Transcription Factor Requirements for In Vitro Formation of ...

MOLECULAR AND CELLULAR BIOLOGY, May 1990, p. 2390-2401 0270-7306/90/052390-12$02.00/0 Copyright C) 1990, American Society for Microbiology

Vol. 10, No. 5

Transcription Factor Requirements for In Vitro Formation of Transcriptionally Competent 5S rRNA Gene Chromatin SARA J. FELTS,t P. ANTHONY WEIL, AND ROGER CHALKLEY* Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received 8 August 1989/Accepted 17 January 1990

The Saccharomyces cerevisiae 5S rRNA gene was used as a model system to study the requirements for assembling transcriptionally active chromatin in vitro with purified components. When a plasmid containing yeast 5S rDNA was assembled into chromatin with purified core histones, the gene was inaccessible to the yeast class Ill gene transcription machinery. Preformation of a 5S rRNA gene-TFIIIA complex was not sufficient for the formation of active chromatin in this in vitro system. Instead, a complete transcription factor complex consisting of TFIIIA, TFIIIB, and TFIIIC needed to be formed before the addition of histones in order for the 5S chromatin to subsequently be transcribed by RNA polymerase III. Various 5S rRNA maxigenes were constructed and used for chromatin assembly studies. In vitro transcription from these assembled 5S maxigenes revealed that RNA polymerase III was readily able to transcribe through one, two, or four nucleosomes. However, we found that RNA polymerase Ill was not able to efficiently transcribe a chromatin template containing a more extended array of nucleosomes. In vivo expression experiments indicated that all in vitro-constructed maxigenes were transcriptionally competent. Analyses of protein-DNA interactions formed on these maxigenes in vivo by indirect end labeling indicated that there are extensive interactions throughout the length of these maxigenes. The patterns of protein-DNA interactions formed on these genes are consistent with these DNAs being assembled into extensive nucleosomal arrays.

The use of in vitro model systems has contributed greatly to our understanding of the protein factors required for specific transcription of both procaryotic and eucaryotic genes (29, 36, 54). As the number of known transcription factors, particularly in eucaryotes, has increased, it has become quite clear that the mechanisms of gene regulation are indeed complex. Transcription, and transcriptional regulation, in eucaryotes is further complicated by the fact that the genes of a cell are not transcribed as naked DNA but are instead transcribed as chromatin. Hence, our understanding of transcription requires not only the reconstitution of transcription complexes with purified factors but also includes the utilization of chromatin templates reconstituted with

binding of TFIIIA and TFIID to their respective promoters (15, 18, 29, 43, 58). We and others have shown that nucleosomes can be efficiently reconstituted on plasmid DNA in vitro using purified histones (11, 41, 42). Nucleoplasmin, an acidic protein first purified from unfertilized Xenopus eggs, permits the formation of bona fide soluble nucleosomes in vitro at physiologic histone-to-DNA ratios and ionic strengths. The simplicity of this system makes it quite attractive for the study of the requirements for the formation of transcriptionally active chromatin. We chose to study transcription of the yeast SS rRNA gene within the context of this in vitro chromatin system. The 5S gene is transcribed by RNA polymerase III, which requires three transcription factors (TFIIIA, TFIIIB, and TFIIIC) to initiate specific SS rRNA synthesis in vitro (29, 44, 53). TFIIIA, TFIIIB (24, 55), and RNA polymerase III (39) have been purified to near homogeneity from yeasts. TFIIIC has also been highly purified from yeasts (6, 13; M. Parsons and P. A. Weil, J. Biol. Chem., in press). For a recent review of work dealing with transcription of nonyeast 5S genes (i.e., Xenopus), see reference 14. Although the yield of purified yeast transcription factors is not yet suitable for in vitro assembly studies, sufficient amounts of separated and partially purified factors are available to perform these experiments. Owing to the problems inherent in interpreting in vitro data obtained with crude chromatin assembly extracts, the assembly of the yeast SS gene with purified histones and nucleoplasmin lends itself well as a model system with which to study the assembly of active chromatin in vitro. The studies presented here show that prebinding of yeast TFIIIA to SS rDNA is not sufficient for the assembly of the SS gene into active chromatin. Instead, a complete transcription factor complex consisting of transcription factors TFIIIA, TFIIIB, and TFIIIC must be formed before nucleosome

nucleosomes and other chromatin-associated proteins. It has been reported that Xenopus 5S rDNA is assembled into inactive chromatin when injected into Xenopus oocytes or added to crude oocyte extracts (16, 27). Further, Gottesfeld and Bloomer (16) showed that simple formation of a Xenopus 5S rRNA gene-transcription factor IIIA (TFIIIA) binary complex was sufficient to form an active chromatin template in these extracts. Similar studies with the adenovirus major late promoter have indicated that the single transcription initiation factor TFIID performs an analogous function for class II promoters (35, 59). Thus, it has been postulated that the binding of a single transcription factor (either TFIIIA or TFIID) to the DNA before the formation of nucleosomes precludes histone octamer deposition over crucial cis-promoter elements of these genes, thereby making them accessible to the rest of the transcriptional machinery at some later time. More recent studies, however, suggest that other factors are required to stabilize the Corresponding author. t Present address: Department of Biochemistry, SJ-70, University of Washington, Seattle, WA 98195. *

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deposition for the 5S gene to be transcribed as chromatin in vitro. Additionally, we utilized this chromatin reconstitutiontranscription system to study in vitro the expression of a family of SS maxigenes. Transcription of this family of 5S maxigenes was examined in vivo and in vitro. RNA polymerase III is able to efficiently transcribe maxigenes containing one to three nucleosomes. In contrast, a maxigene large enough to contain more than four nucleosomes was inefficiently transcribed in vitro. Since polymerase III does not normally transcribe such large genes, we asked whether this polymerase possesses this capacity in the cell. We showed that the 5S maxigenes in vivo are extensively complexed with protein and appear to be transcriptionally competent at a level comparable to that of the endogenous 5S genes. MATERIALS AND METHODS

Plasmid DNAs. pUC5S contains a single copy of the yeast 5S rRNA gene and approximately 220 base pairs (bp) of 5'and 90 bp of 3'-flanking sequences cloned into pUC9. This plasmid was used for most of the in vitro studies shown. Plasmid pBM272 (21) was used to construct centromeric 5S maxigenes for in vivo studies. All cloning was done by standard methods as described by Maniatis et al. (34). Transcription factors. Yeast class III transcription factors were partially purified from yeast whole-cell extracts (23, 55). Buffer C (BC) contained 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.9), 0.2 mM EDTA, and 10 or 20o (vol/vol) glycerol as indicated. Sodium chloride concentrations in BC are abbreviated in the text (BC/100 = BC plus 100 mM NaCl; BCM/100 = BC plus 10 mM MgCl2 and 100 mM NaCl). Yeast whole-cell extracts were applied to a heparin-sepharose column equilibrated with BCM/100. The column was extensively washed with BCM/100. Transcription factors TFIIIB and TFIIIC were eluted with 2 to 3 column volumes of BCM/300. Transcription factor TFIIIA was then eluted with 2 column volumes of BCM/700 and further purified by using Sephacryl S300 (55); we estimate that the final product is 1 to 2% pure. Gel mobility shift assays for TFIIIA were conducted as previously described (55). For further purification and separation of TFIIIB and TFIIIC, the BCM/300 eluate from the heparin column was pooled and dialyzed against BCM/100. The protein was applied to DEAE-Sephadex (A25) equilibrated with BCM/100. TFIIIB was obtained in the column breakthrough fractions. After extensive washing of the column with BCM/100, TFIIIC activity was eluted with BCM/250. Fractions containing TFIIIB activity were pooled, aliquoted, and stored at -70°C. The protein peak of the BCM/250 elution containing TFIIIC activity was dialyzed against BCM/100, aliquoted, and stored at -70°C. Yeast RNA polymerase III was purified (>80% pure) by a modification of the method of Ruet et al. (39). Purification of histones and egg nucleoplasmin. Hyperacetylated core histones were prepared from HTC cells (rat hepatoma cells) and purified essentially as described previously (9). Increasing the load of chromatin per milliliter of bed volume of hydroxylapatite resin (at least 0.5 mg/ml of bed) was found to improve the yield of histones. Because of the unusual evolutionary stability of histones, the use of rat histones with yeast factors is not anticipated to bias the overall conclusions. Nucleoplasmin was prepared from unfertilized Xenopus eggs as described by Sealy et al. (42). In vitro chromatin assembly and transcription reactions.

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Chromatin assembly reactions with pUC5S, histones, and egg nucleoplasmin were conducted essentially as described previously (42). Plasmid DNA was defined to be fully assembled when upon treatment with topoisomerase I and removal of the histones with sodium dodecyl sulfate (SDS) and proteinase K the DNA had the same number of supercoils as plasmid DNA isolated from bacteria. For pUCSS (3,125 bp), this amount of assembly corresponds to the production of 16 to 18 negative supercoils. In the purified assembly system, the amount of histone required to obtain full assembly (i.e., 10lo) corresponds to a histone-to-DNA mass ratio of 0.9, and this amount of histone is defined as 1.0 unit. When lesser amounts of histone were used in the assembly reaction, the extent of assembly was quantitated by counting the average number of supercoils produced and dividing by 17 (the average of 16 and 18 that is fully assembled) and is expressed as a percentage of a complete assembly. For example, a reaction that introduces 12 supercoils into pUCSS is considered to have been assembled to 70% completion. Highly assembled chromatin templates were quantitated by resolving the supercoiled species by electrophoresis in the presence of chloroquine (2.5 ,ug/ml [data not shown] [49]). In all assembly reactions, the amount of egg nucleoplasmin added was based on a nucleoplasminto-histone mass ratio of approximately 1.4. In all assembly reactions, there is some DNA that migrates as relaxed DNA in the agarose gels. This is due to the presence of nicked DNA in the starting DNA preparation and the failure of topoisomerase to close these nicks. This relaxed DNA was found to be assembled into chromatin and to be capable of binding transcription factors and directing SS-specific RNA synthesis when analyzed by sucrose gradient centrifugation (unpublished observations). Nucleoplasmin and histones were preincubated in a small volume (5 RI or less) for 45 min at room temperature. Plasmid DNA (0.3 to 0.4 ,ug of relaxed or supercoiled plasmid; or plasmid complexed with transcription factors and purified over a Bio-Gel A-15M [Bio-Rad Laboratories, Richmond, Calif.] column, see below) was then added for the times indicated in the figures. Supercoiled DNA was relaxed with topoisomerase I (0.5 to 1.0 U/Rg of DNA [Promega Biotec, Madison, Wis.]) for approximately 15 min at the conclusion of the assembly reaction. Final assembly conditions (50-,ul volume) were 15 mM HEPES (pH 8.0), 0.1 mM EDTA, 12% (vol/vol) glycerol, 120 mM NaCl, and 1 mM dithiothreitol. To assay the assembled material for transcriptional activity, 10 or 20 ,ul of the assembly reaction was assayed in a 25-pl reaction volume (saving 40 or 30 ,u1 for agarose gel analysis of the degree of assembly) or the entire assembly reaction was assayed in a 70-,ul volume. Conditions for transcription were as described previously (12). From the known (limiting) amount of TFIIIA added, we estimated that '90% complete in 10 min) in our in vitro chromatin assembly system (Felts, Ph.D. thesis). We therefore wished to test whether transcription factor complexes could form on the yeast SS rRNA gene after variable amounts of nucleosome assembly had occurred in vitro. This is most easily accomplished by varying the histone/DNA ratios in in vitro chromatin assembly reactions. Using this approach, a graduated range of increasingly assembled chromatin templates could be established (Fig. 1A, top panel). After assembly, 5S gene templates were fully soluble since centrifugation of assembly reactions (15,000 x g for 10 min) failed to decrease the amount of DNA in solution in the supernatant (Fig. 1A, lanes p and s [pellet and supernatant]). This maintenance of template solubility was especially important since we have observed

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A FIG. 1. Transcription of chromatin templates assembled to various extents. (A) Plasmid pUC5S was assembled with increasing amounts of histone to yield templates assembled to various extents. Numbers above each pair of lanes indicate supercoil quantitation (see Materials and Methods); chromatin solubility was judged by a 3-min centfifugation at 15,000 x g; p, pellet; s, supernatant; std, standard. After 2 h of assembly, transcription factors and RNA polymerase III were added for an additional 30 min. Top panel, Ethidium bromide staining of assembled material resolved on a 2.2% agarose gel. Bottom panel, Autoradiogram of labeled transcripts from the corresponding assembly reactions resolved on a 10% polyacrylamide gel. (B) Nucleosome-induced inhibition of transcription occurs only in cis. The experimental protocol followed is depicted at the bottom of the figure. 5S rDNA chromatin was assembled to 0, 35, 88, or 100% of maximum by a 2-h incubation of pUC5S DNA with an appropriate nucleoplasmin (NP) to the histone mixture. A 5S maxigene template (p1171 DNA; see Fig. 4) was then added, immediately followed by rNTPs and RNA polymerase (RNAP) III and factors TFIIIA, TFIIIB, and TFIIIC. After a 30-min incubation, 32P-labeled RNAs were purified, resolved in a 10% polyacrylamide gel, and detected by exposure of the dried gel to X-ray film. The resulting autoradiogram is presented. Specific transcripts from the pUC5S and 5S maxigene are indicated by the labels and arrows.

that if any DNA precipitates during chromatin reconstitution, then this DNA is transcriptionally inert (Felts, Ph.D. thesis). The assembled templates were organized into nucleosomal arrays over the range of histone concentrations employed (data not shown). Each assembled template (and a naked DNA control with no histone) was tested for the accessibility of the 5S gene to transcription factors and RNA polymerase III. At the end of the 2-h assembly reaction, yeast transcription factors TFIIIA, TFIIIB, TFIIIC, RNA polymerase III, and nucleoside triphosphates (NTPs) (including [a-32P]GTP) were added to each assembled template. These transcription reactions were incubated for an additional 30 min, and the 32P-labeled RNAs synthesized were purified and resolved on a 10% polyacrylamide gel and visualized by autoradiography (Fig. 1A, bottom panel). Chromatin assembled to approximately 25% of completion (four supercoils; see Materials and Methods for details of quantitation of percent assembly) was about 65% as competent in 5S gene transcription as naked DNA (no histone). In contrast, 5S templates assembled to 50% or higher extents of assembly (eight or more supercoils) were essentially totally inaccessible to the class III gene transcription machinery. The exact dose-response (inhibition) curve was somewhat variable from experiment to experiment. This is probably due to a number of distinct factors such as difficulties in quantitating the exact extent of assembly and the fact that the amounts of the various transcription factor fractions used in these experiments were empirically determined to give the maximal transcription signal. Saturating amounts of factors were not used because adding excess factor fractions

inhibited transcription. These results are similar to the levels of histone that repressed adenovirus major late promoter activity in crude extracts (28, 59). Thus, the presence of only a modest number of nucleosomes of the 5S rRNA genecontaining template either greatly decreased the ability of yeast transcription factors to form a stable transcription initiation complex with this DNA or interfered with the interaction of RNA polymerase III with the binary factorDNA complex. Similar results were obtained when kinetic experiments were performed. DNA assembled with a full complement of histone (i.e., a sufficient amount of histone was added to allow for 100% assembly) but for various lengths of time became unavailable for transcription as soon as eight nucleosomes had been deposited on the DNA. This amount of assembly occurred in less than 5 min (data not shown). A control experiment with naked DNA added to the reaction after full assembly of a previously added template showed that the naked template was actively transcribed even though the assembled DNA was inactive. This indicates that the nucleosomes themselves are acting in cis to inhibit transcription directly (Fig. 1B). Prebinding of TFIIIA is not sufficient for formation of active 5S chromatin. The results described above showed that as soon as six to eight nucleosomes had formed on a plasmid containing 5S DNA, the 5S gene became transcriptionally inactivated. We therefore tested whether prior binding of one or more transcription factors would allow for the establishment of active templates that contained higher nucleosome densities. Previous studies with crude extracts from Xenopuis oocytes had indicated that binding of Xenopus

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FIG. 2. Effect of TFIIIA on the assembly of active 5S chromatin. Plasmid pUC5S was assembled into chromatin in the presence (+) or absence (-) of TFIIIA. The factor was preincubated with the plasmid DNA for 30 min. Assembly reactions were then conducted in the presence of various amounts of histone as in Fig. 1. After 2 h, transcription was assayed by the addition of RNA polymerase III, TFIIIB, and TFIIIC (TFIIIA was also added to the assembly reactions conducted without the factor). Top panel, Agarose gel analysis of the assembled templates. p, Pellet; s, supernatant. Numbers above each pair of lanes indicate supercoil quantitation. Bottom panel, Autoradiogram of the corresponding transcripts.

TFIIIA alone was sufficient to prevent subsequent nucleosome-induced inactivation of Xenopus 5S rRNA gene transcription (17). We therefore examined the ability of yeast TFIIIA to maintain accessibility of the yeast 5S gene to the other transcription factors (TFIIIB and TFIIIC) and RNA polymerase III upon subsequent chromatin assembly in a purified system. Yeast TFIIIA (Fig. 2, +) or factor buffer (Fig. 2, -) was incubated with pUC5S plasmid DNA for 30 min before its addition to the nucleoplasmin-histone mixture. As in Fig. 1, the amount of histone present in the assembly reaction was varied to yield chromatin templates assembled to various extents. After a 2-h assembly, the accessibility of the 5S template was assessed by the addition of transcription factors TFIIIB, TFIIIC, RNA polymerase III, and NTPs (and TFIIIA to - reactions). The analysis of chromatin assembly by induction of supercoils is shown in the top panel of Fig. 2. The DNA in the assembly reactions remained soluble in the presence of TFIIIA. The DNAs in the assembly reactions without histone were completely relaxed by topoisomerase I treatment. The presence of TFIIIA in the assembly reaction mixtures did not appear to have any significant topological effect on the 5S DNA (compare + lanes with - lanes). We noted that in the presence of small amounts of histone, the presence of the TFIIIA seemed to have a small topological effect, but this observation was not uniformly reproducible. In general, the amount of assembly (measured by the formation of supercoils) was proportional only to the amount of histone present in the reaction mixture. The results of the chromatin transcription reactions are shown in the bottom panel of Fig. 2. Transcription reactions programmed with naked DNA templates are shown in the first two lanes. Naked DNA incubated with TFIIIA was slightly less active than the template reacted with the factor without prior incubation. This slight decrease in transcription is presumably due to some loss of factor activity during

the 2-h assembly reaction since incubation of TFIIIA alone also leads to some inactivation (data not shown). Templates assembled to 20 and 50% of maximal nucleosome density were transcriptionally active whether assembled in the absence or presence of the factor. However, as observed in the experiment depicted in Fig. 1, the overall activity of these moderately assembled templates was decreased relative to that of naked DNA. Again, the exact amount of the decrease showed a small experiment-to-experiment variation, but the general trend was fully reproducible. As also observed for the results depicted in Fig. 1, more highly assembled chromatin templates were not transcribed. Therefore, from the results of these two experiments, we conclude that prebinding of TFIIIA alone does not appear to be sufficient to allow for assembly of the yeast 5S gene into transcriptionally active chromatin. These results suggested that the yeast 5S-TFIIIA complex was not stable to the addition of histones. This result might be expected from the work of others with Xenopus TFIIIA (15, 47, but see also reference 38). The stability of the yeast 5S rDNA-yeast TFIIIA complex was examined by gel mobility shift assays. The results of this experiment indicated that yeast TFIIIA is rapidly lost from the 5S gene ICR under the assembly conditions. Only a very small amount of factor remained bound to the DNA 5 min after the addition of histones (data not shown). Control experiments in which nucleoplasmin lacking histone was added to the reaction mixture containing the 5S ICR and TFIIIA showed an efficient binding of the transcription factor under the ionic conditions of the assembly. This binding was unaffected by a range of nucleoplasmin concentrations, and thus, the increased dissociation rate of TFIIIA-ICR complexes under assembly conditions is clearly not mediated by the assembly agent used in these experiments. Preformation of complete transcription factor complex permits assembly of transcriptionally competent 5S chromatin template. Since binding of TFIIIA to 5S rDNA was insuffi-

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FIG. 3. Assembly and transcription of complete preformed factor-5S rRNA gene complexes. Preformed 5S rDNA-TFIIIA-TFIIIBTFIIIC complexes were purified by gel filtration over A-15M resin as described in Materials and Methods. These complexes were then assembled for 2 h in the presence of various amounts of histones. Transcriptional activity was then assayed by the addition of NTPs followed by a 30-min incubation. Top panel, Autoradiogram of Southern hybridization of assembled templates resolved on an agarose gel. p, Pellet; s, supernatant. Bottom panel, Autoradiogram of the corresponding labeled transcripts. Numbers above each pair

of lanes show supercoil quantitation.

cient for the formation of active 5S chromatin, we tested the transcriptional activity of chromatin templates assembled with preformed transcription complexes. Since TFIIIA and TFIIIB do not form a stable complex and TFIIIA and TFIIIC only form a metastable complex with the 5S rRNA gene, we moved directly to a study of the complex of DNA and all three transcription factors. In these studies, yeast transcription factors TFIIIA, TFIIIB, and TFIIIC were incubated with 55 DNA to form stable multi-transcriptionfactor complexes. These complexes were then purified by gel filtration over a Bio-Gel A-15M column. Column chromatography was found to be crucial in working with these stable transcription factor-DNA complexes. Column purification of stable complexes was essential for maintaining nucleoprotein solubility during subsequent chromatin assembly (see Materials and Methods). After column chromatography, the transcription factor-DNA complexes were assembled into chromatin as described above for naked DNA templates. After assembly, RNA polymerase III and NTPs were added to each reaction to test the activity of chromatin templates which had been assembled to various extents. The results of such an experiment are shown in Fig. 3. In contrast to the results described above, 5S rRNA was efficiently synthesized from complete preformed factor-5S gene complexes assembled with nucleosomes up to 85 to 95% of maximum (the precise value varied from experiment

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to experiment). Since preformation of a TFIIIA-5S DNA complex was not sufficient to permit transcription of chromatin templates assembled to these nucleosome densities, we conclude that formation of a complete transcription complex is required before assembly to permit the establishment of active chromatin in vitro. The observation that templates containing complete TFIIIA-TFIIIB-TFIIIC-5S gene complexes and 17 nucleosomes (i.e., 100% assembled) were transcriptionally inactive was intriguing. This result may be explained by the fact that when chromatin is extensively assembled with purified histones in the presence of egg nucleoplasmin, the average nucleosome spacing is approximately 140 bp (Felts, Ph.D. thesis). This degree of spacing is significantly less than that obtained with crude assembly extracts (180 to 200 bp [10]) and is also less than that observed for bulk yeast chromatin (160 bp [30; Felts, Ph.D. thesis]). It is possible that in the in vitro system, nucleosomes become too crowded at higher levels of assembly to permit appropriate transcription when the template is fully assembled. This hypothesis can only be tested once the factor(s) involved in nucleosome spacing becomes known. In our experiments, we therefore avoided the use of these abnormally high inhibitory levels of histone in the assembly reactions. Construction of 5S maxigenes. Our ability to successfully assemble transcriptionally active 5S chromatin in vitro allowed us to extend our experimental approach and test whether or not RNA polymerase III can efficiently transcribe through extended arrays of nucleosomes in vitro. We constructed a series of 5S maxigenes by inserting 1, 2, 4, or 10 copies of a 117-bp BstEII restriction fragment from bacteriophage lambda (sequences 13572 to 13689 bp) into the yeast 5S gene at position +93 (Fig. 4). Insertion of the lambda DNA sequences fortuitously re-forms the DNA sequence of the yeast 5S gene ICR up to position + 100. The family of maxigenes (termed 1171, 1172, 1174, and 1171o) formed by these manipulations were all transcribed efficiently in vitro as naked DNA templates (see below). These same templates were also subcloned into a single-copy yeast vector, pMB272 (21), to study their transcriptional activity and chromatin structure in vivo. This vector contains the URA3 gene for use as a selectable marker as well as ARSl and CEN4 sequences for autonomous replication, plasmid stability, and low copy number of yeasts (3). The general structure of the CEN117, constructs in plasmid pBM272 is diagrammed in Fig. 4. For in vitro studies, the 5S maxigenes were subcloned into pGEM 7f(+) (Promega). The smaller sizes of these subcloned constructs greatly aided the assessment of nucleosome assembly by agarose gel electrophoresis supercoiling assays. RNA polymerase m cannot efficiently transcribe through an extended array of nucleosomes in vitro. The ability of RNA polymerase III to negotiate a long array of nucleosomes was tested in the following way. 5S wild type or 5S maxigene TFIIIA-TFIIIB-TFIIIC-polymerase III complexes were formed (see Materials and Methods) and then assembled into chromatin in the presence of various amounts of histone. The assembled material was transcribed, and the 32P-labeled transcripts produced were purified and resolved on a 10% polyacrylamide gel (Fig. 5A). The data show that the assembled 5S gene and the smaller maxigenes (1171, 1172, 1174) were transcribed efficiently. The slight decrease in transcription observed with increasing assembly was approximately the same for all these templates. In contrast, the amount of RNA synthesized from 11710 was drastically reduced when the template was assembled into chromatin (RNA synthesis

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FIG. 4. Structure of 5S maxigenes and CEN117X constructs. A total of 1, 2, 4, or 10 head-to-tail copies (verified by DNA sequencing) of a 117-bp BstEII fragment from bacteriophage lambda were cloned into the BstEII site at +93 of the yeast 5S gene in pUC5S. EcoRI-BamHI fragments containing these maxigenes were excised from pUC5S clones and then inserted into the yeast centromeric plasmid pBM272, previously digested with EcoRI and BamHI. was reduced to 10% of control values at only 60% assembly and to