Supplementary Information. Synergistic action of RNA polymerases in overcoming the nucleosomal barrier

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1 Supplementary Information Synergistic action of RNA polymerases in overcoming the nucleosomal barrier Jing Jin, Lu Bai, Daniel S. Johnson, Robert M. Fulbright, Maria L. Kireeva, Mikhail Kashlev, Michelle D. Wang Supplementary Figure 1. Experimental setup Supplementary Figure 2. Inhibition of RNAP diffusive translocation by depletion of Mg 2+ Supplementary Figure 3. Heparin, competitor DNA and RNase T1 do not alter the unzipping force signature of an RNAP or a nucleosome Supplementary Figure 4. RNAP pausing pattern within a nucleosome is independent of DNA downstream of the nucleosome Supplementary Figure 5. Comparison of intrinsic pause sites with nucleosome-induced pause sites Supplementary Figure 6. 5 min transcription result obtained from bulk transcription gel analysis and single molecule unzipping method Supplementary Figure 7. Location of RNAP on DNA in a PTC Supplementary Figure 8. An RNAP did not dissociate until it reached the runoff end Supplementary Discussion Calculation of runoff efficiencies Supplementary References Nature Structural & Molecular Biology: doi:1.138/nsmb.1798

2 a Digoxygenin Nick Biotin 61 ~1.3 kbp Anchoring segment ~.8 kbp Unzipping segment b dsdna 61 Optical Trap RNAP microsphere ssdna Coverslip is moved to unzip dsdna Supplementary Figure 1. Experimental setup. Experimental configuration is similar to that previously described 1-4. (a) Each DNA template for single molecule experiments consisted of an anchoring segment and an unzipping segment, which were ligated together leaving a nick at the ligation site (Methods for details). As illustrated by a single-promoter template here, the anchoring segment was dig-labeled at the distal end, while the unzipping segment was labeled with a biotin 5 bp away from the nick. (b) The DNA template was attached at one end to the surface of a glass coverslip via a digoxigenin-antidigoxigenin linkage and at its nick to a microsphere via a biotinstreptavidin linkage. As the coverslip was moved away from the trapped microsphere using a loading-rate clamp, the dsdna was sequentially converted into ssdna upon base pair separation. RNAP and nucleosome are not drawn to scale. Nature Structural & Molecular Biology: doi:1.138/nsmb.1798

3 a RNAP Fraction ºC, 2 min Expected range +19 bp b RNAP Fraction RT, 2 min EDTA quench +22 bp c RNAP Fraction RT, 2 min EDTA quench 37ºC, 3 min +22 bp Position relative to transcription start site (bp) Nature Structural & Molecular Biology: doi:1.138/nsmb.1798

4 Supplementary Figure 2. Inhibition of RNAP diffusive translocation by depletion of Mg 2+. To determine if the depletion of Mg 2+ by EDTA quenching may minimize RNAP diffusive motion along the DNA in an elongation complex, we performed experiments to compare RNAP force rise locations in the PTC formed under three different conditions. The mean position of each distribution is indicated by an arrow. (a) PTC formation for 2 min at 37ºC (Methods) is known to encourage RNAP backtracking. Indeed, the mean position of the RNAP location distribution was found to be ~ +19 bp, outside the expected range of location ( bp). (b) PTC was allowed to form under room temperature (RT) and quenched after 2 min by EDTA (same as Fig. 1e). The mean position of the RNAP location distribution was found to be at ~ +22 bp, within the expected range of location. (c) In order to examine whether depletion of Mg 2+ by EDTA quenching can preserve the RNAP translocation state by minimizing its diffusive motion along the DNA, the experiment began as in Supplementary Figure 2b, and was followed by a further incubation at 37ºC for 3 min. Unzipping experiments revealed that both the mean location and the standard deviation of the RNAP locations were essentially unaltered, indicating that depletion of Mg 2+ by EDTA quenching indeed inhibits RNAP diffusive translocation in an elongation complex. Nature Structural & Molecular Biology: doi:1.138/nsmb.1798

5 a c RNAP near the +2 nt position (%) Force (pn) Force (pn) Competitor DNA + Competitor DNA Reaction time (min) RNAP at +2 Transcription Time 1 s 1 min 5 min + RNase T1 Nucleosome + RNase T1 b d Force (pn) Force (pn) Heparin (mg/ml) Nuc. DNA Heparin Heparin Position relative to dyad (bp) Position relative to dyad (bp) Nature Structural & Molecular Biology: doi:1.138/nsmb.1798

6 Supplementary Figure 3. Heparin, competitor DNA and RNase T1 do not alter the unzipping force signature of an RNAP or a nucleosome. (a) Competitor DNA prevents re-initiation on nucleosomal templates. An experiment was conducted to determine how effective competitor DNA was in preventing re-initiation. The competitor DNA was 25 bp in length and contained a single T7A1 promoter..4 nm single promoter template, in the absence or presence of competitor DNA, was mixed with 2 nm RNAP together with 1 mm ApUTP, ATP, GTP, and CTP at concentrations identical to those used in experiments described in the text. This allowed any RNAP that might initiate from the promoter to advance to at most the +2 nt position. Transcription reactions were quenched by the addition of EDTA at given time points and the percentage of DNA that formed a PTC near the +2 nt position was determined by DNA unzipping. Data are shown as (mean ± s.e.) and smooth curves were drawn for clarity (not fits). In the absence of competitor DNA, a PTC was able to form on the experimental DNA template in ~ 1 min and the percentage formed plateaued at ~ 75% by 3 min. However, in the presence of competitor DNA, only a very small fraction of the experimental DNA templates formed a PTC. Even after 3 min, this fraction was < 5%. Competitor DNA ensured single round transcription in the experiments described in Figures 1-5. To exclude the possibility that competitor DNA dissociated the RNAP and/or nucleosome from experimental DNA templates, RNAP was first walked to the +2 nt position on nucleosomal DNA templates, and after 3 min incubation in the presence or absence of competitor DNA, the percentage of the RNAP/nucleosome remaining on the DNA template was examined using the unzipping method. The presence of competitor DNA had no detectable effect on the binding or unzipping Nature Structural & Molecular Biology: doi:1.138/nsmb.1798

7 signatures of the RNAP/nucleosomes (data not shown). (b) Increasing heparin concentration converted nucleosomes to naked DNA. Heparin has previously been shown to completely or partially remove histones 5. Here native gel analysis confirmed that increasing heparin concentration converted nucleosomal DNA to naked DNA. This was further confirmed by DNA unzipping assays (data not shown). In all experiments presented in the main text requiring histone dissociation, 4 mg ml -1 of heparin was used. (c) RNase T1 does not alter the unzipping force signature of an RNAP or a nucleosome. (Upper) In order to check whether RNase T1 alters RNAP and/or nucleosome unzipping signatures, PTCs were formed on the single promoter nucleosomal templates and the sample was incubated with 5 units ul -1 RNase T1 at room temperature for 3 min. Unzipping data show unaltered signatures for both a PTC and a nucleosome prior to transcription (black). Unzipping pattern of naked DNA with the same sequence is also shown for comparison (grey). (Bottom) Transcription was resumed in the presence of RNase T1 and then stopped by EDTA. The histones were subsequently removed by heparin. Representative traces of unzipping through an elongation complex are shown after indicated transcription times, and they show identical unzipping signatures to those in the absence of RNase T1. Arrows indicate locations of RNAPs. (d) Heparin does not dissociate RNAP or alter its unzipping signature. After a PTC was formed on a nucleosomal template, the sample was incubated with heparin at room temperature for 3 min, and the DNA was unzipped. The unzipping data show that heparin only dissociated histones without dissociating the RNAP or altering its location and/or unzipping signature. Nature Structural & Molecular Biology: doi:1.138/nsmb.1798

8 Runoff 5 min M s 1 s 3 s 1 min 5 min 1 min 3 s 1 s s M M Runoff Dyad 3 Dyad Supplementary Figure 4. RNAP pausing pattern within a nucleosome is independent of DNA downstream of the nucleosome. To determine if the RNAP pausing pattern was dependent on the naked DNA downstream of the nucleosome, we repeated the transcription gel assay shown in Figure 2a (shown again in left panel for comparison) using a DNA template that lacked a segment downstream of the 61 NPE (right panel). A very similar pausing pattern was found, both in terms of pausing sites and the 1 bp periodicity. Transcription times are indicated on the top of each gel image. M stands for 1 bp DNA marker. Nature Structural & Molecular Biology: doi:1.138/nsmb.1798

9 a b Transcription time 1 [NTP] (um), 2 5 Nucleosome Naked DNA Runoff RNAP population (AU) Naked DNA 5uM NTP 1 Dyad scanned region RNAP population (AU) uM NTP s 1 min RNA location relative to dyad (bp) Supplementary Figure 5. Comparison of intrinsic pause sites with nucleosomeinduced pause sites. (a) We compared transcription gels from naked 61 NPE reverse template (left panel) with the corresponding nucleosomal template (right panel). In order to enhance intrinsic pausing on the naked DNA, we lowered the NTP concentration and quenched the reaction after 2 seconds. Right panel is identical to Figure 2b right panel. (b) Quantitative analysis of the gel shown in a. As shown, the intrinsic pausing sites do not display a 1-bp periodicity and in general do not completely coincide with the Nature Structural & Molecular Biology: doi:1.138/nsmb.1798 Nucleosome 2-9 nucleosome-induced pausing sites. -1

10 a b RNAP fraction RNAP population (AU) 2 39 bp Region 1 Region 2 t = 5 min Transcription gel scan RNA location relative to dyad (bp).3 49 bp Single molecule.2.1 c RNAP fraction bp Single molecule + RNase T RNAP active site relative to dyad (bp) Nature Structural & Molecular Biology: doi:1.138/nsmb.1798

11 Supplementary Figure 6. 5 min transcription result obtained from bulk transcription gel analysis and single molecule unzipping method. All experiments were conducted using the DNA template shown in Figure 1d. The transcription reaction was performed for 5 min. The mean location of each distribution is indicated by a dashed line. (a) An intensity scan of the gel shown in Figure 2a. The 3 RNA location is specified relative to the dyad. The predominant peak positions indicate RNAP pause sites. (b) Distribution of RNAP active site location as determined by the unzipping method. The active site location is specified relative to the dyad. The displacement between the mean location of the active site and that of the 3 end of the RNA indicates the RNAP backtracking distance. (c) Distribution of RNAP active site location in the presence of RNase T1. Nature Structural & Molecular Biology: doi:1.138/nsmb.1798

12 a b Relative to transcription start site (bp) RNAP remaining near the +2 nt position (%) RNAP 2 RNAPs, Leading RNAP 2 RNAPs, Trailing RNAP Transcription time (min) Expected RNAP location Transcription time (min) 1 RNAP 2 RNAPs, Trailing RNAP Nature Structural & Molecular Biology: doi:1.138/nsmb.1798

13 Supplementary Figure 7. Location of RNAP on DNA in a PTC. (a) Location of RNAP on DNA in a PTC. To determine the location of an RNAP on the DNA template when a PTC was formed with an RNA length of +2 nt, we unzipped DNA through the PTC on both the single promoter template (Fig. 1b) and the two-promoter template (Fig. 4a) prior to NTP addition and at different time points after NTP addition. EDTA was used to quench the reaction before single molecule data were taken. Only RNAP detected near the +2 nt position were examined in order to reveal the time course of escape from the stalled state. Data points are represented as (mean ± s.e.). The smooth curves were drawn for clarity (not fits). Percentages of RNAP remaining near the +2 nt position on the single-promoter template and the two-promoter template were plotted as a function of transcription time. The majority of the RNAP resumed transcription immediately (< 1 s, the shortest measurement time) after 1 mm NTP was added. Only < 5% of the RNAP remained near + 2 nt after 5 min in all cases. (b) Experiments were done at the same condition as in b. The location of RNAP remaining was plotted against transcription time. Before NTP addition, the mean location of the force rise was found to be ~ 3 bp upstream of the expected location (grey dashed line at +22 bp). As the transcription time increased, the fraction remaining near + 2 nt decreased, and the mean location of the remaining RNAPs shifted upstream away from the expected location for both RNAPs on the single promoter template and trailing RNAPs on the two-promoter template. This indicates that more extensively backtracked RNAPs took longer to exit a backtracked state. Nature Structural & Molecular Biology: doi:1.138/nsmb.1798

14 6 6 RNAP runoff (%) 4 2 Bulk Single molecule 4 2 Naked DNA (%) Transcription time (min) Supplementary Figure 8. An RNAP did not dissociate until it reached the runoff end. We examined RNAP runoff and dissociation from the DNA template as a function of transcription time. The runoff percentage was determined from the transcription gel shown in Figure 2a. The naked DNA percentage was determined from single molecule experiments such as those shown in Figure 1f. The excellent agreement between these data indicates that RNAP only dissociated at the runoff end. This finding was also supported by previous studies that separately assayed RNA associated with, versus released from, a DNA template 6. In those studies, the released RNA, resulting from RNAP dissociation, was only full-length transcript. Previous studies also showed that front-to-back collision between two E. coli RNAPs did not induce RNAP dissociation 7. Nature Structural & Molecular Biology: doi:1.138/nsmb.1798

15 Supplementary Discussion Calculation of runoff efficiencies In order to calculate the runoff efficiency shown in Figure 5, we needed to correct for two small but significant contributions: templates without a nucleosome at the 61 NPE or without the formation of a PTC. Using the DNA unzipping method, we determined conditions under which the probability of PTC formation at a promoter ( P RNAP ) was at least 9% and the probability of a template containing a nucleosome ( P nuc ) was also at least 9%. For the single-promoter template, at a given transcription time, if the measured probability of a template being naked was P 1RNAP, raw, then the corrected runoff efficiency was calculated as P ( 1! P P ) 1RNAP 1RNAP, raw RNAP nuc =. PRNAP Pnuc P! For the two-promoter template, at a given transcription time, if the measured probability of a template being naked was P, containing a single RNAP P 1, and containing two RNAPs P 2, then the corrected runoff efficiency of the leading RNAP was calculated as leading P P = 1!, P P 2 2 RNAP nuc and of the trailing RNAP: P trailing = P! (1! P nuc )! 2P RNAP 2 RNAP P P P nuc nuc (1! P RNAP ) P 1RNAP Nature Structural & Molecular Biology: doi:1.138/nsmb.1798

16 Supplementary References 1. Koch, S.J., Shundrovsky, A., Jantzen, B.C. & Wang, M.D. Probing protein-dna interactions by unzipping a single DNA double helix. Biophys J 83, (22). 2. Jiang, J. et al. Detection of high-affinity and sliding clamp modes for MSH2- MSH6 by single-molecule unzipping force analysis. Mol Cell 2, (25). 3. Shundrovsky, A., Smith, C.L., Lis, J.T., Peterson, C.L. & Wang, M.D. Probing SWI/SNF remodeling of the nucleosome by unzipping single DNA molecules. Nat Struct Mol Biol 13, (26). 4. Hall, M.A. et al. High-resolution dynamic mapping of histone-dna interactions in a nucleosome. Nat Struct Mol Biol 16, (29). 5. Bancaud, A. et al. Nucleosome chiral transition under positive torsional stress in single chromatin fibers. Mol Cell 27, (27). 6. Kireeva, M.L. et al. Nucleosome remodeling induced by RNA polymerase II: loss of the H2A/H2B dimer during transcription. Mol Cell 9, (22). 7. Toulme, F., Guerin, M., Robichon, N., Leng, M. & Rahmouni, A.R. In vivo evidence for back and forth oscillations of the transcription elongation complex. EMBO J 18, (1999). Nature Structural & Molecular Biology: doi:1.138/nsmb.1798