COMMUNICATION:
Phage Display of RNA Binding Zinc Fingers from Transcription Factor IIIA*

(Received for publication, January 31, 1997, and in revised form, March 5, 1997)

Westley J. Friesen and Martyn K. Darby Dagger

From the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Zinc fingers in transcription factor IIIA (TFIIIA) contribute differentially to RNA and DNA binding affinity. We investigated whether the same putative alpha -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.


INTRODUCTION

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, alpha -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 alpha -helical by comparison with the Zif268 crystal structure (17). The significance of these putative alpha -helix amino acids for RNA binding is less clear.

We have investigated whether amino acids in the putative alpha -helices of zinc fingers are important for RNA and DNA binding affinity.


EXPERIMENTAL PROCEDURES

Alanine Substitution Mutant Construction

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 Purification

Zinc 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-beta -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 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.

RNA and DNA Binding Assays

A 66-base pair AvaI fragment of the Xenopus borealis oocyte 5 S rRNA gene internal control region was end-labeled with alpha -[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 alpha -[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.

Table I.

Equilibrium dissociation constants for alanine-substituted zinc finger polypeptides


Zinc finger polypeptide Amino acids substituted with alanine (-1, +2, +3, +6) 5 S rRNA (KD ± S.E.)a Reduction in 5 S rRNA binding affinity (KDmutant/KDtz4-7)

nM -fold
t4-7 0.10  ± 0.02 1.0
t4-7A4 KNQV 7.70  ± 0.75 77.0
t4-7A5 LSRR 0.06  ± 0.01 0.6
t4-7A6 TTLK 4.30  ± 1.17 43.0
t4-7A7 RSFE 0.10  ± 0.03 1.0
tz4-7-1A4 K 3.65  ± 0.46 36.5
tz4-7 236A4  NQV 0.68  ± 0.18 6.8
tz1-3b 2.27  ± 0.48 1.0
tz1-3A1b KWKA 9.78  ± 2.77 4.3
tz1-3A2b SHHR 9.26  ± 1.42 4.1
tz1-3A3b TANK 3.43  ± 0.30 1.5

a Errors represent S.E. for linear regression of four independent Scatchard plots, not necessarily the error in absolute KD, which may be significantly larger (see "Results and Discussion").
b Proteins retain 6-histidine tag.

Phage Display and Selection

Four codons (-1, +2, +3, and +6 of the alpha -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.


RESULTS AND DISCUSSION

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 alpha -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.


Fig. 1. 5 S rRNA binding activity of alanine substitution mutants of tz4-7. A, complexes between tz4-7 alanine substitution mutants (black boxes are zinc fingers with alanine substitutions at -1, +2, +3, and +6 of the alpha -helix) were formed with 5 nM 32P-labeled 5 S rRNA and increasing concentrations of each protein and resolved by non-denaturing gel electrophoresis. Concentration of each protein is as follows: 6 nM (lane 1), 12 nM (lane 2), 25 nM (lane 3), 50 nM (lane 4), 100 nM (lane 5), 200 nM (lane 6). Lanes marked F indicate control reactions without added protein. Open triangles indicate the position of unbound 5 S rRNA. B, representative Scatchard plots for each substitution mutant were derived from titrations of constant protein with variable 5 S rRNA, not from the protein titrations above which are included to illustrate the number of shifted bands. KD values derived from at least three plots of each mutant are summarized in Table I. black-square, tz4-7; square , tz4-7A4; black-triangle, tz4-7A5; black-diamond , tz4-7A6; open circle , tz4-7A7.
[View Larger Version of this Image (27K GIF file)]



Fig. 2. 5 S rRNA and DNA binding activity of alanine substitution mutants of tz1-3. A, complexes were formed by incubation of recombinant protein with 32P-labeled 5 S rRNA (7.6 nM) or 32P-labeled DNA fragment from the X. borealis internal control region (1 nM) in binding buffer at room temperature and electrophoresed on polyacrylamide gels. Concentration for each protein: lane 1, 0.6 nM; lane 2, 2 nM; lane 3, 12 nM; lane 4, 61 nM; lane 5, 305 nM; lane 6, 1500 nM. Lanes marked F indicate control reactions without added protein. Zinc fingers with alanine substitutions are indicated by black boxes. B, representative Scatchard plots for 5 S rRNA binding by tz1-3 mutants. Bound and free fractions of 5 S rRNA were determined at RNA concentrations generating a single-shift. tz1-3 (black-square), tz1-3A1 (black-diamond ), tz1-3A2 (open circle ), and tz1-3A3 (square ). C, complexes formed with double alpha -helix mutants of tz1-3 and 5 S rRNA. 32P-Labeled 5 S rRNA (2 nM) was incubated with the following protein concentrations: 1.5 nM (lane 1), 6 nM (lane 2), 24 nM (lane 3), 95 nM (lane 4), 380 nM (lane 5). The position of free 5 S rRNA is indicated with an open triangle.
[View Larger Version of this Image (34K GIF file)]


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 alpha -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 Lys-1, Asn+2, Glu+3, and Val+6 is responsible for this effect. In the context of tz1-7, a Glu+3 right-arrow 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 right-arrow 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.

RNA and DNA Binding by Zinc Fingers 1-3

Alanine substitution at four putative alpha -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 alpha -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 tz4-7

To 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 alpha -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.


Fig. 3. 5 S rRNA binding by phage displayed zinc fingers 4-7. A, structure of the degenerate zinc finger 4 construct as a fusion with bacteriophage fd cpIII. B, chart showing the bacteriophage eluted from immobilized 5 S rRNA as a fraction of the bacteriophage incubated at each round of selection (black bars) and phage recovered from control experiments for elution from microtiter plates without immobilized RNA (white bars). In round 4, selection was carried out in the presence of the indicated molar excess (over immobilized 5 S rRNA) of yeast tRNA competitor. C, electrophoretic mobility shifts of bacterially expressed and affinity-purified phage-selected zinc fingers and 5 S rRNA. The amino acids at -1, +2, +3, and +6 in the zinc finger alpha -helix are shown below each protein name. Bold type signifies identity with wild type TFIIIA. 5 S rRNA (3 nM) was incubated with the following concentration of each mutant protein: 0.6 nM (lane 1), 1.5 nM (lane 2), 4.6 nM (lane 3), 14 nM (lane 4), 42 nM (lane 5), 125 nM (lane 6). F indicates control incubation without protein.
[View Larger Version of this Image (32K GIF file)]


Table II.

Sequence of zinc finger 4 on 5 S rRNA binding phage


Clone Zinc finger 4 alpha -helix positiona
Theoretical probability of occurrence (× 105)b
 -1 +2 +3 +6

wt K N Q V 1.03
19/24 K F Q A 1.03
20 K F Q S 1.54
14 K A H V 2.06
5 K S L V 9.26
23 K S S V 9.26
13/22 K S R A 9.26
11 K R A A 6.17
2 K S S L 13.88
3 K S S S 13.88
I K S V S 9.26
6/21 K S L S 13.88
7 K S S A 9.26
15 K S I A 3.09
16/17 K S L T 9.26
18 K S L G 9.26
10 K R L A 9.26
4/8 K G L A 6.17
12 K G R D 3.09
9 K T G Q 2.06
Consensus K S L A
24/24 14/24 9/24 10/24
U6 S W R F 4.63
U7 D T L W 3.09
U10 G S S R 13.88

a Bold type indicates identity to native TFIIIA sequence.
b Maximum and minimum probabilities are 41.65 × 10-5 and 0.51 × 10-5, respectively.

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 Lys-1 was confirmed by construction of a point mutant Lys-1 right-arrow 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 alpha -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 alpha -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 alpha -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.


FOOTNOTES

*   This work was supported in part by National Science Foundation Grant MCB-9206873.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.
Dagger    To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, 233 South 10th St., Philadelphia, PA 19107. Tel.: 215-503-4504; Fax: 215-923-0249; E-mail: M_Darby{at}lac.jci.tju.edu.
1   The abbreviations used are: TFIIIA, transcription factor IIIA; PCR, polymerase chain reaction; DTT, dithiothreitol; MES, 4-morpholineethanesulfonic acid.

ACKNOWLEDGEMENTS

We thank E. Rebar and C. Pabo for providing fd.tet.7000 and E. coli strains MC1061 and K91.


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