Functionally Relevant Histone-DNA Interactions Extend Beyond the Classically Defined Nucleosome Core Region*

We demonstrate that core histones can affect the accessibility of a DNA element positioned outside of the classically defined nucleosome core region. The distance between a well positioned nucleosome and the binding site for the 5 S-specific transcription factor TFIIIA was systematically varied and the relative binding affinity for TFIIIA determined. We found that core histone-DNA interactions attenuate the affinity of TFIIIA for its cognate DNA element by a factor of 50–100-fold even when the critical binding region lies well outside of the classically defined nucleosome core region. These results have implications for the validity of parallels drawn between the accessibility of general nucleases to DNA sequences in chromatin and the activity of actual sequence-specific DNA binding factors.

In eukaryotes, the primary repeating subunit of chromatin is the nucleosome (1,2). Each nucleosome contains about 200 bp 1 of DNA, an octamer of core histone proteins, (H2A/H2B/H3/ H4) 2 , and a single linker histone. An obvious function of chromatin structure is to bring about the ordered compaction of DNA within the nucleus. In addition, in vitro and in vivo experiments have shown that nucleosomes also play a functional role in both activating and repressing gene activity (2)(3)(4). However, in general, the presence of histone-DNA interactions greatly reduces the DNA binding activity of sequencespecific factors that mediate gene transcription (5,6). Thus, the presence of a nucleosome may effectively reduce or eliminate the occupancy of crucial DNA elements at the concentrations of trans-acting factors found in vivo.
A major question is how transcription occurs within the context of nucleosomes. Recent work has uncovered several strategies employed in eukaryotic nuclei for alleviating nucleosome repression (2,4). Active expression of certain genes is dependent upon energy-intensive active disruption of histone-DNA contacts by activities such as the SWI⅐SNF, NURF, RSC, and CHRAC complexes (7)(8)(9) or posttranslational modifications of the histone tail domains (10,11). Promoter mapping experiments have shown that in many instances cognate DNA elements of critical transcription factors are located precisely in the linker region between positioned nucleosomes (5,12,13).
In vitro experiments using model chromatin templates have proven useful to investigate the ability of trans-acting factors to bind cognate elements assembled into nucleosomes (5,14,15). Some DNA binding factors such as the glucocorticoid and thyroid hormone receptors can make DNA contacts apparently compatible with core histone-DNA interactions and are thus able to bind to DNA on the surface of the nucleosome (15)(16)(17). However, in general the affinities of such trans-acting factors such as TFIIIA, TBP, or Gal4 for their cognate DNA sequences are reduced by several orders of magnitude when the target sequences are associated with the core histone octamer (5, 7, 18 -20). Polach and Widom (6) have demonstrated that this restrictive influence is modeled by a set of linked equilibria in which histones and DNA binding factors compete for binding to the same DNA. Their quantitative analysis shows that histone competition is most severe near the center of the particle and decreases as one moves toward the "edge" of the nucleosome core region. However, the full extent of the repressive influence of a single nucleosome core has not been accurately determined. The reach of restrictive histone-DNA interactions is generally considered to be equivalent to ϳ146 base pairs, the amount of DNA within the nucleosome core region. However, the nucleosome core particle DNA is defined by protection from the endo-and exo-nucleolytic activities of micrococcal nuclease digestion, not DNA binding of trans-acting factors.
In the present paper, we examined the extent of influence of core histone-DNA interactions within a single nucleosome core on the binding of the transcription factor, TFIIIA. We show that the entire cognate element for TFIIIA must be moved well beyond the edge of the nucleosome core region to recover high affinity binding by this factor. The results suggest that histone-DNA interactions 15-20 base pairs beyond the edge of the nucleosome core region have significant consequences that may be relevant for the activation of 5 S RNA genes in vivo.

EXPERIMENTAL PROCEDURES
Construction of Modified 5 S DNAs-The HpaII-XbaI fragment flanked by asymmetric BstXI linkers from pJHXI-BstXI, which contains the Xenopus borealis somatic 5 S RNA gene (22), was prepared and modified by standard three-and four-primer polymerase chain reaction methods. The primers were designed to modify the 5 S DNA fragment to obtain inserts of 5-35 base pairs in increments of 5 bp between positions ϩ50 and ϩ51 (with respect to the transcription start nucleotide of 5 S RNA gene, ϩ1). Inserts were designed as tandem repeats of the wt sequence such that the ϩ5 insertion resulted in uninterrupted 5 S sequence from Ϫ102 to ϩ55 appended to sequences ϩ51 to ϩ132; the ϩ10 construct has uninterrupted 5 S DNA from Ϫ102 to ϩ60 appended to sequences ϩ51 to ϩ132, and so on. A fragment containing a 20-base pair insert was not obtained because of the inability to be propagated in bacterial cells. The modified fragments were then restricted with BstXI and cloned into pBSIIsk(ϩ) (Stratagene). The HpaII-XbaI fragments were radiolabeled at the 5Ј end of either strand with T4 polynucleotide kinase and [␥-32 P]ATP by standard methods.
Nucleosome Core Reconstitutions-Nucleosome cores were reconstituted onto radiolabeled DNA fragments either by exchange with stripped chromatin or by dialysis from high salt with purified chicken erythrocyte histones (22). Under no circumstances did we detect differences in results due to the reconstitution methodology employed. In the histone exchange method, a large molar excess (ϳ50-fold) of stripped chromatin was mixed with radiolabeled DNA, and NaCl concentration was adjusted to 1 M. The mix was then incubated for 1 h at room temperature. The salt concentration was then diluted to 0.8, 0.6, 0.2, 0.1, and 0.05 M NaCl by adding 10 mM Tris-HCl, pH 8.0. For the salt dialysis method, nonspecific carrier DNA (ϳ10 g), radiolabeled DNA (ϳ0.25 g), and purified core histones (mass ratio histone/DNA ϳ0.8) were mixed in a total volume of 200 l in 2.0 M NaCl. The sample was dialyzed for at least 1.5 h against several buffers containing 10 mM Tris-HCl, pH 8.0, 10 mM ␤-mercaptoethanol, 1 mM EDTA, and decreasing NaCl concentrations of 1.2, 1.0, 0.8, and 0.6 M. A final dialysis was performed for 12 h against 10 mM Tris-HCl, pH 8.0. After reconstitution, only naked DNA, (H3/H4) 2 tetramer-DNA complexes and nucleosomes were detectable by nucleoprotein gel electrophoresis.
Micrococcal Nuclease Mapping-HpaII-XbaI DNA fragments were internally labeled at the BbvI site as described (23). Nucleosomes or (H3/H4) 2 tetramer-DNA complexes were reconstituted as described and then digested for 5 min at room temperature with 0.75, 0.4, 0.2, and 0.1 unit of micrococcal nuclease (Worthington). Ca 2ϩ concentration was adjusted to 0.5 mM concomitantly with addition of micrococcal nuclease. Digestion was terminated with addition of EDTA and SDS. The DNA was recovered by ethanol precipitation and resolved by electrophoresis in non-denaturing 6% polyacrylamide gels. DNA fragments of 147 bp were gel-extracted and restricted with EcoRV to map the termini of the micrococcal nuclease digestion products (24). The digestion products were then resolved by electrophoresis in denaturing gels (25).
Hydroxyl Radical Footprinting-DNA fragments were end-labeled either at the HpaII site or at the XbaI site. Nucleosome cores reconstituted onto HpaII-XbaI-labeled fragments were treated with hydroxyl radicals as described, and reactions were quenched with the addition of glycerol to a final concentration of 5% (22). Samples were then directly loaded onto preparative 0.7% agarose gels and electrophoresed for 1.5-2 h at 120 V. Radiolabeled bands were identified by autoradiography of the wet gel; DNA was recovered from the gel and analyzed by denaturing 6% polyacrylamide gel electrophoresis and autoradiography (25). Specific DNA markers were produced by Maxam-Gilbert cleavage at guanine Gases.
Purification of TFIIIA-The7 S storage particle and TFIIIA were purified as described by Smith et al. (26). Briefly, immature ovary homogenate was fractionated on glycerol gradients, bound to DEAEcellulose, and eluted on a salt gradient. The7 S particle fractions (1-10 mg of protein) were adjusted to 0.1 M KCl in 50 mM HEPES, pH 7.5, 5 mM MgCl 2 , 1 mM dithiothreitol, 10 mM ZnCl 2 , 20% glycerol (buffer A). RNase A was mixed with7 S particle (50 mg of RNase A per mg of protein starting material), incubated for 5 min, and then mixed with an equal volume of buffer A containing 0.1 M KCl and 10 M urea. The mixture was loaded onto a Bio-Rex 70 column, and TFIIIA was eluted with increasing concentrations of KCl. TFIIIA was eluted between 0.8 and 1 M KCl. The protein was Ͼ95% pure (26).

RESULTS
We have used hydroxyl radical footprinting to reveal the precise location of histone-DNA interactions within a well positioned nucleosome assembled on a DNA fragment containing a 5 S RNA gene from X. borealis ( Fig. 1) (27). Interestingly, the assembly of this DNA fragment into a nucleosome prevents binding of the 5 S-specific transcription factor TFIIIA to the internal promoter of the 5 S gene (18). As previously reported, histone-DNA interactions can be detected well beyond the edge of the nucleosome core region located between positions Ϫ70 and ϩ79 as defined by micrococcal nuclease digestion (18,27). Importantly, these contacts extend through the entire internal promoter and include the crucial ϩ80 to ϩ92 region necessary and sufficient for TFIIIA binding ( Fig. 1) (28,29). To assess more accurately the significance of these histone-DNA interactions with regard to TFIIIA binding, we designed constructs to gradually move the TFIIIA-binding site away from the positioned nucleosome.
To obtain the constructs needed for this study, it was first necessary to locate the DNA element that drives sequence-dependent positioning of the histone octamer. Sequence-dependent conformational variations in B-DNA structure such as variation in minor groove width are reflected in the hydroxyl radical cleavage patterns of naked DNA (30,31). The hydroxyl radical cleavage pattern of naked Xenopus 5 S DNA contains regions of strikingly periodic and uniform modulation ( Fig. 1), reminiscent of the cleavage pattern obtained with stably curved DNA sequences (31,32). Such DNA structures have been shown to efficiently direct nucleosome positioning (31,33). Importantly, quantitative densitometry clearly shows that these periodic cleavage modulations directly align with the much larger modulations within the hydroxyl radical cleavage pattern of 5 S DNA assembled into a nucleosome (Fig. 2). Thus, a discrete region within the 5 S DNA has a conformation that yields a hydroxyl radical cleavage pattern similar in nature, but not in magnitude, to the severely curved DNA found within the nucleosome. In addition, the position of this element corresponds to the approximate intranucleosomal location of positioning elements identified by mutational and deletion analysis (34,35). Our interpretation is that the hydroxyl radical cleavage pattern of naked 5 S DNA allows the identification of sequence-dependent DNA structure likely to function as the nucleosome positioning element within the 5 S DNA fragment (Fig. 2).
The putative nucleosome positioning element we identified is located approximately between positions Ϫ30 to ϩ50 within the 5 S DNA sequence. This element abuts the TFIIIA-binding site within the internal promoter region located approximately from ϩ50 to ϩ95 (Fig. 2) (28). Thus, we designed constructions of the 5 S DNA fragment to gradually separate the TFIIIA and nucleosome-binding sites by placing insertions between nucleotides ϩ50/ϩ51 (Fig. 3). Modified 5 S DNAs were obtained by standard polymerase chain reaction methods. The length of the inserts was increased in increments of 5 bp to a maximum of 35 bp, except that a plasmid bearing an insert of ϩ20 bp could not be propagated in bacterial cells (Fig. 3). Inserts were designed as tandem repeats of the original sequence in order to provide as little disruption of the 5 S sequence as possible (see "Experimental Procedures").
We first analyzed if the insertions had any effect on the formation or positioning of nucleosomes reconstituted in vitro.
Reconstitutions were carried out such that about 50% of the labeled nucleoprotein complexes were (H3/H4) 2 tetramer-DNA complexes, and the other 50% were nucleosomes (Fig. 4). The presence of both complexes was desired in order to provide an internal nucleoprotein control for TFIIIA binding (see below). The identity of each nucleoprotein complex was confirmed by co-migration of purified reconstituted tetramer or octamer in agarose gels (data not shown; see Ref. 22). We next determined the position of the nucleosomes on our 5 S DNA constructs by mapping the limits of the 146-base pair nucleosome core DNA produced during micrococcal nuclease digestion. Micrococcal nuclease digestion was carried out as described under "Experimental Procedures," and the DNA products were resolved on non-denaturing polyacrylamide gels (Fig. 5). In agreement with the nucleoprotein gel, all constructs were efficiently reconstituted into canonical nucleosomes as evidenced by the production of the146-bp nucleosome core particle DNA during micrococcal digestion.
The precise position of the nucleosome was determined by recovering the 146-bp DNA from the gel, cleavage with EcoRV at position ϩ31 within 5 S DNA, and analyzing the products on a sequencing gel (Fig. 6). Note that because the DNA is labeled internally at position Ϫ23, only one of the two fragments produced by EcoRV digestion of the nucleosome core DNA will be observed (see "Experimental Procedures"). The digestion products indicate that all constructs have nucleosome positioning identical to the unmodified wt fragment. Specifically, two major bands are observed of ϳ100 and 130 nucleotides in length (Fig. 6A). The ϳ100-nucleotide band corresponds to the major nucleosome position mapped previously on the wt 5 S DNA fragment (23,24). The 130-bp band was unexpected based on earlier mapping studies of octamer-5 S DNA complexes (18,24). Since an octamer located at this position would be expected to bind TFIIIA even on the wt 5 S DNA construct (see below), we next determined whether this band might be due to the Cleavage data for the naked and nucleosomal 5 S DNA fragments similar to that shown in Fig. 1 were quantitated as described and corrected peak intensities for all positions smoothed by a three-nucleotide running average and plotted as cleavage frequency (arbitrary units) versus nucleotide position as indicated (27). The position of the 5 S gene (black arrow), the internal promoter (stippled box), and 5 S DNA sequences within the enzymatically defined nucleosome core region (oval) are indicated. ICR, internal promoter region. tetramer-DNA complexes present within the reconstitutions. We therefore carried out a reconstitution with only the (H3/ H4) 2 tetramer and the wt 5 S fragment. Micrococcal nuclease digestion of these complexes yields two predominant doublestranded DNA products of about 100 and 146 bp in length (data not shown). Interestingly, EcoRV digestion and analysis of the 146-bp product reveals that only bands ϳ130 bp in length are produced (Fig. 6A, lanes 8). One explanation for this result is that this 146-base pair product is an intermediate formed during micrococcal digestion of tetramer-DNA complexes in which trimming has occurred only on the downstream end of the complex. In this case, the predicted position of the downstream edge of the tetramer would be exactly coincident with the position previously mapped in footprinting experiments (24). Nonetheless, the data indicate that the presence of the inserts does not alter the position of the nucleosome with respect to the nucleosome positioning element among the 5 S DNA fragments (Fig. 6B).
We next carried out hydroxyl radical footprinting of the different nucleosome constructs to confirm the mapping results and to obtain a more accurate assessment of the location of histone-DNA interactions (27). Complexes were prepared with DNAs containing a radioactive end label on either strand, cleaved with hydroxyl radicals, and the nucleosomes resolved on a preparative nucleoprotein gels. The DNA products were purified and analyzed on sequencing gels (Fig. 7). As evident when the DNA was radioactively labeled at the downstream end of the 5 S fragment, the insertions had the effect of moving the nucleosome footprint progressively further away, by discrete 5-bp increments, from the TFIIIA-binding site (Fig. 7). Conversely, when the label was incorporated at the upstream end of the 5 S DNA fragments, all nucleosome footprints occurred at the same position with respect to the end of the fragment containing the nucleosome positioning element and were qualitatively identical to that observed on the wt fragment (results not shown; see Fig. 1).
The above results indicate that the nucleosome is present at the same location with respect to the nucleosome positioning element in every construct, and thus histone-DNA interactions are gradually retracted from the TFIIIA-binding site in a 3Ј-5Ј direction. To determine the effect on 5 S promoter accessibility, we performed nucleoprotein gel shift assays. Complexes reconstituted on each of the 5 S constructs were incubated with increasing amounts of purified TFIIIA in binding buffer, and the resulting products were resolved in agarose gels. As the TFIIIA concentration increased, the intensity of the free DNA band for each construct was reduced, and a new band corresponding to TFIIIA⅐DNA complex appeared (Fig. 8). Importantly, quantitative titrations with the naked DNA fragments alone showed that the TFIIIA binding affinity was identical for all 5 S constructs (results not shown). Thus, the mid-point in the titration for TFIIIA binding to the naked DNAs in the nucleoprotein experiments occurred when the free concentration of active TFIIIA was ϳ1 ϫ 10 -9 M and was used as an internal reference (36,37). By using the naked DNAs as a reference, an approximate relative dissociation constant for TFIIIA binding to all nucleoprotein complexes was determined by quantitative densitometry with corrections for residual den- sities of products and reactants on gels such as are shown in Fig. 8 (38). As expected, we observed TFIIIA binding to tetramer-DNA complexes in all cases (18). However, in the case of tetramers assembled with the wt 5 S DNA fragment, the affinity of TFIIIA was reduced approximately 4-fold relative to the affinity of TFIIIA for naked 5 S DNA (Table I). Likewise, the affinity of TFIIIA for the ϩ5, the ϩ10, and the ϩ15 complexes was similarly reduced compared with the naked DNA. However, we found that TFIIIA binding affinities for the ϩ25tetramer complex and the remaining complexes were approximately equivalent to that of the naked 5 S DNA (Table I).
A much different situation was found for TFIIIA binding to 5 S DNA assembled into a complete nucleosome. No binding was detected for reconstitutions on the wt, ϩ5, ϩ10, ϩ15, and ϩ25 templates (see nucleosome band, Fig. 8). In these cases we estimate the affinity of TFIIIA to be reduced at least 50 -100fold relative to naked 5 S DNA. However, some binding is detected for nucleosomes reconstituted on the ϩ30 construct, and a complete shift of the nucleosome band is observed for the ϩ35 construct (Fig. 8). Densitometric analysis indicated that the relative binding affinities of TFIIIA for the ϩ30 and ϩ35 nucleosomes to be approximately 5-and 3-4-fold lower than that of the naked 5 S DNA, respectively (Table I). DISCUSSION We have tested if histone-DNA interactions outside of the classically defined nucleosome core region can significantly modulate the binding of a eukaryotic transcription factor, TFIIIA. This protein has a modular structure, with nine individually folded zinc finger domains (2). These nine fingers are combined into three distinct structural domains containing fingers 1-3, 4 -6, and 7-9 (39,40). Previous work with truncated forms of TFIIIA revealed that 90% of the binding free energy is contributed by interaction of the first three fingers of this protein with 5 S sequences between ϩ80 and ϩ92 in the 5 S gene (28,29). Thus, a polypeptide containing fingers 1-3 binds DNA with an affinity only 3-4-fold less than the intact protein. About half of the remaining stability is contributed by finger 5 and half by fingers 7-9 (41). Thus, blockage of the binding of fingers 4 -9 would cause a loss in affinity of only about 3-fold.
In the case of (H3/H4) 2 tetramer⅐wt 5 S DNA complex, we FIG. 7. Hydroxyl radical footprinting of nucleosomes reconstituted with modified 5 S DNA fragments. Nucleosomes were reconstituted with each of the 5 S DNA fragments labeled at the XbaI site, downstream of the 5 S RNA gene (see Fig. 3). The complexes were subjected to hydroxyl radical cleavage, octamer-DNA complexes isolated by nucleoprotein gel electrophoresis, and the cleavage patterns analyzed on sequencing gels as described. Lanes 1-6 show Maxam-Gilbert guanine base-specific reactions done with the ϩ5, ϩ10, ϩ15, ϩ25, ϩ30, and ϩ35 5 S fragments, as markers. Lanes 7-13 show the hydroxyl radical footprints of nucleosomes assembled with the wt, ϩ5, ϩ10, ϩ15, ϩ25, ϩ30, and ϩ35 fragments, respectively. Schematic indicates the location of the 5 S RNA gene coding sequence (black arrow); the binding site for TFIIIA (hatched box), the region of DNA in contact with core histone proteins as determined by the footprinting analysis; and the nucleosomal dyad (black oval). The positions of the inserts and the radioactive end label are also indicated. Note that the position of the histone octamer (oval) is shown only for the wt 5 S DNA.  observe a relative affinity of TFIIIA that is ϳ3-4-fold less than that of naked 5 S DNA. Our interpretation is that H3/H4 tetramer-DNA contacts in this complex restrict the interaction of fingers 4 -9 with 5 S DNA while not interfering with the binding of fingers 1-3 (Fig. 9). This hypothesis is supported by hydroxyl radical footprinting experiments that show that H3/H4 tetramer-DNA contacts extend to about position ϩ75 within the 5 S sequence. Thus these contacts completely overlap the binding sites of fingers 4 -9, while leaving exposed the region ϩ80 to ϩ92 which is contacted by fingers 1-3 (18,29,39). Moreover, DNase I footprinting of tetramer⅐wt 5 S DNA⅐TFIIIA triple complexes suggests that whereas fingers 1-3 are bound to their cognate sequences, the remaining fingers may not be bound to the DNA in a manner analogous to their interaction within the naked 5 S DNA⅐TFIIIA complex (19). The binding data for all 5 S constructs suggest that tetramer complexes formed on the ϩ5, ϩ10, and ϩ15 5 S fragments all behave in a way qualitatively similar to the wt complex (Fig. 9). Thus, the tetramer complex must be "moved" 15-20 base pairs further away from the TFIIIA-binding site before all nine zinc fingers of the protein can freely bind DNA.
The results indicate that the binding site for fingers 1-3 is blocked when the wt 5 S DNA fragment is assembled into a nucleosome (Figs. 8 and 9). These histone-DNA contacts cause the affinity of TFIIIA to be reduced Ͼ50 -100-fold. Interestingly, we find that the nucleosome must be moved over 30 base pairs further away from the TFIIIA-binding site before detectable high affinity binding is restored. However, in this case the relative affinity of TFIIIA for the ϩ30 and ϩ35 nucleosomes suggests that only fingers 1-3 are able to specifically interact with DNA within these complexes (Table I). This hypothesis will be tested in future experiments using hydroxyl radical footprinting to precisely map protein-DNA contacts in the ϩ35 nucleosome⅐TFIIIA triple complex. Thus the data indicate that the crucial ϩ80 to ϩ92 sequences must be moved approximately 90 -100 base pairs away from the dyad axis of the nucleosome before fingers 1-3 can freely bind this region (Fig.  9). Clearly, histone-DNA contacts, or at least the presence of the core histones, can moderate the binding activity of elements well beyond the edge of the enzymatically defined 146-bp nucleosome core region.
The present results are in good agreement with a very recent demonstration of the dependence of TFIIIA binding on the translational position of a nucleosome on the X. laevis somatic 5 S RNA gene (42). In this paper, most of the reconstituted 5 S constructs containing multiple translational positions were unable to bind TFIIIA. However, one construct in which the nucleosome was positioned just upstream of the internal promoter region did bind TFIIIA but only at concentrations much higher than that required to fully load the naked 5 S DNA (the data presented do not allow a more accurate assessment of the relative dissociation constants of TFIIIA for nucleosomal and naked DNA).
A previous quantitative analysis of the effects of histone-DNA interactions within the nucleosome core region concluded that these interactions effectively attenuated sequence-specific binding affinities by ϳ10 2 -10 5 -fold, from the edge to the center of nucleosome, respectively (6). The present paper extends these results by showing that sequence-specific binding affinities in the region encompassing about 20 bases to either side of the nucleosome core region are attenuated by approximately 50 -100-fold for at least some DNA binding factors. The biological significance of this observation is that if such effects are relevant in vivo, very little of the so-called "linker DNA" would be as accessible as might be indicated by nuclease digestion experiments. Nucleases in general do not need as intimate contact with the DNA as sequence-specific DNA-binding proteins. In fact, many nucleases can access the phosphodiester backbone at selected sites and effect cleavage at reasonable rates even when the DNA is centrally located within the nucleosome (1, 2). Thus, general nuclease cleavage accessibility might not be a good general model for the "openness" of linker DNA.
The data indicate that core histone-DNA interactions can have significant effects on accessibility of linker DNA. Furthermore, these results suggest that the region of linker DNA between two neighboring nucleosomes, which is as accessible as naked DNA, is effectively much smaller than previously thought for some transcription factors. Thus strategies in which cis-acting elements are places between nucleosome may require much more precise positioning than was previously evident or may require concurrent nucleosome disruption activities or histone modifications, such as are thought to be relevant for intra-core DNA binding activities.
Previous experiments have shown that nucleosome positioning on the 5 S DNA fragment at least partly recapitulates the nucleosome positioning observed in vivo (43). Experiments with reconstituted templates showed that the presence of a nucleosome on the X. borealis somatic 5 S rDNA gene efficiently inhibits transcription in Xenopus oocyte extracts, whereas the template could be transcribed in presence of incomplete nucleosome core composed by H3/H4 tetramer (44,45). Transcription in these experiments is correlated with the apparent ability of TFIIIA to bind to the reconstituted templates (18). The results presented here suggest that nucleosome sliding to expose the TFIIIA-binding site within a linker region between nucleosomes might not be sufficient to allow activation of 5 S genes in vivo since little unrestricted linker DNA is expected to exist between nucleosomes spaced at intervals of approximately 200 base pairs (45). Thus, other factors, such as histone acetylation must play an important role in activation of these genes, concomitant with displacement of histone H1 (19, 46, 47). For tetramer complexes (Tet) assembled with the wt and ϩ5 fragments, only fingers 1-3 of TFIIIA can bind to the DNA, whereas fingers 1-6 are bound to DNA in the ϩ10 and ϩ15-tetramer complexes. All fingers of TFIIIA are presumed bound in tetramer complexes assembled with the ϩ25, ϩ30, and ϩ35 5 S DNA fragments. Nucleosomes assembled with the wt, ϩ5, ϩ10, ϩ15, and ϩ25 5 S DNA fragments (Oct) are refractory to TFIIIA binding, whereas fingers 1-3 are bound within the ϩ30 and ϩ35-nucleosome complexes. The translational positions of the tetramers and nucleosomes corresponding to each of the 5 S DNA fragments are indicated. The nine zinc fingers of TFIIIA (small ovals) and the location of fingers 1 and 9 in the model are indicated.