©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Fos Leucine Zipper Variants with Increased Association Capacity (*)

(Received for publication, April 26, 1995; and in revised form, July 17, 1995)

Dominique Porte Pascale Oertel-Buchheit Michèle Granger-Schnarr Manfred Schnarr(§)

From the Institut de Biologie Moléculaire et Cellulaire du CNRS, UPR 9002, 15 rue René Descartes, F-67084 Strasbourg Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Fos wild-type leucine zipper is unable to support homodimerization. This finding is generally explained by the negative net charge of the Fos zipper leading to the electrostatic repulsion of two monomers. Using a LexA-dependent in vivo assay in Escherichia coli, we show here that additional antideterminants for Fos zipper association are the residues in position a within the Fos zipper interface. If the wild-type Fos zipper is fused to the DNA binding domain of the LexA repressor (LexA-DBD), no excess repression is observed as compared with the LexA-DBD alone, in agreement with the incapacity of the wild-type Fos zipper to promote homodimerization. If hydrophobic amino acids (Ile, Leu, Val, Phe, Met) are inserted into the five a positions of a LexA-Fos zipper fusion protein, substantial transcriptional repression is recovered showing that Fos zipper homodimerization is not only limited by the repulsion of negatively charged residues but also by the nonhydrophobic nature of the a positions. The most efficient variants (harboring Ile or Leu in the five a positions) show an about 80-fold increase in transcriptional repression as compared with the wild-type Fos zipper fusion protein. In the case of multiple identical substitutions, the overall improvement is correlated with the hydrophobicity of the inserted side chains, i.e. Ile geq Leu > Val > Phe > Met. However at least for Val, Phe, and Met the impact of a given residue type on the association efficiency depends strongly on the heptad, i.e. on the local environment of the a residue. This is particularly striking for the second heptad of the Fos zipper, where Val is less well tolerated than Phe and Met. Most likely the a(1) residue modulates the interhelical repulsion between two glutamic acid side chains in positions g(1) and e(2). Most of the hydrophobic Fos zipper variants are also improved in heteroassociation with a Jun leucine zipper, such that roughly half of the additional free energy of homodimerization is imported into the heterodimer. A few candidates (including the Fos wild-type zipper) deviate from this correlation, showing considerable excess heteroassociation.


INTRODUCTION

Many transcription factors associate with their DNA binding site as either homodimers or heterodimers, making use of the so-called bZIP DNA binding motif. These proteins share the common feature of a basic region adjacent to a ``leucine zipper'' which is essential for dimerization (for a recent review, see (1) ). Most of them can form homodimers, and a subset forms heterodimers with other leucine zipper proteins. Leucine zippers are attractive targets to interfere with different cellular functions or dysfunctions. Granger-Schnarr et al.(2) have shown recently that expression of a leucine zipper fused to the LexA DNA binding domain in transformed NIH3T3 fibroblasts leads to the recovery of an untransformed phenotype. In this special case the target was a member of the AP-1 family of transcription factors.

These factors are expressed in a variety of cell types, usually at low basal levels, and can be induced in response to a wide set of stimuli, including growth factors, oncogenes, cytokines and UV irradiation (for reviews, see (3) and (4) ). AP-1 consists of two groups of bZIP proteins, the Fos-related proteins (c-Fos, Fos B, Fra1, Fra2), and the Jun proteins (c-Jun, JunB, JunD). A Fos protein has to dimerize with a Jun protein in order to bind to DNA, whereas Jun forms both homodimers and heterodimers with other Jun and Fos proteins. Jun and Fos proteins interact further with several other transcription factors, giving rise to a cross-talk between different signal transduction pathways by the formation of alternative dimeric species. Jun and/or Fos interact via their leucine zippers with members of the ATF/CREB(5) , C/EBP(6) , Maf (7, 8) , and Rel families (9, 10) as well as MyoD(11) , FIP (12) and LRF-1(13) . Regions outside the leucine zipper seem to play a role in the interaction with the glucocorticoid receptor(14) . The composition of these mixed oligomers may dictate an altered DNA binding specificity (5, 15, 16, 17) or lead to the repression of transcription(11) .

The dimerization domains of Jun and Fos proteins are alpha-helical structures characterized by a periodic repeat of leucine every 7th amino acid, i.e. every two helical turns, which forms a parallel coiled-coil structure(18, 19) . The seven amino acids of each repeat are referred to by the letters a to g (a` to g` referring to the opposite helix). The d positions are occupied by the highly conserved leucine residues and non-polar residues usually occur also at the a positions. Both a and d positions contribute to the formation of the hydrophobic interface which accounts for most of the free energy of dimerization. Additional stability can be provided by interhelical salt bridges between oppositely charged residues at positions e and g.

The preferential assembly of Jun and Fos as heterodimers has been shown to be mostly specified by the residues occupying the g and e positions(20) . Among these residues two pairs of oppositely charged amino acids in positions gand eaccount for most of the additional free energy of heterodimerization(21) . The Jun protein forms functional homodimers while the Fos protein does not. Beside electrostatic repulsion, it has been suggested that Fos homodimerization might also be hampered by a lack of hydrophobic residues in the apositions(22) . In these positions the c-Fos protein, as well as the other members of the Fos family, harbors indeed mostly polar residues, i.e.two threonine, two lysine, and one isoleucine residues.

To address the question if the nonhydrophobic nature of these residues is indeed a major antideterminant for Fos zipper dimerization, we have generated a restricted random collection of Fos zipper mutants harboring Leu, Ile, Val, Phe, or Met in the five a positions. The dimerization capacities of these Fos zipper variants were assayed in a bacterial in vivo test using protein fusions between these zippers and the LexA DNA binding domain(23, 24) , which is devoid of intrinsic dimerization activity both free in solution (25, 26) and bound to operator DNA(27) . We have used this assay previously to study Jun zipper variants mutated in the e and g positions(28) . Similar approaches made, respectively, use of the phage (29) and the phage 434 (30) repressor DNA binding domain.

Here we show that a Fos leucine zipper can considerably improve the repressor activity of the LexA DNA binding domain, if the a positions of the coiled-coil are occupied by hydrophobic residues. The observed improvement is correlated with the hydrophobicity of the inserted side chain (Ile geq Leu > Val > Phe > Met) and a Fos zipper variant can reach the efficiency of the Jun leucine zipper when all the a positions are occupied by leucine and isoleucine residues. However the effect of a given residue depends strongly on the heptad where it has been inserted. For example, Val is more penalizing than Phe or Met if inserted in the a position of the second heptad of the Fos zipper. Most likely the a residue in this heptad modulates the interhelical repulsion between the two glutamic acid side chains in positions gand e(21) . Finally, we show that the most efficient homoassociating Fos zipper variants are also improved in heteroassociation with the wild-type Jun zipper. For most of the hydrophobic Fos zipper variants, one observes a correlation between homoassociation and heteroassociation such that roughly half of the additional free energy of homoassociation is conserved upon interaction with the wild-type Jun zipper.


MATERIALS AND METHODS

Bacterial Strains and Plasmids

Escherichia coli strains JL806 (lexA71::Tn5(Delta(lacIPOZYA)169 sulA11(recAop-lacZ cI ind)/F`::Tn3 lacI^qlacZDeltaM15::Tn9), JL1436 (as JL806 but sulA::lacZ cI ind and Tn9 instead of Tn3) and the plasmid pJLW70 bearing the lexA gene were kindly provided by John Little (31, 32) .

Plasmid pMS404 bearing the wild-type LexA-c-Fos zipper fusion was constructed by insertion of a BstEII restriction site into the XmnI site of pJWL70 using a short oligonucleotide. The C-terminal domain of LexA was deleted by BstEII/PstI digestion and the remaining N-terminal domain (residues 1-87) was fused to the Fos zipper. The DNA coding for this peptide (see Fig. 1A) was chemically synthesized and fused to the N-terminal part of lexA via the BstEII site, which contributes a valine in position 88.


Figure 1: Amino acid and nucleotide sequence of the Fos leucine zipper (A) and of the Jun leucine zipper (B) fused to the LexA DNA binding domain (amino acids 1-87). The nucleotide sequence has been chosen such that rare codons in E. coli are avoided and that several unique enzyme cleavage sites are present in the zipper coding sequences to allow for recombination of mutated subfragments.



Plasmid pDP107 bearing the LexA-c-Jun zipper fusion was obtained by insertion of a XhoI restriction site within the XmnI site of pJWL70. The C-terminal part of LexA was deleted by a XhoI/PstI digestion, and the remaining N-terminal domain was fused to the c-Jun zipper sequence shown in Fig. 1B. The XhoI site contributes a serine residue in position 88.

Mutagenesis of the aPositions of the c-Fos Zipper

Hydrophobic amino acids were introduced within the a positions of the c-Fos leucine zipper using two degenerated synthetic oligonucleotides. One oligonucleotide corresponds to the coding strand and the second to the noncoding strand of the c-Fos zipper sequence in order to allow for double strand DNA synthesis by annealing and extending the complementary 3` ends of the two oligos. The coding strand contains an AgeI restriction site close to the 5` end, the noncoding strand bears a XhoI restriction site (see Fig. 1A). Degenerated codons were introduced at the five a positions (using NTN or NAN for the oligonucleotides corresponding, respectively, to the coding or the complementary strand) which allows to introduce selectively the hydrophobic residues Leu, Ile, Val, Phe, or Met. The two oligonucleotide families (overlapping by 12 base pairs) were annealed, and the 3` ends were extended with T7 polymerase essentially as described(33) . A collection of c-Fos zipper variants was constructed by cloning AgeI-XhoI fragments into a similarly digested pMS404 plasmid resulting in a set of plasmids called pMS500. Recombinant DNA was isolated and the LexA-c-Fos variants were identified by sequencing the entire gene, including the promoter sequence.

The single mutations in an all-leucine context (Fig. 4) were obtained either by the insertion of a small synthetic oligonucleotide cassette into the gene of the randomly obtained mutant protein (Fos) or via the recombination of several randomly obtained variants shown in Table 1(series pMS500) using the appropriate restriction enzymes.


Figure 4: Positional effects of single mutations (Ile, Val, Phe, or Met) in the context of an all-leucine hydrophobic interface. The repression of the lacZ gene (strain JL1436, 10M IPTG) conferred by LexA-Fos zipper proteins varies according to the a position, where the mutated residue has been incorporated. The penalty index p of each mutant protein relative to the parental Fos zipper (see ) is given in brackets behind the number of the experimentally determined beta-galactosidase units (black bars). The gray bars are adjusted penalty values obtained upon optimization of the fits of log(K) versus log(K) plots. For the LLMLL and LLLML variants such an adjustment was not possible because the combinatorial mutant set (Table 1) contains no methionine in positions a(3) and a(4).





In Vivo Measurement of the Association Capacities of the Mutant Proteins

The homo- and heteroassociation capacities of the different fusion proteins were determined in vivo as the repressor activity that can be conferred to the DNA binding domain of the LexA protein. The repressor activity is determined as the amount of beta-galactosidase units that can be measured in appropriate E. coli indicator strains (JL1436 or JL806). Overnight cultures, grown in the absence or in the presence of various concentrations of IPTG (^1)were diluted 100-fold in Luria broth containing the same IPTG concentration. Assays and units were as described(34) . The homoassociation capacity of the mutant proteins was measured using the pMS500 pUC like plasmids. In these constructs, the expression of the mutant proteins is under the control of a lacUV5 promoter.

For the measurement of the heteroassociation capacity, a two-plasmid system was used allowing for a low expression of the proteins in order to minimize or to prevent the formation of homodimers. The expression of the lexA-c-junZIP gene was decreased by the introduction of a mutation within the ribosome binding site. This mutation (REG9) has been shown to reduce the level of LexA expression about 4-fold (35) and was introduced by an exchange of the ClaI/MluI restriction fragment of pDP107 (see above) for the ClaI/MluI restriction fragment of REG9 giving rise to plasmid pDP204. The lexA-c-junZIP gene was further transferred to a plasmid with lower copy number (pACYC184) which enables it to coexist with vectors that carry the colE1 origin of replication. The ClaI/DraI restriction fragment of pDP204 (750 base pairs) was fused to the pACYC184 vector after HincII/ClaI digestion (2550 base pairs), giving rise to plasmid pDP301.

The expression of the lexA-c-fosZIP wild-type and mutant genes was also decreased by exchanging the ClaI/MluI restriction fragment of pMS404 and of 15 candidates of the pMS500 series for the ClaI/MluI restriction fragment of REG9 giving rise to pMS604 and to the pMS600 set of plasmids for the wild-type and the mutant proteins, respectively.

Assay of the Steady-state Level of the LexA-Fos Zipper Fusion Proteins

Crude extracts were prepared from the E. coli strain JL1436 in the presence of 5 times 10M IPTG according to(35) . Lysates were subjected to electrophoresis in 12.5% SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride Immobilon-P membrane. The fusion proteins were detected with purified rabbit antibodies directed against the N-terminal domain of LexA using an enhanced chemiluminescence detection system (Amersham ECL, RPN2106), preflashed films for detection, and a Shimadzu CS-9000 densitometer for quantification.

Construction and Purification of the Full-length Fos Mutant Proteins

The rat c-fos gene harboring six additional histidine codons to allow for nickel affinity chromatography (36) was excised from pDSFos using SphI and BamHI restriction and subcloned into a SphI/BamHI-digested M13mp18 vector. To allow for easy leucine zipper subcloning, a silent EcoRI site was introduced after the fifth leucine of the Fos zipper using site-directed mutagenesis (Amersham Sculptor kit) with the following oligonucleotide: 5`-AGAAAAGCTGGAATTCATCCTGGCGG. In a second round of mutagenesis, the Thr in position awas changed to leucine using the oligonucleotide: 5`-GAACTGCTGGACACC. The following variants were obtained after subcloning into the parental pDS vector, pDSFos(Eco), giving rise to a His-tagged Fos protein with a wild-type leucine zipper sequence, pDSFos(Eco) and pDSFos(Eco), harboring the wild-type Thr residue in position aand, respectively, four isoleucine or leucine residues in positions a, a, a, and aand finally pDSFos(Eco) harboring leucine residues in all five apositions.

The Fos proteins (385 amino acids) were purified by nickel affinity chromatography in the presence of guanidine hydrochloride(36) .

Electrophoretic Mobility Shift Assays

10-µl samples of protein/DNA mixtures, containing 1.15 times 10M of Fos protein and about 5 times 10M of a 21-base pair DNA duplex harboring an AP-1 binding site, were incubated in a buffer containing 50 mM Tris-HCl, pH 8.0, 5 mM MgCl(2), 5 mM DTT, 1 mM EDTA, 10% glycerol, 0.1% Nonidet P-40, 2 µg of bovine serum albumin, and 0.1 µg of poly(dI-dC) and subsequently run on a 4% polyacrylamide gel (with 1:37 bisacrylamide). The AP-1 oligonucleotide was as follows.


RESULTS

Association of Jun and Fos Leucine Zippers Probed by Repressor Activity Conferred to the LexA DNA Binding Domain

The gene fragments coding for the Fos or the Jun leucine zippers have been assembled from synthetic oligonucleotides (see Fig. 1) and were fused to the LexA DNA binding domain (LexA-DBD) as described under ``Materials and Methods.'' The LexA part corresponds to residues 1-87 of the E. coli LexA protein(37) . The Fos leucine zipper comprises residues 162-200 of c-Fos, and the Jun leucine zipper encompasses residues 277-315 of the human c-Jun protein. The fusion proteins are referred to as LexA-c-FosZIP and LexA-c-JunZIP, respectively. The expression of both proteins is driven by the same IPTG-inducible promoter (lacUV5) and, for this part of the results, both are cloned into identical expression vectors.

The ability of these domains to associate is tested as their capacity to restore the repressor activity of the truncated LexA protein (devoid of its own dimerization domain) in an in vivo test using appropriate E. coli indicator strains(32) , one of which is schematized in Fig. 2A. The JL1436 host strain is deficient in the chromosomal lexA gene and bears a lacZ gene driven by the LexA-dependent sulA promoter, whereas in the JL806 strain, the lacZ gene is under the control of the stronger (also LexA-regulated) recA promoter. The repressor activity of the fusion protein is determined as the residual beta-galactosidase activity that can be measured at different levels of expression of the proteins obtained upon varying the IPTG concentration. Characteristic repression curves are shown in Fig. 2B. In the absence of repression (expression vector alone), we measured about 4200 beta-galactosidase units regardless of the IPTG concentration. When the truncated form of LexA (DBD alone) was expressed, a significant repression of the lacZ gene was observed only upon overproduction of the protein, reaching about 2300 beta-galactosidase units at 10M IPTG. With the entire LexA protein, the lacZ gene is nearly completely turned off even in the absence of IPTG. In the absence of IPTG the c-Jun zipper is able to restore the DNA binding capacity of the truncated LexA protein at least partially, whereas the c-Fos zipper does not. Upon overproduction of the fusion protein, a level of repression identical to that obtained with LexA was reached with the LexA-c-JunZIP protein, whereas with its c-FosZIP counterpart the repression was the same as with the LexA DNA binding domain alone. These results are in agreement with previous results showing that (unlike the Jun zipper) the Fos zipper is unable to promote homodimerization.


Figure 2: A, schematic representation of the JL1436 E. coli indicator strain. This strain harbors a single copy of the lacZ gene under the control of the promoter/operator region of the sulA gene which is under the control of the LexA repressor. The strain is deficient in the chromosomal lexA gene and transcription of the lacZ gene will be repressed only, if the LexA deficiency is complemented by a plamid producing a functional LexA-leucine zipper fusion protein. The production of these fusion proteins is further regulated by the E. colilac repressor (produced from an F` factor) and is thus IPTG inducible. Strain JL806 is essentially identical except that the lacZ gene is under the control of the recA promoter/operator region. B, representative repression curves as a function of IPTG concentration, using strain JL1436. The unrepressed state (⊡, pBR322) gives rise to about 4200 beta-galactosidase units. Transformation with a plasmid coding either for the LexA DNA binding domain alone (up triangle) or for LexA-c-FosZip (bullet) gives rise to the same degree of repression at elevated IPTG concentration. Transformation with a plasmid coding for a LexA-c-Fos (circle) fusion protein (i.e. all a positions of the Fos zipper are replaced by leucine residues) gives rise to a degree of repression similar to a LexA-c-JunZip protein (). The intact wild-type LexA protein (times) remains the strongest repressor at least at low IPTG concentration.



Construction of Hydrophobic Combinatorial Fos Zipper Variants

In order to introduce randomly hydrophobic residues within the five a positions of the Fos leucine zipper, we used two complementary and partially overlapping synthetic oligonucleotides spanning the zipper part of the LexA-c-FosZIP protein. The codons corresponding to each a position of the five heptads were degenerated at the first and the third base of the codon, i.e. NTN or NAN for the coding strand and the complementary strand, respectively. As described earlier(29) , this approach allows the random introduction of Leu, Ile, Val, Phe, or Met. After annealing and synthesis of the complementary strand, the mutated c-Fos zippers were ligated to the LexA DNA binding domain. The recombinant plasmids were isolated, and the fusion genes were entirely sequenced in order to check for additional mutations within the lexA part or the regulatory sequence. A total of 250 mutants was sequenced giving rise to 57 different Fos zipper variants. The other mutants represent duplicates (58) and variants (135) harboring additional mutations.

Indicator Strain JL1436 versus JL806

The 57 different LexA-c-FosZIP mutant proteins were characterized for their homoassociation capacities within the JL1436 and JL806 indicator strains. The difference between the two strains resides both in the strength of the promoter and the operator in front of the lacZ gene. The recA promoter (JL806) is stronger than the sulA promoter (JL1436), giving rise to 12,000 instead of 4200 beta-galactosidase units in the unrepressed state. Fig. 3shows that transcriptional repression by the different LexA-c-FosZIP variants in the two indicator strains is reasonably well correlated (R^2 = 0.9). In the following most of the experiments were done with the JL1436 strain, because in this strain, the useful repression range is broader for two reasons. First, the LexA-DBD alone allows already for a repression of 80% at 10M IPTG in JL806, but only of 45% in JL1436. Second, it is virtually impossible to repress the recA promoter completely even with very high amounts of LexA wild-type repressor.


Figure 3: Correlation between the repression of the lacZ gene in the two indicator strains JL1436 (sulA promoter) and JL806 (recA promoter) at 10M IPTG: hydrophobic LexA-Fos zipper variants (box) harboring only Leu, Ile, Val, Phe, or Met in position a (see Table 1), LexA-c-FosZip (), LexA DNA binding domain (bullet).



Fos Zipper Mutants with Hydrophobic Residues in the Five aPositions

In Table 1each mutant protein is identified according to the amino acid found in the five successive a positions, with TTKIK being the wild-type amino acids. For each protein, the beta-galactosidase units given are those measured at 10M IPTG. Except one (VVFFV), all the other combinatorial mutant proteins increase the repressor capacity of the truncated LexA protein, although to various extents. As shown previously in the case of the Jun leucine zipper (28) , Fos zipper variants with an improved hydrophobic interface can thus efficiently replace the LexA dimerization domain.

Given that for all the fusion proteins the LexA-DBD is unchanged, the increased repressor activity is likely to reflect the efficiency with which the different leucine zippers are able to associate, provided that the proteins are produced in similar amounts. Therefore for each mutant protein, the relative in vivo amount was measured (at least in duplicate) by an immunoblot analysis using antibodies against the LexA part of the chimeric proteins as described under ``Materials and Methods.'' The intracellular steady-state level of all the mutant proteins were found similar or identical to the parental LexA-c-FosZIP protein.

Table 1shows the collection of Fos zipper variants classified according to the amount of beta-galactosidase units measured in strain JL1436. The smaller the number of units, the tighter the transcription of the indicator gene is repressed by the LexA-Fos zipper fusion proteins. Under these conditions the LexA-DBD alone and the LexA-Fos wild-type zipper fusion (TTKIK in Table 1) give rise to about 2300 units. The most strongly improved variants contain mostly Ile or Leu side chains in the five a positions, giving rise to only 28 units in the case of five Ile residues, i.e. an 82-fold improvement in repressor efficiency as compared with the Fos wild-type zipper fusion protein.

The different hydrophobic side chains are not equivalent in promoting Fos zipper association. Phenylalanine and methionine residues, especially, are mostly found within weakly improved Fos zipper variants, suggesting that these amino acids are not as efficient as leucine, isoleucine, or valine side chains (see Table 1). In order to establish this hierarchy more precisely Fos zipper variants were created bearing the same hydrophobic amino acid within the five a positions (Table 1, bottom right). As anticipated from the random collection we find the following hierarchy: Ile geq Leu > Val > Phe > Met. This order may be understood in terms of the hydrophobicity of these side chains (see Discussion).

The Different aPositions Are Not Equivalent

The random mutations in Table 1suggest further that the five a positions of the Fos zipper are not equivalent. For example a comparison of the mutant proteins LIVLL (52 beta-galactosidase units) with the nearly identical LVILL (198 beta-galactosidase units) suggests that a valine residue in the second heptad may be less favorable than in the third heptade. To investigate the positional effects of a given type of residue more systematically, a set of 20 single mutations has been created in the context of an all-leucine hydrophobic interface. These variants contain a single Ile, Val, Phe, or Met in any of the five heptades.

As shown in Fig. 4, Ile is at least as efficient as Leu, since in none of the five a positions it influences the dimerization capacity of the Fos host zipper. This is not the case for the other three amino acids, which all, to various degrees, diminish the repressor activity of the LexA-Fos protein.

As suspected from the random combinatorial variants, we observe pronounced position-dependent effects. The a position of the first heptad is less sensitive to mutations than the four others, since only minor changes are observed even when ais occupied by the relatively unfavorable residues Phe and Met. This may be understood by the fact that ais not packed between two leucine layers as the other aresidues (see Fig. 8B).


Figure 8: Correlation between the square root of the relative dissociation constant for homoassociation (deduced from the beta-galactosidase units in Table 1) and the relative dissociation constant for heteroassociation (deduced from the beta-galactosidase units in Fig. 7). bullet represents the wild-type Fos zipper, box the outsiders VMIIM and VVIFV, and the other variants from Fig. 7.




Figure 7: Repression of the lacZ gene (JL1436, no IPTG) due to heteroassociation of LexA-Fos zipper variants with the LexA-Jun zipper protein. The expression of the fusion proteins was chosen such that a single fusion protein (gray bars) gives rise to essentially no repression, whereas coexpression of a Fos zipper variant with the Jun wild-type zipper fusion protein (black bars) recovers repression. The unrepressed state (4200 units ± 10%) is indicated by two dashed lines.



In position athe hierarchy (Ile geqLeu >Val >Phe >Met) mentioned above is inverted, i.e.in this position Val is less favorable than Phe and Met. This atypical behavior is most likely linked to the two glutamic acid residues in positions gand e`, which tend to pack over the aside chain as discussed below. In aand a, Val is well accepted, whereas in ait is again unfavorable, albeit less than in a. Phe and Met are always unfavorable as compared with the parental Foszipper, albeit with some modulations. Phe is particularly unfavorable in aand Met in a. Hu et al.(29) also observed positional effects in aand dpositions in a study of the GCN4 leucine zipper.

The Effect of Multiple Mutations Is Essentially Additive

The interpretation of the in vivo repression data in terms of dimerization efficiency is potentially complicated by the fact that the nature of the a (and d) position residues may modulate the number of strands within an alpha-helical coiled-coil. Ile in position a and Leu in d favors a classical two-stranded geometry(38) , and Ile in both a and d leads to the formation of a triple-stranded coiled-coil (39) , whereas Leu in a and Ile in d leads to the formation of a tetrameric coiled-coil(38) .

No crystal structure is available for a leucine zipper harboring Leu in both a and d. Zhu et al.(40) have shown by size exclusion chromatography for a coiled-coil with Leu residues at positions a and d that in the reduced form these peptides adopt a dimeric (two-stranded) coiled-coil structure. Harbury et al.(38) showed (also by size exclusion chromatography) that a GCN4 leucine zipper variant harboring Leu at positions a and d apparently forms trimers in the reduced state, but dimers and higher oligomers in the oxidized (disulfide-bonded) state. This behavior is not easily understood in comparison with an isoleucine zipper (Ile in a and d), which forms also trimers in the reduced state, but only hexamers in the oxidized state(38) .

To address the question if a Fos leucine zipper variant harboring Leu at positions a and d would have a strong tendency to form higher order species, we have constructed and purified Fos protein variants harboring mostly or exclusively leucine or isoleucine in position a (for details see ``Materials and Methods''). Fig. 5shows that contrary to the Fos protein harboring a wild-type leucine zipper (lane 4), the Fos variants Fos (lane 1), Fos (lane 2), and Fos (lane 3), harboring a hydrophobic interface, are able to interact with a 21-base pairs AP-1 binding site (TGACTCA).


Figure 5: Electrophoretic mobility shift assay showing that full-length Fos protein variants harboring a hydrophobic leucine zipper interface are able to interact with a 21-mbase pair DNA duplex harboring an AP-1 binding site (TGACTCA): Fos (lane 1), Fos (lane 2), Fos (lane 3); whereas a Fos protein harboring the wild-type leucine zipper (lane 4) does not bind to this oligonucleotide. The protein concentration was 1.15 times 10M and the DNA concentration about 5 times 10M. For the assay conditions, see ``Materials and Methods.''



Relevant for the question we ask here is the finding that the gel mobility of the two complexes with a leucine interface (Fos and Fos) is the same as that with an isoleucine interface (Fos). This behavior strongly suggests that the dominant species for both proteins should be a protein dimer, since Ile in position a (with Leu in position d) is known to favor the formation of leucine zipper dimers as shown by equilibrium ultracentrifugation(38) . Gel retardation is in fact very sensitive to the oligomeric state of a protein bound to a small DNA duplex. For example the migration of a dimeric variant of lac repressor (a protein of 360 amino acids, i.e. of similar size as the Fos protein) is different from that of the tetrameric repressor in a complex with a 40-base pair operator fragment (41) .

For the following reason the data in Table 1also argue in favor of the dimer as the dominant oligomerization state of the LexA-c-FosZip variants in vivo; if the repressor effect of the multiple combinatorial mutations in Table 1could be understood in terms of the sum of the corresponding single mutations (placed in a Leu hydrophobic interface, see Fig. 4), we may argue that multiple oligomerization states should not play a major role in the establishment of the repressor efficiency (Table 1), since (contrary to the single mutations) the combinatorial mutations are not particularly leucine-rich, but contain also Ile (a dimer-driving residue in position a), as well as Val, Met, and Phe.

To test this hypothesis we have proceeded in the following four steps. 1) The beta-galactosidase units of the 20 different single mutations were normalized to the all-leucine zipper (Fos) to obtain a relative dissociation constant K = K/K(L) for each single mutation in this context. In the case of the single mutations these relative dissociation constants may be considered as a penalty index p, associated with the mutation of a given leucine to isoleucine, valine, phenylalanine, or methionine.

Relative Dissociation Constants May be Determined from the beta-Galactosidase Units(42

According to the following equation,

where is the fractional occupancy of the sulA operator by one of the LexA-mutant Fos zipper hybrid proteins, (L) the fractional occupancy by the LexA-c-Fos protein, Z the number of beta-galactosidase units of a single mutant hybrid protein (see Fig. 4), Z the corresponding value for the Fos hybrid protein (40 units), and Z(D) the number of beta-galactosidase units of the unrepressed state (4183 units).

2) For each of the multiple mutations in Table 1we calculated a theoretical relative dissociation constant with respect to the Fos hybrid protein,

as the product of the corresponding K values, since if the effects of the single mutations are additive in terms of loss in free energy, the corresponding relative equilibrium constants have to be multiplied.

3) For each of the multiple mutations in Table 1we calculated further an experimental relative dissociation K constant with respect to the Fos hybrid protein according to the following equation.

4) The theoretical relative dissociation constants were plotted as a function of the experimental relative dissociation constants on a log-log scale to guarantee an even distribution of the data points along the 2 orders of magnitude covered by the experimental relative dissociation constants. These log (K) versus log (K) plots were finally submitted to a linear least squares fitting procedure.

Fig. 6shows such a log-log plot of the theoretical Kversus the experimental K value. A linear least squares fit of these data gives the following straight line: logK = 1.07 logK - 0.15, with R^2 = 0.83.


Figure 6: Correlation between the theoretical relative dissociation constant (see ) and the experimental relative dissociation constant K calculated according to . For the calculation of K the penalty values of the five isoleucine variants were set to unity, since the deviation from the parental Fos mutant zipper are within experimental error (see Fig. 4).



The slope (1.07) is close to the theoretically expected value of 1, and the intersection of the y axis is close to zero. This behavior shows that the experimental data scatter almost evenly around the expected correlation line. On average the multiple leucine zipper mutations may thus be explained by the additive effect of the individual mutations in the context of an all-leucine hydrophobic interface. This behavior suggests indeed that the repressor efficiency of the Lex A-Fos zipper fusion proteins in vivo is dominated by a single oligomerization state. This dominant state is most likely a Fos zipper dimer, since those leucine zippers which are rich in Ile in position a have been shown to form exclusively dimers(38) .

The correlation between K and K can be further improved by adjusting the K values. Adjustment was achieved by a stepwise variation of each K value and subsequent recalculation of the correlation between K and K. Using an adjusted K data set (see gray bars in Fig. 4), the correlation (not shown) can be increased to R^2 = 0.90. Major improvement is achieved upon increasing the K values of MLLLL and LFLLL and diminishing that of LLLFL. Most of the other K values (especially those of the valine single mutations) are close to their optimal value in terms of their fitting efficiency.

Improving the Homoassociation Capacity of a Fos Leucine Zipper Can Improve Its Heteroassociation Capacity with a Jun Leucine Zipper

It has been suggested that one of the decisive parameters for a leucine zipper to heterodimerize rather than to homodimerize resides in its incapacity or in its poor capacity to homodimerize(20) . Since the Fos zipper variants shown in Table 1have acquired the capacity to associate forming most likely dimeric coiled-coil structures, it was interesting to study their heteroassociation capacity with a Jun leucine zipper using the LexA-mediated repression system described above. In a first step it was necessary to reduce the expression of the LexA-zipper fusion proteins such that transcriptional repression due to homoassociation was negligible thus allowing the measurement of heteroassociation only. This was achieved by using a down-mutation of the lacUV5 promoter (35) for both LexA-c-FosZip and LexA-c-JunZip. LexA-c-JunZip was additionally subcloned into a compatible plasmid having a lower copy number and finally, transcriptional repression was measured in the absence of IPTG to assure maximal repression of the fusion genes by the lac repressor (see Fig. 2).

Fig. 7shows the results obtained with a representative subset of the combinatorial Fos zipper variants of Table 1. Under these conditions the LexA-c-JunZip protein is not able to repress the lacZ gene in JL1436 and also most of the LexA-c-FosZip variants fall into the range of the unrepressed state, i.e. 4183 units ± 10%. Only two c-FosZip variants (LLLLL and ILLLL) show a slight residual repression due to homodimerization.

The coexpression of LexA-c-JunZip and LexA-c-FosZip leads to the recovery of transcriptional repression. The two wild-type zipper sequences together produce an about 3-fold repression, whereas coexpression of LexA-c-JunZip with the Fos variant leads to a 10-fold repression under these conditions. Interestingly the four Fos zipper variants in Fig. 7being most improved in heteroassociation with the Jun zipper (i.e. LLLLL, IILII, LIVVL, and ILLLL) are also the most efficient homoassociating species of this set of Fos zipper variants.

Improved Homoassociation and Heteroassociation Are Correlated

We asked further if it is possible to establish a quantitative correlation between improved heteroassociation and improved homoassociation. One may expect that the change in free energy of a Fos zipper variant within a heterodimer with the Jun wild-type zipper should be about half of the change in free energy of the same variant within the corresponding homodimer, because in the heterodimer only one of the partners is mutated. If this holds true, one might expect a linear relationship between the square root of the K values for homodimerization (derived from ) and the corresponding K values for heterodimerization (derived from the same equation, using this time the heterodimerization of the Fos hybrid protein for normalization, i.e.Z(L) = 406 units).

Fig. 8shows that 13 of the 15 Fos zipper variants are indeed reasonably close to a straight line in such a plot (i.e. (K)^0.5 = 0.67 K + 0.29, R^2 = 0.83). In principle this straight line should go through zero and have a slope of 1. A minor adjustment of the exponent (i.e. 0.56 instead of 0.5 in the case of a square root) leads indeed to a fit which fulfills these two requirements better (i.e. (K) = 0.85 K + 0.01, R^2 = 0.83).

Two Fos zipper variants (especially VVIFV, but also VMIIM) are fairly far apart from this hetero- versus homoassociation correlation. Both variants heteroassociate more efficiently than they should do in view of their homoassociation capacity. The same holds true even more strikingly for the Fos wild-type zipper (see Fig. 8, full circle), which has the by far weakest homoassociation capacity of all the variants shown in Fig. 7, but an intermediate heteroassociation capacity as compared with the mutant Fos zippers. These data suggest that one or several of the wild-type Fos zipper amino acids in position a (Thr, Thr, Lys, Ile, Lys) should be favorable for the formation of heterodimers with the Jun zipper. The two lysine residues (Lys) in positions aand aare likely candidates, since the crystal structure of the Fos/Jun DNA complex (19) shows that these residues form hydrogen bonds with two glutamine side chains in positions gand gof the Jun zipper. The conservation of the five aresidues Thr, Thr, Lys, Ile, Lys in c-Fos, Fos B, Fra1, and Fra2 (1) would be consistent with a possible role of aresidues in Fos/Jun zipper assembly.


DISCUSSION

The Fos and Jun leucine zippers have been extensively studied in the past using both genetic and biophysical approaches. Most of the mutagenesis work has been focused on loss-of-function mutations, which established the importance of the conserved leucine side chains for dimerization and DNA binding using either single or multiple hydrophobic (valine, phenylalanine) or helix breaking substituents(22, 43, 44, 45) .

A gain-of-function mutation has been described in the case of the Fos zipper (46) where the replacement of Glu-168 (which is the g residue of the first heptad) by lysine enables a truncated Fos protein to interact with DNA as a homodimer. For technical reasons the interaction of this variant with Jun could not be evaluated. It seems, however, unlikely that Fos E168K would be also improved in Jun binding, since in the Fos-Jun heterodimer this mutation would juxtapose two lysine residues.

Homoassociation of Improved Hydrophobic Fos Zipper Variants

Here we describe Fos zipper variants with a modified hydrophobic interface, which are improved both in homoassociation and in heteroassociation with the Jun zipper as inferred from the recovery of repressor activity in the context of LexA-Fos zipper fusion proteins. We may conclude from these studies that the homoassociation of the wild-type Fos zipper is not only limited by the repulsion of the negatively charged residues in positions g and e(20, 21, 46, 47) but also by the nonhydrophobic nature of the a positions. Just as the Fos E168K variant (46) is able to overcome the handicap of the poor hydrophobic interface, the hydrophobic Fos zipper variants described here are able to overcome the repulsion of the negatively charged g and e residues. The most efficient Fos zipper variants are those harboring only Ile and Leu in the a positions (see Table 1). One may wonder if such a rather uniform hydrophobic interface is sufficiently specific to hold the two leucine zippers in register. Apparently slippage is not a major problem for these variants, since the full-length Fos proteins harboring leucine or isoleucine in position a bind specifically to DNA. Slippage would almost certainly prevent DNA binding, since the two basic domains would be out of frame.

The degree of improvement of these variants in homoassociation is, however, not uniform and depends both on the nature of the hydrophobic amino acid substituents and on the position within the Fos zipper. If one compares Fos zipper variants harboring only Leu or Ile or Val or Phe or Met in the five a positions, then the improvement may be correlated with the hydrophobicity of the side chains (see Fig. 9A). No correlation exists between homoassociation improvement and alpha-helix propensity of these residues, since all relevant helicity scales (compiled in (48) ) attribute a higher alpha-helix propensity to Met (the least efficient homoassociation-inducing residue in our experiments) as compared with Ile (the most efficient residue in our experiments).


Figure 9: A, correlation between the homoassociation capacity of Fos zipper variants harboring only either leucine (L), isoleucine (I), valine (V), phenylalanine (F), or methionine (M) in the five a positions with the hydrophobicity of these side chains as inferred from the free energy of transfer from cyclohexane to water(45) . B, representation of a Fos homodimer (with variable a positions) as two parallel helical cylinders as adapted from Hu et al.(29) . Coupling between g residues in one helix with e` residues in the opposite helix is indicated by white bars.



The hydrophobicity scale used for the plot in Fig. 9A is from Radzicka and Wolfenden (49) and corresponds to the free energies of transfer of the amino acid side chains from cyclohexane to water. On this scale Leu, Ile, Val, Phe, and Met are the five most hydrophobic amino acid side chains. The compilation of 61 leucine zipper-containing transcription factors (1) reveals that 24% of the a positions are occupied by Val, 21% by Asn, 11% by Leu, and 10% by Ile. The residues being on average most efficient in promoting Fos zipper homodimerization (Ile geq Leu > Val) are thus also beside the most common a residues in this leucine zipper compilation.

The correlation between hydrophobicity and repressor efficiency shown in Fig. 9A corresponds to five simultaneous mutations in position a. Since the five heptades of the Fos zipper are not identical these data correspond to an average over different local environments.

Residues in Position a Are Likely to Modulate g e` Coupling

However at least for Val, Phe, and Met the impact of a given residue type on the repressor efficiency depends strongly on the heptad, i.e. on the local environment of the different a residues (see Fig. 4). This is particularly striking for the a residue in heptade 2, where Val (233 units) is clearly more penalizing than Phe (70 units) and Met (139 units). This conclusion may be drawn both from single mutations in the context of an all-leucine interface and from the random mutations in Table 1.

What are the possible reasons for this inversion from the general Val > Phe > Met hierarchy to Phe > Met > Val in heptade 2? The most likely explanation is that in heptade 2 the a residue modulates the interhelical repulsion of the two glutamic acid side chains in positions gand e`(where the primed residue corresponds to the opposite helix, see Fig. 9B), since in the case of the Fos zipper mutants, the cavity into which an aresidue has to fit is the same for a, a, a, and a. In a parallel coiled-coil the packing of the side chains in the dimer interface is such that an aresidue is surrounded by four amino acid side chains of the opposite helix(50, 51) . In the Fos zipper an aresidue is always surrounded by a glutamic acid side chain (in position g`), two leucines (in d`and d`), and the opposite a`residue, which is conserved in the case of a homodimer (see Fig. 9B).

Whereas the cavity is the same (including the g residues), the e residues are variable from one heptade to the other. Host-guest studies have shown that electrostatic repulsion between glutamic acid residues in positions gand e` is particularly destabilizing for the host leucine zipper(21) . The atypically behaving aresidue lies just between these residues and may conceivably modulate the negative interaction between them. Valine might bring the two glutamic acid side chains close together and thus increase electrostatic repulsion. This hypothesis might also explain a puzzling result of Krylov et al.(52) who showed that the g` e`coupling energy for glutamine pairs is surprisingly favorable if packed over valine residues in position a. Physical model building based on the co-ordinates of the GCN4 leucine zipper (50) showed us that two glutamine residues (or two protonated glutamic acid side chains) in gand e`of the next heptade might be brought sufficiently close together to form potentially two interhelical hydrogen bonds. Independently of the precise molecular mechanism, our results strongly suggest that aresidues, especially valine, may modulate interhelical g` e`coupling.

It is worthwhile to notice that a mutation of valine in position aof the Jun leucine zipper to phenylalanine gave rise to a more active Jun protein(53) , whereas the substitution of other aresidues (Asn in a, Ala in a, and Val in a) with phenylalanine was penalizing. In the Jun leucine zipper, alies also between equally charged residues (two lysines in gand e`), which may also give rise to electrostatic repulsion, albeit with a smaller thermodynamic cost as in the case of two glutamic acids(21, 52) .

Heteroassociation of Improved Fos Zipper Variants with the Jun Leucine Zipper

A representative subset of hydrophobic Fos zipper variants has been further tested for heteroassociation with a wild-type Jun zipper under conditions where neither protein alone conferred significant transcriptional repression. The rule that emerges from a correlation between homo- and heteroassociation data is that for most tested variants (13 of 15) improved homoassociation is correlated with heteroassociation such that roughly half of the additional free energy of homoassociation may be imported into the Fos/Jun zipper complex (Fig. 8).

For these Fos zipper variants, one may conclude that the general stability rather than the specificity of Fos/Jun zipper assembly is affected. However we would not expect that these Fos zipper variants would be able to dimerize efficiently with any leucine zipper, since the major specificity determinants for Fos/Jun zipper assembly in positions e and g are conserved(20, 21) .

However there are a few interesting exceptions from this correlation. Especially the wild-type Fos zipper does not fit to this correlation. It has an intermediate heteroassociation activity in this assay, but confers no homoassociation at all, suggesting that the Fos wild-type a positions (TTKIK) are extremely unfavorable for homoassociation, but acceptable or favorable for an interaction with the Jun leucine zipper. Another exception from the correlation in Fig. 8is the VVIFV variant. This hydrophobic Fos zipper variant is also better in heteroassociation than expected from the homoassociation data. A possible reason for this behavior is that the underlined valine residues in positions aand aare destabilizing for a Fos zipper in the context of the equally charged glutamic acid residues in positions g, e, and g

Several canonical Fos zipper a variants (i.e. those for which homo- and heteroassociation are correlated) and the ``excess heteroassociation'' variants are currently under study for their transformation suppressor activity in ras-transformed fibroblasts.


FOOTNOTES

*
This work was supported by research grants from Rhone-Poulenc Rorer, from INSERM, from the Association de Recherche contre le Cancer, and from the Ligue Nationale contre le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-88-41-70-64; Fax: 33-88-60-22-18.

(^1)
The abbreviation used is: IPTG, isopropyl-beta-D-thiogalactopyranoside.


ACKNOWLEDGEMENTS

We thank Dr. J. W. Little (Tucson, AZ) and Dr. T. Curran (Nutley, NJ) for some of the strains and plasmids used in this work. Matthias John kindly established the drawing shown in Fig. 9B.


REFERENCES

  1. Hurst, C. H. (1994) Protein Profile 1,123-152 [Medline] [Order article via Infotrieve]
  2. Granger-Schnarr, M., Benusiglio, E., Schnarr, M., and Sassone-Corsi, P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,4236-4239 [Abstract]
  3. Busch, S. J., and Sassone-Corsi, P. (1990) Trends Genet. 6,36-40 [CrossRef][Medline] [Order article via Infotrieve]
  4. Angel, P., and Karin, M. (1991) Biochim. Biophys. Acta 1072,129-157 [CrossRef][Medline] [Order article via Infotrieve]
  5. Hai, T., and Curran, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,3720-3724 [Abstract]
  6. Hsu, W., Kerppola, T. K., Chen, P.-L., Curran, T., and Chen-Kiang, S. (1994) Mol. Cell. Biol. 14,268-276 [Abstract]
  7. Kataoka, K., Noda, M., and Nishizawa, M. (1994) Mol. Cell. Biol. 14,700-712 [Abstract]
  8. Kerppola, T. K., and Curran, T. (1994) Oncogene 9,675-684 [Medline] [Order article via Infotrieve]
  9. Stein, B., Baldwin, A. S., Jr., Ballard, D. W., Greene, W. C., Angel, P., and Herrlich, P. (1993) EMBO J. 12,3879-3891 [Abstract]
  10. Nolan, G. P. (1994) Cell 77,795-798 [Medline] [Order article via Infotrieve]
  11. Bengal, E., Ransone, L., Scharfmann, R., Dwarki, V. J., Tapscott, S. J., Weintraub, H., and Verma, I. M. (1992) Cell 68,507-519 [Medline] [Order article via Infotrieve]
  12. Blanar, M. A., and Rutter, W. J. (1992) Science 256,1014-1018 [Medline] [Order article via Infotrieve]
  13. Hsu, J.-C., Bravo, R., and Taub, R. (1992) Mol. Cell. Biol. 12,4654-4665 [Abstract]
  14. Kerppola, T., Luk, D., and Curran, T. (1993) Mol. Cell. Biol. 13,3782-3791 [Abstract]
  15. Benbrook, D. M., and Jones, N. C. (1990) Oncogene 5,295-302 [Medline] [Order article via Infotrieve]
  16. Ivashkiv, L. B., Liou, H.-C., Kara, C. J., Lamph, W. W., Verma, I. M., and Glimcher, L. H. (1990) Mol. Cell. Biol. 10,1609-1621 [Medline] [Order article via Infotrieve]
  17. Chatton, B., Bocco, J. L., Goetz, J., Gaire, M., Lutz, Y., and Kedinger, C. (1994) Oncogene 9,375-385 [Medline] [Order article via Infotrieve]
  18. O'Shea, E. K., Rutkowski, R., Stafford, W. F., and Kim, P. S. (1989) Science 245,646-648 [Medline] [Order article via Infotrieve]
  19. Glover, J. N. M., and Harrison, S. C. (1995) Nature 373,257-261 [CrossRef][Medline] [Order article via Infotrieve]
  20. O'Shea, E. K., Rutkowski, R., and Kim, P. S. (1992) Cell 68,699-708 [Medline] [Order article via Infotrieve]
  21. John, M., Briand, J. P., Granger-Schnarr, M., and Schnarr, M. (1994) J. Biol. Chem. 269,16247-16253 [Abstract/Free Full Text]
  22. Ransone, L. J., Visvader, J., Sassone-Corsi, P., and Verma, I. M. (1989) Genes & Dev. 3,770-781
  23. Hurstel, S., Granger-Schnarr, M., Daune, M., and Schnarr, M. (1986) EMBO J. 5,793-798 [Abstract]
  24. Fogh, R. H., Ottleben, G., Rüterjans, H., Schnarr, M., Boelens, R., and Kaptein, R. (1994) EMBO J. 13,3936-3944 [Abstract]
  25. Schnarr, M., Pouyet, J., Granger-Schnarr, M., and Daune, M. (1985) Biochemistry 24,2812-2818 [Medline] [Order article via Infotrieve]
  26. Schnarr, M., Granger-Schnarr, M., Hurstel, S., and Pouyet, J. (1988) FEBS Lett. 234,56-60 [CrossRef][Medline] [Order article via Infotrieve]
  27. Kim, B., and Little, J. W. (1992) Science 255,203-206 [Medline] [Order article via Infotrieve]
  28. Schmidt-Dörr, T., Oertel-Buchheit, P., Pernelle, C., Bracco, L., Schnarr, M., and Granger-Schnarr, M. (1991) Biochemistry 30,9657-9664 [Medline] [Order article via Infotrieve]
  29. Hu, C. H., O'Shea, E. K., Kim, P. S., and Sauer, K. T. (1990) Science 250,1400-1403 [Medline] [Order article via Infotrieve]
  30. Pu, W. T., and Struhl, K. (1993) Nucleic Acids Res. 21,4348-4355 [Abstract]
  31. Little, J. W., and Hill, S. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,2301-2305 [Abstract]
  32. Lin, L. L., and Little, J. W. (1989) J. Mol. Biol. 210,439-452 [Medline] [Order article via Infotrieve]
  33. Oliphant, A. R., Nussbaum, A. L., and Struhl, K. (1986) Gene (Amst.) 44,177-183 [CrossRef][Medline] [Order article via Infotrieve]
  34. Miller, J. H. (1972) Experiments in Molecular Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  35. Oertel-Buchheit, P., Lamerichs, R. M. J. N., Schnarr, M., and Granger-Schnarr, M. (1990) Mol. & Gen. Genet. 223,40-48
  36. Abate, C., Luk, D., and Curran, T. (1990) Cell Growth Differ. 1,455-462 [Abstract]
  37. Markham, B. E., Little, J. W., and Mount, D. W. (1981) Nucleic Acids Res. 9,4149-4161 [Abstract]
  38. Harbury, P. B., Zhang, T., Kim, P. S., and Alber, T. (1993) Science 262,1401-1407 [Medline] [Order article via Infotrieve]
  39. Harbury, P. B., Kim, P. S., and Alber, T. (1994) Nature 371,80-83 [CrossRef][Medline] [Order article via Infotrieve]
  40. Zhu, B. Y., Zhou, N. E., Kay, C. M., and Hodges, R. S. (1993) Protein Sci. 2,383-394 [Abstract/Free Full Text]
  41. Li, L., and Matthews, K. S. (1995) J. Biol. Chem. 270,10640-10649 [Abstract/Free Full Text]
  42. Oertel-Buchheit, P., Porte, D., Schnarr, M., and Granger-Schnarr, M. (1992) J. Mol. Biol. 225,609-620 [Medline] [Order article via Infotrieve]
  43. Gentz, R., Rauscher, F. J., Abate, C., and Curran, T. (1989) Science 243,1695-1699 [Medline] [Order article via Infotrieve]
  44. Schuermann, M., Neuberg, M., Hunter, J. B., Jenuwein, T., Ryseck, R. P., Bravo, R., and Müller, R. (1989) Cell 56,507-516 [Medline] [Order article via Infotrieve]
  45. Turner, R., and Tijan, R. (1989) Science 243,1689-1694 [Medline] [Order article via Infotrieve]
  46. Nicklin, M. J. H., and Casari, G. (1991) Oncogene 6,173-179 [Medline] [Order article via Infotrieve]
  47. Schuermann, M., Hunter, J. B., Hennig, G., and Müller, R. (1991) Nucleic Acids Res. 19,739-746 [Abstract]
  48. Blaber, M., Zhang, X. J., and Matthews, B. W. (1993) Science 260,1637-1640 [Medline] [Order article via Infotrieve]
  49. Radzicka, A., and Wolfenden, R. (1988) Biochemistry 27,1664-1670
  50. O'Shea, E. K., Klemm, J. D., Kim, P. S., and Alber, T. (1991) Science 254,539-544 [Medline] [Order article via Infotrieve]
  51. Ellenberger, T. E., Brandl, C. J., Struhl, K., and Harrison, S. C. (1992) Cell 71,1223-1237 [Medline] [Order article via Infotrieve]
  52. Krylov, D., Mikhailenko, I., and Vinson, C. (1994) EMBO J. 13,2849-2861 [Abstract]
  53. Smeal, T., Angel, P., Meek, J., and Karin, M. (1989) Genes & Dev. 3,2091-2100

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.