-terminal fragment from TFIIIA is most marked for fingers 1 and 2. This result differs from the effect of broken finger mutants of full-length TFIIIA in which a zinc-coordinating histidine is changed to asparagine to disrupt zinc binding. The change in KD for DNA affinity for a single broken finger 1 or 2 is modest (2.5- and 7-fold, respectively) compared with four alanine substitutions in a single finger (22). However, the effects of mutations in individual zinc fingers may be amplified in the context of a short fragment where compensatory binding by many other fingers is not possible.
Phage Display of Zinc Fingers tz4-7To determine which amino
acids in zinc finger 4 were able to support RNA binding, we used phage
display to screen a large number of mutant tz4-7 sequences (23).
Native codons for amino acids 1, +2, +3, and +6 in zinc finger 4 were
replaced with a degenerate sequence, NN(G/C), that encodes all 20 amino
acids and the amber stop (Fig. 3A). Enrichment of 5 S rRNA
binding phage from the zinc finger 4 library over 4 rounds of selection
is shown in Fig. 3B. The final round included competitor
tRNA at 10-, 100-, or 1000-fold molar excess over 5 S rRNA to reduce
nonspecific RNA binding. DNA sequencing of 24 clones from the selection
round using a 1000-fold excess tRNA revealed complete conservation of lysine at the
1 position of the zinc finger 4
-helix. The sequence of 24 clones at the selected positions is shown in Table
II. Identity with wild type at positions other than
1
were found in 5 sequences, at Glu+3 and Val+6.
However, the majority of clones had only Lys
1 in common
with wild type, suggesting an important zinc finger structural or RNA
contact role for this amino acid. Serine was present in 14 of 24 clones
at +2, suggesting a role for a polar amino acid at this position.
Serine is present in many zinc finger proteins at +2, and within the
known zinc finger-DNA structures, serine makes contacts with G and T
(11, 13, 24). Serine can be represented by three codons in the
degenerate codon NNS and therefore may be selected on the basis of its
frequency in the library. However serine was not predominant at any
other position.
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RNA binding activity of selected zinc fingers was confirmed by
expression and purification of His-tagged zinc finger mutants from
E. coli. In protein titration assays, the binding of
selected zinc fingers is comparable to wild-type tz4-7 (Fig.
3C). The significance of Lys1 was confirmed by
construction of a point mutant Lys
1
Ala (tz1-4
1A4) in zinc finger 4 and a 3-position alanine substitution of amino
acids +2, +3, and +6 (tz4-7 236A4) (Table I). Replacement of
Lys
1 resulted in a 37-fold reduction in affinity, whereas
alanine substitution of +2, +3, and +6 reduced affinity 7-fold (Table I), suggesting of these four positions Lys
1 makes the
greatest contribution to RNA affinity.
Comparison of putative -helical regions in TFIIIA from amphibian
species shows greatest identity in zinc finger 4, suggesting a
conserved function for these amino acids. Lys
1 is the
only amino acid in zinc finger 4 that is also conserved between TFIIIA
from frog, human, and the related 5 S rRNA-binding protein, p43
(25-27). Our results suggest an important contribution is made to RNA
binding by this finger tip residue in zinc finger 4. Alanine
substitution in zinc finger 6 also caused a significant reduction in
RNA affinity of the tz4-7 fragment. In zinc finger 6 the corresponding
finger tip amino acid at
1 is threonine. The triplet amino acid
sequence, Thr
1-Trp+1-Thr+2,
containing this amino acid is also conserved between TFIIIA and p43 and
has been shown by others to be important for RNA binding in the context
of zinc fingers 1-7 (3). Zinc fingers 5 and 7 may make fewer contacts
with 5 S rRNA or use amino acids other than
1, +2, +3, and +6. Our
experiments do not distinguish these hypotheses.
Our results suggest zinc finger recognition of RNA may be similar to
DNA in that some contacts made by the -helix are identical to DNA
contacts made by C2H2 zinc fingers and DNA.
Loop E and the junction of helices I, II, and V are the major secondary
and tertiary structural features of 5 S rRNA crucial for specific TFIIIA binding (28-30). Zinc finger 4 lies in the proximity of loop E
(31). In finger 4, Lys
1 could make base contacts as a
hydrogen bond donor with guanosine in the loop E region where the major
groove is distorted and more accessible to the zinc finger
-helix
(29). Alternatively, Lys
1 may contribute to the overall
affinity for RNA through a salt bridge with the phosphate backbone,
while base-specific contacts are made by other zinc fingers. We believe
this report is the first demonstration that RNA binding can be
successfully assayed by phage display of zinc fingers. Phage display of
RNA binding zinc fingers with more extensive degeneracy in individual
zinc fingers should reveal the primary structural requirements for RNA
binding by the extensive C2H2 class of zinc
finger proteins.
We thank E. Rebar and C. Pabo for providing fd.tet.7000 and E. coli strains MC1061 and K91.