1. Nature of the Nucleosomal Barrier to RNA Polymerase II
Maria L. Kireeva, Brynne Hancock, Gina H. Cremona, Wendy Walter, Vasily M. Studitsky, Mikhail Kashlev 
Molecular Cell 
Volume 18, Issue 1, Pages (April 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1
The Nucleosome Triggers Conversion of Transient Pauses to Transcription Arrests
(A) Experimental setup. TEC9 is ligated to nucleosomes assembled on the 204 bp (N1 and N2) or 164 bp (N1short) DNA fragment.
(B) Sites of nucleosome-induced arrest are independent of nucleosome position and coincide with sites of transient pausing on the free DNA. TEC9 ligated to the nucleosome was treated with Fnu4HI, which digests only free DNA, or with EcoRI, which cleaves off the N2 nucleosome, walked to TEC64, and chased with 1 mM NTP in TB150. Transcription of the DNA digested with Fnu4HI and EcoRI results in the formation of a 126 nt RNA and 82 nt RNA, respectively. TEC64 obtained on the free DNA template was incubated with increasing concentrations of NTPs. Three major clusters of pauses/arrests relative to the positions of the nucleosome are shown by the blue boxes between the panels.
(C) The nucleosome promotes arrest of the stalled TECs. TEC9 ligated to 164 bp DNA or N1short (which was purified from the free DNA) was walked to TEC45, 55, 64, 72, and 78. TEC64 obtained on the N1short nucleosome was chased for 15 min with the indicated concentrations of NTP in TB40 (left). TECs were incubated in TB40 in the absence of NTP for the indicated time (10 or 120 min), and the extent of the arrest was determined by incubation of the TEC with 5 μM NTP subset for 5 min (right).
(D) The nucleosome-specific and sequence-specific arrests vary at different positions within the nucleosome. The experiment was done as described for (C), right. The hatched bars and blue bars indicate the fraction of the TECs that failed to extend the RNA after 2 hr halting on the free DNA and on the nucleosome, respectively. The arrest on the nucleosome was normalized to the arrest in the same position on the nucleosome-free DNA: arrested TECnucleosome normalized to the DNA = 1 − (active TECnuclesome/active TECDNA).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

3. Figure 2 The Nucleosome Promotes Extensive Backtracking of Pol II
TEC64 labeled at positions +56, +57, and +63 was obtained on N1short or free DNA. To obtain TECs arrested on the nucleosome-free DNA, TEC64 was incubated for 5 min with 3 μM NTP and washed (TEC64+ arrested). TEC64 on N1short was incubated for 5 min with 1 mM NTP in TB40 and either left with NTPs (TEC64, NTP) or washed (TEC64+ arrested). The resulting TECs were treated with 0.3 μM TFIIS for 10 min in TB40. The products of TFIIS-induced endonucleolytic cleavage were released from the immobilized pol II into the supernatant. Odd lanes (S) are loaded with one-half of the supernatant. Even lanes (P) are loaded with the remaining supernatant and the entire pellet. An equal distribution of the RNA product between the S and P fractions indicates its complete release from the immobilized pol II.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

4. Figure 3
Limiting the Extent of Backtracking Reduces the Nucleosome-Induced Arrest of the TEC
(A) Experimental setup. Backtracking is blocked by an oligo complementary to the transcript or by truncation of the RNA.
(B) An antisense oligo decreases the catalytic inactivation of the TEC on a nucleosomal template. TEC64 was obtained as in Figure 1. The last walking step was done in the presence or absence of 10 μM antisense oligo complementary to positions +39–+48 of the transcript, then the TEC was washed and chased with 1 mM NTP in TB150 for 15 min with or without the same oligo.
(C) Truncation of the 5′ end of the RNA suppresses inactivation of the TEC. TEC64 labeled at positions +56, +57, and +63 as described in Figure 2 was obtained on N1 + N2 nucleosomes, treated with 10 U of RNase T1, washed, and chased with 1 mM NTP in TB150 for 15 min. Red arrows in (B) and (C) indicate arrested TEC64.
(D) Cotranscriptional removal of the 5′ end portion of the RNA promotes pol II passage through the nucleosome. TEC9 with the template DNA strand labeled at the 5′ end was ligated to the N1short nucleosome or 164 bp DNA and chased in TB40 with 1 mM NTP with or without 0.5 unit/μl RNase T ng of the 160 bp DNA was added to the reaction to promote release of the transcribed template from pol II after 15 min incubation with NTPs. Although transcription was completed after 5 min incubation with NTP, the DNA release continued for 15 min on both free DNA and nucleosome templates due the slow dissociation of the TEC that has reached the DNA end.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

5. Figure 4 Block of Backtracking Leads to Nucleosome Rearrangement
TEC9 with 5′ end-labeled RNA was formed on the N1short nucleosome. TEC64 was formed on the N1short nucleosome as described in Figure 2, and one half of TEC64 was treated with 10 U of RNase T1. TECs were extensively washed with TB40 and digested with SacI, ApaI, Fnu4HI, or EaeI in TB40 containing 0.2 mg/ml BSA for 30 min. The enzymes were removed by washing with 1 ml of TB40 and 1 ml of TB1000, and TECs were chased with 1 mM NTP in TB1000. The run offs from the full-length and digested templates were quantified by using a phosphorimager, and the fraction of the digested template was calculated. The cyan bars show accessibility of the DNA in TEC9; the blue bars, in TEC64; and the green bars, in TEC64 treated with RNase T1. The data represent the average of three independent experiments, and error bars show SD.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

6. Figure 5
Truncation of DNA Upstream of a TEC Stalled within a Nucleosome Does Not Affect the Pattern or Efficiency of Nucleosome-Induced Arrest
(A) TEC9 was assembled with the template and nontemplate DNA strands carrying the BsrBI recognition site ligated to N1short or nucleosome-free DNA, and both ends of the DNA were labeled. TEC9 was walked to TEC64 and treated with RNase T1. One-half of TEC was treated with 20 U of BsrBI for 40 min.
(B) TEC64 was obtained exactly as in (A), but instead of the DNA labeling, the RNA was labeled as described in Figure 2. The chase was done with 1 mM NTP for 10 min in TB40 or TB150.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

7. Figure 6 TFIIS Promotes Transcription of the Nucleosomal Template
TEC9 with the RNA labeled at the 5′ end was ligated to N1 + N2, walked to the starting position, and eluted from Ni-NTA agarose.
(A) TEC45 was chased with 1 mM NTP in TB40 with or without 0.1 μM TFIIS. The reaction was stopped after 0.5, 1.5, 5, 15, and 45 min.
(B) TFIIS reactivates TECs arrested on the nucleosomal template. TEC64 was incubated with 1 mM NTP in TB40 for 5 min (lanes 2 and 7) to form TECs arrested within the nucleosome, and 1 μM TFIIS was added to the chase reaction for the additional 10 min (lanes 4 and 9). In a separate reaction, 1 mM NTP was added together with TFIIS to TEC64 (lanes 5 and 10).
(C) The effect of TFIIS does not depend on the source of histone proteins used for the nucleosome reconstitution. TEC45 was obtained on nucleosome reconstituted by chromatin exchange (native nucleosomes), or by Nap1-mediated assembly from yeast recombinant histones (recombinant nucleosomes) and chased with 1 mM NTP in TB40 or TB300 in the presence of 0.3 μM TFIIS.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

8. Figure 7
The Nucleosomal Barrier to Pol II: Nucleosome Stabilizes Backtracked Conformation of the TEC
See details in the text.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions