chkYB-2 binds to several single-stranded motifs on the noncoding strand of the RSV LTR and acts as a potent activator of RSV LTR-driven transcription in avian fibroblasts. To localize the polypeptide region that is responsible for site-specific binding to single-stranded DNA, we performed deletion mutagenesis of YB-2 cDNA. Figure A is a line diagram of the mutants that were created by using standard recombinant DNA methods. Except for one nested deletion, YB-2 (Δ158-222), all constructs were either NH2-terminal or COOH-terminal deletions. The YB-2 (75-230) mutant has deletions at both termini.
FIG. 1 (A) Diagram of chkYB-2 mutant constructs. Wild-type (w/t) chkYB-2 (298 aa) is shown schematically. The filled-in region represents the highly conserved CSD. The various amino-terminal and carboxyl-terminal deletion mutants, and one internal deletion mutant (more ...)
Wild-type and mutant YB-2 polypeptides expressed in E. coli as MBP fusion proteins were column purified on amylose, and aliquots electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels (data not shown). In all cases, a protein of the expected size was obtained. Wild-type and mutant YB-2 proteins were examined for DNA binding activity by carrying out gel shift assays, using a radiolabeled single-stranded response element from the RSV LTR (LTR −106/−135 [Table ]), shown in this report to be the maximum affinity binding site of chkYB-2.
In chkYB-2, the CSD extends from amino acids (aa) 86 to 155 (Fig. B). The first construct that we made was YB-2 (Δ158-222). The internal deletion of 66 aa in the carboxyl-tail domain created a mutant with intact amino-terminal and CSDs. If the CSD is sufficient for DNA binding, this mutant polypeptide would be expected to bind DNA. As seen in Fig. (lane 9), no binding to DNA was observed in an in vitro DNA binding assay. DNA binding was tested under different salt conditions, using up to 2 μg of the protein prepared from several independent clones, as well as after cleavage of the MBP fusion with factor Xa (data not shown). No binding was detected under any of these conditions. There are two possible explanations for the total abrogation of DNA binding upon the deletion of these 66 aa. This 66-aa stretch is immediate to the carboxyl side of the carboxyl boundary (aa 155) of the CSD. Given the proximity to the well-structured CSD, this deletion could have resulted in a major functional deformity in the protein leading to loss of DNA binding. An alternative explanation for the nonfunctionality of this mutant in terms of DNA binding is that the deleted aa 158 to 222, or at least some of them, are part of the minimum DNA binding domain, which in chkYB-2 could extend beyond the strict confines of the conserved residues of the CSD.
FIG. 2 Localization of the domains responsible for DNA binding. chkYB-2 deletion mutants were expressed as MBP fusion proteins in E. coli and partially purified on amylose columns. The ability of these mutant polypeptides to bind DNA was assessed by carrying (more ...)
To map the carboxyl boundary of the DNA binding activity of chkYB-2, we constructed the YB-2 (1-169) and YB-2 (158-298) mutants and assayed their ability to bind DNA. YB-2 (158-298) expresses only the carboxyl-tail domain of YB-2, the entire CSD and amino terminus having been deleted. As seen in Fig. (lane 13), the YB-2 (158-298) mutant did not bind DNA. This result indicated that the carboxyl half of the protein cannot independently bind DNA. Although the residues in the 158–222 region are critical for DNA binding, they of themselves do not confer DNA binding activity in the absence of the CSD.
Mutant YB-2 (1-169) retains the CSD and, unlike YB-2 (Δ158-222), has 14 residues to the immediate carboxyl side of the CSD intact. As seen in Fig. (lane 5), this polypeptide bound DNA, albeit with a 10-fold-lower affinity than the wild-type YB-2 protein (lanes 3 and 4, containing factor Xa-cleaved and MBP fusion proteins, respectively). The partial restoration of DNA binding activity upon preservation of residues 156 to 169 supports our belief that the domain subserving DNA binding in YB-2 extends beyond the CSD. Construction of mutants with their carboxyl termini at different positions between aa 169 and 298 is currently under way. DNA binding assays with these proteins would help in defining precisely the carboxyl boundary of the polypeptide displaying full restoration of DNA binding activity.
We were curious to know if all of the 14 residues in the short carboxyl tail of YB-2 (1-169) were necessary for DNA binding activity or whether small truncations in this region would be tolerated. Progressive C-terminal deletions of YB-2 (1-169) yielded the mutants YB-2 (1-167) and YB-2 (1-162). Gel shift assays were carried out with these mutant polypeptides to determine their ability to bind DNA. As seen in Fig. (lanes 6 and 7), YB-2 (1-167) bound less avidly than YB-2 (1-169) and YB-2 (1-162) bound even less than YB-2 (1-167), showing that progressive deletions were deleterious to DNA binding. This effect was clearly seen when DNA binding was totally lost with the YB-2 (1-155) mutant (lane 8). The deletion in YB-2 (1-155) removes the entire carboxyl-tail domain of YB-2 up to the carboxyl boundary of the CSD. The total absence of DNA binding with the YB-2 (1-155) protein is similar to the behavior of the YB-2 (Δ158-222) mutant. Taken together, these results indicate that in chkYB-2, the CSD is necessary but not sufficient for DNA binding activity.
Interestingly, in gel shift assays, the DNA-protein complexes formed by the YB-2 (1-169), (1-167), and (1-162) mutants migrated more slowly than the complexes formed by the full-size protein. The YB-2 (1-162) protein in fact forms two complexes, one a faster-migrating complex similar to the wild-type YB-2 protein and the other a slower-migrating complex similar to those formed by the YB-2 (1-169) and (1-167) proteins. This was surprising, considering the fact that on SDS-polyacrylamide gels the migration of these polypeptides was proportional to their molecular weights. This pattern was reproduced even when DNA binding assays were carried out with factor Xa-cleaved protein, ruling out any artifacts introduced by the MBP moiety. The formation of multimeric complexes is a likely explanation for the above observation. Further experiments are, however, required to demonstrate unequivocally if these mutant proteins do indeed exist as multimers, either in solution or upon binding DNA.
We also constructed YB-2 mutants lacking portions of their N termini. The mutant YB-2 (121-298) disrupts the CSD and as expected showed negligible DNA binding ability (Fig. , lane 12). The mutant YB-2 (75-298), while carrying a large deletion at its amino terminus, still retains the entire CSD and could be expected to bind DNA. However, this mutant protein also showed minimal ability to bind DNA. A possible explanation for this could be the proximity of the deletion to the CSD. Given the behavior of this mutant, it was not surprising that the double-deletion mutant YB-2 (75-230) demonstrated no ability to bind DNA (Fig. , lane 10). We also tested the ability of the YB-2 (1-169) mutant to bind a series of unrelated single-stranded and double-stranded DNA oligonucleotides to which wild-type YB-2 had not bound. No binding was observed (data not shown). We also examined the ability of this mutant to bind the different RSV LTR mutant oligonucleotides shown in Table . The relative binding affinity of YB-2 (1-169) to these mutants (data not shown) always paralleled the results obtained with the full-size protein, indicating that although YB-2 (1-169) binds with less affinity than the wild-type YB-2, there is no relaxation in the sequence specificity. The components of the YB-2 protein molecule that are involved in sequence-specific recognition probably reside within the 1–169 region.
In summary, the DNA binding studies with the chkYB-2 mutants described above indicate that the CSD is important for DNA binding and that the carboxyl-terminal charge-zipper domain has no independent ability to bind DNA. The CSD mediates sequence-specific recognition as well as binding to single-stranded DNA. It is also evident that unlike the bacterial cold shock proteins wherein the CSD alone is adequate for DNA binding, the residues that make up the CSD in chkYB-2 are necessary but not sufficient for DNA binding. Apparently, the residues to the carboxyl side of CSD, even if not part of the binding domain, contribute to the generation of stable complexes with DNA, at least in vitro.
Some Y-box proteins are known to bind DNA more avidly in the presence of magnesium (18
). Magnesium also appears to play a role in the nucleic acid interactions of several other RNA binding proteins (27
). We carried out gel shift assays to examine the effects of different concentrations (0 to 20 mM) of several divalent cations (Mg2+
, and Zn2+
), as well as spermidine, a polyvalent cation. The results of these gel shift assays are shown in Fig. . Addition of magnesium chloride to final concentrations of 3 to 10 mM in the binding reaction increased DNA binding more than 10-fold, with maximum effect seen at 5 mM; 20 mM MgCl2
, however, had an inhibitory effect. A similar effect was noted with spermidine. While 3 or 5 mM CaCl2
stimulated binding severalfold, concentrations of 10 mM and above were inhibitory. MnCl2
at 3 and 5 mM promoted binding, although less than for the other ions. As found for CaCl2
concentrations of 10 mM or more were inhibitory. In contrast to the stimulatory effects of these cations, the addition of even 3 mM ZnCl2
was inhibitory to the formation of DNA-protein complexes, with higher ionic strengths essentially eliminating binding. Figure (lane 2) shows the binding of 6 ng of chkYB-2 protein to the radiolabeled LTR oligonucleotide −106/−135, in the absence of any divalent cation. Lanes 3 and 4 show the remarkable increase in DNA binding upon the addition of 5 mM MgCl2
or spermidine, respectively. Lanes 6 and 7 show the stimulation of binding in the presence of 3 mM CaCl2
and 3 mM MnCl2
, respectively. ZnCl2
at 3 mM inhibited binding (lane 8). This inhibition was, however, neutralized upon the addition of either 3 mM MgCl2
(lane 9) or 3 mM each MgCl2
and spermidine (lane 10) to the reaction.
FIG. 3 Effects of cations on DNA binding. The RSV LTR oligonucleotide LTR (−106/−135) was end labeled, and its binding to 6 ng of chkYB-2 fusion protein was assayed in gel shift experiments carried out as described in Materials and Methods. Binding (more ...)
The exact significance of the effect of a cationic environment on chkYB-2–DNA interactions is not known. We are not aware of any specific metal ion binding motifs on the chkYB-2 protein. We were curious to know if these results could be reproduced with any of the YB-2 mutants that we have made. We tested the DNA binding activity of the YB-2 (1-169) mutant protein either in the absence of cations or in the presence of MgCl2, spermidine, or CaCl2. As shown in Fig. , the addition of 5 mM MgCl2 (lane 3), 5 mM spermidine (lane 4), or 3 mM CaCl2 (lane 5) significantly promoted DNA binding compared to DNA binding carried out in the absence of any of these ions (lane 2). Lanes 2 to 5 contained 10 ng of the protein. The same effect was repeated when 50 ng of the protein was used in each binding reaction (lanes 7 to 10).
FIG. 4 Effects of cations on chkYB-2 (1-169) binding to DNA. The oligonucleotide LTR −106/−135 was end labeled, and DNA binding assays were carried out with the chkYB-2 (1-169) mutant polypeptide. Binding mixtures were incubated at room temperature (more ...) Effects of point mutations in the core binding site for chkYB-2.
We have shown earlier that the E4 region in the RSV LTR is important for maximal enhancer activity (40
). We also reported that the recognition motif for chkYB-2, the octamer 5′-GTACCACC-3′, is located in this region. Also, transfection experiments using E4-deleted LTR constructs and chkYB-2 antisense oligonucleotides had demonstrated that the ability of chkYB-2 to act as an activator was mediated primarily through this octanucleotide motif. Our earlier work had shown that this protein bound with various affinities several different single-stranded oligonucleotides spanning the RSV LTR. We aligned the sequences of all these oligonucleotides to which chkYB-2 had bound and looked for a consensus sequence. This comparison revealed that the 12-mer 5′-TCGTACCACCTT-3′ is the common motif. This is essentially the previously described octamer 5′-GTACCACC-3′ extended by two nucleotides each in the 5′ and 3′ directions.
The 21-mer oligonucleotide E4C1, bearing this motif and corresponding to the region from −103 to −123 on the noncoding strand of the RSV LTR, was hence used as the wild-type binding motif, and systematic point mutations spanning the entire motif were introduced (Table ). End-labeled oligonucleotides, adjusted for specific activity, were then used in gel shift assays. A summary of the binding results is presented in Table . The gel shift assay shown in Fig. is representative of some of the oligonucleotides used. It is evident from these results that the binding of YB-2 to its recognition motif was abolished upon the introduction of any mutation in the core octamer, except for the mutant oligonucleotide E4C2 M101, where replacement of G with a C at position 3 appeared to be well tolerated. The other exception was the mutant E4C2 M106, where replacement of C with a T at position 9 did not affect binding. However, when the adjacent C was also replaced by a T, to yield the double mutant E4C2 M111, binding was abolished. Nucleotides at positions 11 and 12 did not appear to be critical, as shown by binding equivalent to wild-type binding by the mutant E4C2 M107. Binding to oligonucleotides with mutations at positions 1 and 2 (E4C2 M108 and E4C2 M109, respectively) was significantly less than binding to E4C1. These results indicate that the single-stranded DNA binding protein chkYB-2 binds its ligand in a sequence-specific manner and that maximum binding affinity requires the presence of at least the 5′-TCGTACCACC-3′ decamer motif.
FIG. 5 Mutational analysis of the chkYB-2 binding motif in RSV LTR. A series of point mutations was introduced in the single-stranded DNA oligonucleotide E4C1 (see Table for sequences of oligonucleotides). E4C1 represents the −103 to (more ...)
In our earlier report (40
), we had remarked on the ability of chkYB-2 to bind more than one site on the RSV LTR and had suggested that the appearance of multiple, slower-migrating complexes in gel shift assays carried out with the full LTR as the probe was probably due to occupancy of the other sites by additional molecules. To confirm this effect directly, we designed the 30-mer oligonucleotide X2/E4C1 (Table ), which is a direct repeat of the YB-2 binding motif. As shown in the gel shift assay (Fig. , lane 7), chkYB-2 bound avidly to this oligonucleotide, forming two DNA-protein complexes. The slower-migrating complex is a minor component and probably represents more than one molecule of YB-2 complexed to DNA.
An examination of the sequence of the noncoding strand of the RSV LTR, immediately to the 3′ side of the octamer motif 5′-GTACCACC-3′ (−112 to −119), revealed the presence of an almost identical 5′-CTACCACC-3′ (−123 to −130) motif. Also, the gel shift assays described above had shown that the replacement of the nucleotide G in the motif with a C, as in the mutant E4C2 M101, did not decrease the affinity of chkYB-2 binding (Table ). Hence, the −112 to −130 region of the RSV LTR can be viewed as providing two potential sites for high-affinity binding by chkYB-2. To examine the affinity of chkYB-2 to DNA bearing such a double motif, we synthesized the oligonucleotide LTR −106/−135 (Table ). Unlike the oligonucleotide E4C1 (extending from −103 to −123), which has only the first octamer motif, this new oligonucleotide, by extending from −106 to −135, incorporates both the 5′ and 3′ octamer motifs. The gel shift assay (Fig. ) showed that the ability of chkYB-2 to bind LTR −106/−135 (lane 8) was severalfold greater than that observed with E4C1 (lane 1). As found for X2/E4C1, another slower-migrating complex was also seen as a minor component. Surprisingly, the size of the major protein-DNA complex formed with the double-motif oligonucleotide was comparable to the one found with the single-motif DNA. This finding suggests that only a monomer was complexing with the LTR −106/−135 oligonucleotide to form the major retarded species. These results suggest that it would be more accurate to consider these 5′-GTACCACC-3′ repeats as providing two half-sites, rather than two full motifs, for chkYB-2 binding. It is also evident that binding to this native double-octamer motif on the RSV LTR is comparable to the binding observed with the synthetic construct X2/E4C1, carrying a perfect repeat of the 12-mer 5′-TCGTACCACCTT-3′ motif (lane 7). These experiments with X2/E4C1 and LTR −106/−135 were done with limiting amounts (6 ng) of the chkYB-2 protein. When these experiments were repeated with progressively larger amounts of protein, there was only a slight increase in the amount of the slower-migrating complex (data not shown). Since the formation of the faster-migrating species is an apparent prerequisite for the formation of the second, more slowly migrating species, these probably represent one and two molecules of chkYB-2, respectively. However, considering the low rate at which the slower-migrating complex forms, the binding of the second molecule is apparently not cooperative in nature. Curiously, chkYB-2 demonstrated good binding to another 30-mer oligonucleotide that had the same sequence as the oligonucleotide LTR −106/−135 except that it was synthesized in the antiparallel direction (Fig. , lane 9). We are not aware whether this apparent ability to bind the recognition motif without regard to its polarity has been reported earlier for other single-stranded DNA binding proteins.
Considering the above results, which we obtained with the LTR −106/−135 oligonucleotide, we wished to explore by mutational analysis whether point mutations introduced in one of the motifs adversely affected the ability of chkYB-2 to interact with the second motif. Toward this end, we designed mutant oligonucleotides (E4M series [Table ]) representing the −109 to −135 region, such that identical point mutations were introduced in either the 5′ or 3′ octanucleotide motif alone or in both motifs simultaneously. One set of mutant oligonucleotides was designed based on the negligible protein binding that we had observed with the E4C2 M103 mutant (Fig. , lane 2). The same A→G change was made in either the 5′ or 3′ motif or both motifs (E4M103-1C, E4M103-2C, or E4M103-3C, respectively). The other set of mutants was based on the E4C2 M106 mutant, wherein a C→T change apparently did not affect the formation of DNA-protein complexes (Fig. , lane 6). The oligonucleotides E4M106-1C, E4M106-2C, and E4M106-3C thus represent a C→T change in the 5′, 3′, and both 5′ and 3′ octanucleotide motifs, respectively. The results of chkYB-2 binding to these mutant constructs compared to the wild type are presented in Fig. a. chkYB-2 showed reduced affinity for both M103-1C and M103-2C, indicating the contribution of both motifs for maximal binding. Not surprisingly, negligible binding was observed with the double mutant M103-3C. In contrast, binding to the E4M106 set of mutants was essentially unaffected.
FIG. 6 Contribution of both chkYB-2 recognition octamers to RSV LTR-driven transcription. (A) All mutant oligonucleotides (see Table for nomenclature and sequences) were end labeled and equalized for specific activity. Gel shift assays were carried (more ...)
The experiments described thus far helped in defining the requirements of the chkYB-2 binding motif. However, the exact relevance of results obtained from in vitro DNA binding assays to chkYB-2 interactions with the RSV LTR in vivo had to be determined. One approach was to correlate the mutations that abrogated protein binding in vitro with alterations in the in vivo transactivating potential of RSV LTR constructs carrying the same mutations in the YB-2 binding motif. Toward this end, we constructed a series of mutant RSV LTR reporter vectors. Mutations were made only within the −112 to −130 region described earlier as the site of two motifs for chkYB-2 binding. Furthermore, the point mutations introduced in these six constructs reflect exactly the changes made in either the 5′ or 3′ octamer motif or both binding motifs while designing the E4M series of mutant oligonucleotides.
Chicken embryo fibroblasts were chosen for the transfection experiments, as chkYB-2 is expressed abundantly in these cells and has been demonstrated to activate RSV LTR-driven transcription in these cells, primarily through its interaction with recognition motifs present within the −112 to −130 region (40
). Cells in the mid-log phase of growth were transfected with 1 μg of each of these plasmids, along with 1 μg of the internal control plasmid, pSVGal. CAT assays were performed with protein extracts normalized to β-galactosidase activity. A representative autoradiogram (Fig. b) and the average results of three identical experiments (Fig. c) are presented. These results reveal that point mutations in either the 5′ or 3′ motif (M103-1CAT or M103-2CAT, respectively) reduced the transcriptional activity of the RSV LTR by about 20 to 25%. The decrease in transcriptional activity upon the introduction of point mutations in the two separate motifs is synergistic, as evidenced by the much greater reduction in transcriptional activity of the double mutant (M103-3CAT) compared with either single mutant alone. The activity of this double mutant (mutations are at positions −114 and −125) was, however, much higher than that observed with E4 Del CAT, an RSV LTR construct carrying a 14-nucleotide deletion (−114 to −127) that encompasses both chkYB-2 binding motifs. A possible interpretation of this finding is that although in vitro chkYB-2 bound negligibly to this double mutant (M103-3C [Fig. a]), in vivo, low-affinity interactions with this mutant YB-2 motif probably occur and contribute to transactivation of the LTR, albeit less efficiently. Even these low-affinity interactions apparently cannot take place when both motifs are completely deleted, as in E4 Del CAT. Alternatively, this result could reflect the fact that protein-DNA interactions, other than those mediated by chkYB-2, contribute to transactivation from this deleted region. Figure c also shows that compared to the M103-CAT constructs, the M106-CAT constructs did not show significant reductions in transcriptional activity, with even the double mutant M106-CAT displaying transcriptional activity comparable to wild-type RSV LTR.
In summary, the results presented above show a correlation between the transcriptional activities of RSV LTR constructs carrying point mutations in the chkYB-2 binding motifs and the relative affinities of the corresponding mutant oligonucleotides as assayed by DNA-protein complex formation in vitro. Additionally, our results also show that (i) both of the chkYB-2 recognition motifs contribute to RSV LTR-driven transcription and (ii) the interaction of chkYB-2 with these adjacent motifs is probably just additive and not cooperative in nature, since the reduction in transcription observed from the double mutant (M103-3CAT) was not more than the cumulative reduction in transcriptional activities of the single mutants. These results are also in agreement with our interpretation of the chkYB-2 binding assays with LTR −106/−135, which suggested that the −112 to −130 region is best viewed as providing a single high-affinity binding site for chkYB-2, with each octamer motif behaving as a half-site.