RSC-dependent elongation requires a histone acceptor. (A) In vitro transcription with RSC in the presence or absence of acceptor DNA. Reactions containing 1 ng (0.3 nM) of C-tailed nucleosome template, 0.9 nM pol II, and 0, 0.2, or 2 nM RSC were incubated with ATP in the presence or absence of 10 ng pGEM3Z601R for 60 min. Nucleoside triphosphates containing ³²P-CTP were added for 15 min and the products were resolved on a 7 M urea 10% acrylamide gel. A PhosphorImage is shown. FL indicates the full-length transcript and arrows point to the nucleosomal arrests. Quantitation of the full-length product is indicated below each lane. (B) Nucleosome remodeling/eviction reactions with RSC in the presence or absence of acceptor DNA. Reactions containing 0.3 nM of ³²P-labeled mononucleosome template and 0, 0.6, 2, or 6 nM RSC were incubated with 2 mM ATP in the presence or absence of 10 ng pGEM3Z601R for 60 min, poly(dI:dC) was added, and the products were resolved by 4.5% native PAGE. An autoradiograph is shown. Quantitation of free DNA is shown below the gel. (C) In vitro transcription assay screening various histone chaperones. Transcription reactions with 0.9 nM pol II contained, from left to right, no addition, 2 nM RSC, and 2 nM RSC with either 42 nM NAP1, 53 nM SPT6, 49 nM FACT, 10 ng pGEM3Z601R, or 44 nM ASF1. The mean amount of FL RNA from two independent experiments is shown below each lane. A bar graph display of the data is shown in Fig. S3B.

NAP1-dependent elongation requires RSC, but not nucleosome eviction. (A) In vitro transcription reactions were performed with 0, 0.2 , 0.7, or 2 nM RSC and 14 nM (+) or 43 nM (+++) NAP1. Quantitation of full-length RNA is shown below the gel. (B) Time course of in vitro transcription with 2 nM pol II, 2 nM RSC, and 43 nM NAP1. Quantitation of full-length (FL) RNA from three independent experiments is indicated below the gel. A bar graph of the quantitation is displayed in Fig. S3B. (C) Nucleosome remodeling assay with 0.6 nM RSC and 1.3 or 13 nM NAP1. No acceptor DNA was added. The question mark (?) indicates a NAP1-dependent product. (D) A nucleosome eviction assay with NAP1 was performed with 10 ng acceptor DNA and 0.6–17 nM RSC. NAP1, 4.8–130 nM, was titrated into reactions containing 17 nM RSC. Quantitation of free DNA is shown below the gel.

NAP1 stimulates RSC-dependent hexasome formation. (A) Remodeling assay with NAP1 using nucleosomal substrates containing labeled DNA (*DNA), H3 (*H3), or H2A (*H2A) where indicated. Reactions contained 2 nM RSC, 43 nM NAP1, and 10 ng acceptor DNA where indicated. The hexasome migrates below the remodeled nucleosome (“Hex”). Because of differences in the labeling efficiency, a darker exposure of H3 is shown. (B) Quantitation of the histone to DNA ratios of the remodeled nucleosome and hexasome from A and C. The means from three independent experiments are displayed with the standard deviations as error bars. The ratios were calculated by dividing the intensity for each remodeled nucleosome or hexasome by the intensity of untreated nucleosome (Rem. Nuc/Nuc or Hex/Nuc). The value obtained for either *H3 or *H2A was then normalized to the value obtained for DNA as measured by Cy5 or ³²P label. P values were calculated using a two-tailed Student’s t test. (C) Remodeling assay as in A using Cy5 labeled DNA with ³²P-H3 (*H3) or ³²P-H2A (*H2A). Left panels measure fluorescence intensities of Cy5 DNA template, whereas right panels are PhosphorImages of *H3 or *H2A. As in A, a darker exposure of H3 is shown. (D) Remodeling assay with 0.3, 1, 4, or 16 nM H2A-H2B dimer added to reactions containing 2 nM RSC and 43 nM NAP1.

Hexasome is maintained during transcription. (A) In vitro transcription reactions with C-tailed nucleosomal templates ³²P-labeled on DNA, *H3, or *H2A. The reactions contained unlabeled nucleoside triphosphates (NTPs), with 2 nM pol II, 2 nM RSC, 43 nM NAP1, and/or 10 ng acceptor DNA, where indicated. After addition of poly(dI:dC), the products were resolved by 4.5% native PAGE. A PhosphorImage of the gel is shown. As in Fig. 3, a darker exposure of H3 is shown. (B) In vitro transcription time course (5, 15, or 45 min) as in Fig. 2B with ³²P-labeled nucleosomal templates (*DNA, *H3, or *H2A) (Txn time, transcription time). (C) Quantitation of A. The histone to DNA ratios for the pol II:Nuc band (which migrates slightly faster when remodeled by RSC) was quantified for each condition. Calculations were performed as in Fig. 3C. P values were calculated as in Fig. 3B. (D) Quantitation of B. Histone to DNA ratios for the pol II:Hex band were calculated as in Fig. 3B. P values were calculated using an ANOVA test.

Hexasomes are continually present. In vitro transcription reactions were performed as in Fig. 4B using ³²P-labeled DNA templates. After transcription for the indicated times, C-tail competitor was added and the products were separated by 4.5% native PAGE. A PhosphorImage is shown.