bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 RNA I fidelity, speed and processivity depend on the 2 interplay of its lobe binding subunits 3 4 Philipp E. Merkl#1 , Michael Pilsl1, Tobias Fremter, Gernot Längst, Philipp Milkereit*, 5 Joachim Griesenbeck* and Herbert Tschochner* 6 7 Universität Regensburg, Biochemie-Zentrum Regensburg (BZR), Lehrstuhl Biochemie III, 8 93053 Regensburg, Germany 9 10 1 contributed equally 11 # Current address: Dept. of Microbiology and Immunobiology, Harvard Medical School, 12 77 Avenue Louis Pasteur, Boston, MA 02115 13 * Corresponding authors: H.T ([email protected]), J.G. 14 ([email protected]), P.M. [email protected]) 15 bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

16 Abstract 17 Eukaryotic RNA I and III (Pol I and III) consist of core subunits, which are 18 conserved in RNA polymerase II (Pol II). Additionally, Pol I and III have specific subunits, 19 associating with the so-called ‘lobe’ structure first described within Pol II. In Pol I of the 20 yeast S. cerevisiae, these are Rpa34.5, and the N-terminal domains of Rpa49 and 21 Rpa12.2, here referred to as the lobe-binding module (lb-module). We analyzed 22 functions of the lb-module in a defined in vitro transcription system. Cooperation 23 between lb-module components influenced transcription fidelity, elongation speed, and 24 release of stalled Pol I complexes to continue elongation. Interestingly, lb-module 25 containing Pol I and III, but not Pol II, were able to transcribe nucleosomal templates. 26 Our data suggest, how the Pol I specific subunits may contribute to accurate and 27 processive transcription of ribosomal RNA genes. 28 bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

29 Introduction 30 The eukaryotic RNA polymerases I, II and III (Pol I, II, III) have a similar architecture and 31 contain ten conserved subunits which form the catalytic core. Furthermore, they share a 32 more distantly related heterodimer at the periphery of the , designated as the 33 stalk, formed by Rpa14/Rpa43, Rpb4/Rpb7 and Rpc17/Rpc25 in Pol I, Pol II and Pol III, 34 respectively (see as review (Khatter et al., 2017)). In the yeast S. cerevisiae Pol I and Pol 35 III have additional subunits associated. The heterodimer Rpa34.5/Rpa49 of Pol I 36 corresponds to Rpc37/Rpc53 of Pol III (Khatter et al., 2017) and associates together 37 with the N-terminal part of Rpa12.2 to the Pol I lobe structure forming a lobe binding 38 module (lb-module). Based on structural homology, localisation on the core enzyme and 39 function Rpa34.5/Rpa49 are considered as counterparts of the Pol II transcription 40 initiation factors TFIIE and TFIIF (Kuhn et al., 2007, Jennebach et al., 2012, Geiger et al., 41 2010). The heterodimer Rpa34.5/Rpa49 consists of a dimerization module formed by 42 Rpa34.5 and the N-terminal part of Rpa49 (full length Rpa34.5 and aa 1-110 of Rpa49), 43 the A49 linker (aa 105-187 of Rpa49) and the C-terminal part of Rpa49 (Rpa49CT, aa 44 187-415). The dimerization module binds to the Pol I lobe domain on the side of the 45 active center which is opposite to the stalk (Jennebach et al., 2012, Fernandez-Tornero 46 et al., 2013, Engel et al., 2013) and occupies a similar position as the TFIIF dimerization 47 module in association with Pol II (yeast Tfg1/Tfg2) (Geiger et al., 2010, Kuhn et al., 48 2007). The C-terminal part of Rpa49 forms a tandem winged helix (tWH) similar as in 49 the small TFIIE subunit Tfa2 (Geiger et al., 2010) (Okuda et al., 2000) or in RPC37 50 (Hoffmann et al., 2015). Although this domain cannot be unambiguously localized in 51 current structures of Pol I, there is evidence for a preferential localization at the DNA 52 binding cleft of Pol I at the interface between the stalk and the upstream DNA 53 (Jennebach et al., 2012, Pilsl et al., 2016b, Engel et al., 2013, Tafur et al., 2016). 54 Interplay between the Pol II factors TFIIF and the RNA cleavage factor TFIIS was 55 reported to promote transcript elongation and increase transcription through 56 in a synergistic manner (Schweikhard et al., 2014, Luse et al., 2011). 57 Similar to the Pol II counterparts the Pol I subcomplex Rpa34.5/Rpa49 was suggested to 58 enhance RNA elongation and cleavage (Kuhn et al., 2007, Liljelund et al., 1992, Beckouet 59 et al., 2008, Geiger et al., 2010) and to stabilize the initiation complex (Tafur et al., 2016, 60 Jennebach et al., 2012), presumably contributing to a high rate of Pol I loading (Albert et 61 al., 2011). Interestingly, polymerase-dependent RNA cleavage is stimulated by the 62 Rpa34.5/Rpa49 dimerization module (Geiger et al., 2010). This is important to extend bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

63 arrested and backtracked elongation complexes by removal of a displaced RNA 3´end 64 from the active center of the enzyme (Lisica et al., 2016). The stimulatory effect was 65 explained as an indirect effect resulting from its proximity to subunit Rpa12.2 66 (Jennebach et al., 2012), whose C-terminal part is required for cleavage (Kuhn et al., 67 2007). Rpa49CT binds DNA and supports both -dependent transcription and 68 extension of RNA from a transcription bubble (Pilsl et al., 2016a, Geiger et al., 2010). 69 Based on its position on the Pol I enzyme it was speculated to assist promoter escape by 70 keeping the Pol I clamp structure closed and stabilizing the Pol I- interaction with DNA 71 upstream of the start site (Sadian et al., 2017, Tafur et al., 2016). In vivo, expression of 72 Rpa49CT, but not of Rpa34.5/Rpa49NT is required for normal growth as well as for 73 recruitment of Pol I to the rRNA gene (Beckouet et al., 2008). 74 The Pol I lb-module contains not only the dimerization module Rpa34.5/A49NT but also 75 the N-terminal part (Rpa12C, aa 1- aa 85) of subunit Rpa12.2. Rpa12.2 is homologous 76 to the Pol II transcription elongation factor TFIIS. Deletion of Rpa12.2 results in growth 77 inhibition at elevated temperature, sensitivity to nucleotide-reducing drugs, inefficient 78 transcription termination and hampers the assembly of the Pol I enzyme (Nogi et al., 79 1993, Van Mullem et al., 2002, Prescott et al., 2004). The lack of the complete Rpa12.2 80 may also lead to the loss of Rpa34.5/Rpa49, as judged by immunoprecipitation of Pol I 81 from a rpa12 strain (Van Mullem et al., 2002). If only the C-terminal part of Rpa12.2 is 82 lacking, Rpa34.5/Rpa49 remain Pol I-associated. Whereas, deletion of the C-terminal 83 Rpa12.2 domain showed no significant growth phenotype under the tested conditions, 84 deletion of the N-terminus led to growth phenotypes indistinguishable from that of a 85 rpa12 strain (Van Mullem et al., 2002). The lack of a growth phenotype of an rpa12C 86 strain was surprising, since it is important for RNA cleavage activity when transcription 87 is stalled (Kuhn et al., 2007, Lisica et al., 2016) (Van Mullem et al., 2002). Recent 88 structural investigations indicated that the C-terminal part of Rpa12.2 is displaced from 89 the of the enzyme when Pol I is bound to the transcription initiation factor 90 Rrn3 (Pilsl et al., 2016a, Engel et al., 2016, Tafur et al., 2016). Furthermore, it was 91 proposed that Rpa12.2 is an intrinsic destabilizer of elongating Pol I (Appling et al., 92 2018) and might release - at least under specific conditions - Rpa34.5/Rpa49 from the 93 lobe (Tafur et al., 2018). In summary, it can be concluded that Rpa12.2 in addition to 94 Rpa34.5/Rpa49 is important for the elongating Pol I. The lobe associated N-terminal 95 domain of Rpa12.2 is thereby crucial for Pol I assembly, whereas the C-terminal domain 96 is dynamic and supports RNA cleavage. bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

97 Here, we investigated the function of the Rpa34.5/49 heterodimer and its interplay with 98 Rpa12.2 using defined promoter-dependent and -independent in vitro transcription 99 systems. In particular our studies emphasize the roles of the dimerization domain and 100 Rpa49CT for transcription fidelity, Pol I speed and resumption of stalled Pol I complexes. 101 We also tested the possibility that these subunits and Rpa12.2 contribute to 102 transcription of nucleosomal templates in vitro and compared the three nuclear RNA 103 polymerases in their efficiency to transcribe such templates. 104 bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

105 106 Results 107 108 Domains of the Pol I-specific heterodimer Rpa34.5/Rpa49 influence elongation 109 rate and Pol I processivity 110 111 To study the influence of Pol I lb-module subunits on in vitro transcription wildtype and 112 mutant Pol I were affinity-purified through the ProtA-tagged subunit Rpa135 using the 113 same stringent salt conditions (Merkl et al., 2014). Purified WT Pol I contained all 14 114 subunits, Rpa49 Pol I lacked the heterodimeric subunit Rpa34.5/Rpa49, Rpa12 Pol I 115 lacked subunit Rpa12.2 and the heterodimer Rpa34.5/Rpa49, and Rpa12C Pol I 116 contained the N-terminal part of Rpa12.2 and the Rpa34.5/Rpa49 heterodimer (Suppl 117 Fig. 1A, B), in good correlation with previous analyses (Van Mullem et al., 2002). To 118 analyse the effects of Rpa34.5/Rpa49 or its subdomains on in vitro transcription, the 119 recombinant heterodimer or protein fragments of the truncated heterodimer were 120 purified (Suppl. Fig. 1C) and added to affinity purified WT Pol I or Pol I depleted in lb- 121 module forming subunits. 122 123 The activity of wildtype Pol I and Pol I mutants in elongation in vitro was previously 124 analysed using various DNA/RNA hybrid scaffolds (Kuhn et al., 2007, Geiger et al., 2010) 125 which might differ from transcription bubbles generated by promoter-dependent Pol I 126 transcription. Therefore, we compared transcription activity of different Pol I 127 in promoter-dependent elongation assays using templates with the Pol I promoter 128 followed by 169nt of DNA lacking guanines in the sense strand (G-less cassette) which 129 were immobilized at the 3’ end on magnetic beads (Fig. 1 A). Using this template 130 transcription could be specifically initiated at the promoter in a minimal system 131 consisting of Pol I and recombinant Rrn3 and CF. If the transcription buffer contained 132 only UTP, 32P-CTP, and ATP, but no GTP, ternary complexed were stalled at the end of 133 the G-less cassette (Fig. 1B, lanes 1, 6). Ternary arrested complexes were washed with 134 buffers containing ATP, CTP and UTP to remove unincorporated 32P-CTP and unbound 135 proteins. After addition of GTP (chase), time course experiments were performed to 136 follow transcript extension (Fig. 1B/C). The amounts of fully extended transcripts 137 divided by the amount of arrested transcripts at time point zero was used as a measure 138 for Pol I processivity. Using WT Pol I more than 90% of stalled transcripts could be bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

139 extended and reached the full-size product. In contrast, using a Rpa49 Pol I only 140 around 60% of the arrested transcripts were fully extended after 2 minutes chase 141 indicating reduced Pol I processivity. The escape of Rpa49 Pol I from the arrest after 142 the G-less cassette was apparently not impaired, since for both, WT and Rpa49 Pol I, 143 we observed that less than 12% of the transcripts could not be further elongated after 144 NTP addition. It is thus likely, that Rpa49 processivity was affected downstream of the 145 G-less cassette after resuming elongation. Looking at the migration pattern of 146 radiolabelled RNAs at early timepoints indicated a slightly slower elongation speed of 147 Rpa49 Pol I if elongation was performed at room temperature in the presence of 200 148 M NTPs (see arrows in Fig. 1B)(see text to Fig. 3). 149 150 151 Pol I mutants lacking the lobe-binding-module or the C-terminal part of Rpa12.2 152 impair Pol I processivity in distinct ways 153 154 To see how components of the Pol I lb-module influence Pol I processivity, ternary 155 complexes of WT, Rpa12, Rpa12C and Rpa49 substituted with Rpa49CT were 156 arrested at the end of the G-less cassette for 20 min. Addition of 200 M NTPs to the 157 washed ternary complexes led to full extension of less than 50% of the arrested 158 transcripts at the G-less cassette in the three Pol I mutants tested, whereas most 159 transcripts could be completely extended by WT Pol I (Fig 2A and B). This indicated that 160 Pol I processivity was impaired when the integrity of the lb-module was affected 161 (Rpa12 Rpa49 + Rpa49CT), or the C-terminal part of Rpa12.2 was missing (Fig, 2 A 162 and B). When elongation was performed with reduced NTP concentration (20 M NTPs) 163 Pol I processivity was reduced in all Pol I preparations (Suppl. Fig. 2). However, whereas 164 in WT Pol I still almost 60% of the arrested transcripts could be fully extended, 165 processivity in mutant Pol I enzymes was even more impaired. After 60 second of chase 166 not more than 30 % of the arrested transcripts reached the full size. This underlines the 167 importance of the lb-module, in particular the dimerization domain Rpa34.5/Rpa49NT 168 and subunit Rpa12.2, for efficient elongation. Interestingly, the way how processivity 169 was affected differed for the mutant enzymes. In the presence of Rpa12C only about 170 40% transcripts stalled at the G-less cassette could complete chain elongation after 60 171 seconds chase, whereas more than 85% of the arrested transcripts could be extended, bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

172 when WT, Rpa12.2, and Rpa49 + Rpa49CT polymerases resumed chain elongation 173 (Fig. 2C and Suppl. Fig. 2). This could mean that in Rpa12C Pol I, which contains 174 Rpa34.5/Rpa49, cleavage is required for efficient restart of transcription. In contrast 175 Rpa12.2 which is also impaired in cleavage but lacks Rpa34.5/Rpa49 was more 176 efficient in the elongation of arrested transcripts. Rather efficient resumption of 177 elongation could be also observed for Rpa49 + Rpa49CT Pol I which contained Rpa12.2 178 but no dimerization module. Accordingly, it is possible that the dimerization module 179 keeps Pol I in an arrested and backtracked state until the displaced 3´end of the 180 transcript can be cleaved. Consistently, in the absence of the dimerization module 181 resumption of elongation is possible, no matter if Rpa12.2 is present or not. On the other 182 hand, Rpa12.2 and Rpa49 mutants were able to resume elongation of most arrested 183 transcripts at the G-less cassette but could not complete full extension. This could be due 184 to structural challenges when transcription of a long template leads to torsions and/or 185 the generation of long DNA-RNA hybrids interfering with the elongating enzyme. 186 Another interesting finding in this set of experiments was that the mutant polymerases 187 Rpa12.2C, and Rpa49 + Rpa49CT did not stop RNA synthesis precisely at the G-less 188 cassette, suggesting that wrong nucleotides were incorporated (Fig. 2A, see also results 189 below and Fig. 4). 190 191 The tWH of Rpa49CT accelerates Pol I speed 192 The migration pattern of radiolabelled transcripts at early timepoints after the NTP 193 chase indicated a slightly slower elongation speed of Rpa49 Pol I than WT Pol I if 194 elongation was conducted at room temperature in the presence of 200 M NTPs (see 195 arrows in Fig. 1B). Furthermore, addition of Rpa49CT to Rpa49 resulted in an apparent 196 faster Pol I movement compared to WT Pol I when elongation was performed in the 197 presence of 20 M NTPs (Suppl. Fig. 2, compare time points at 30 sec). Therefore, we 198 studied the impact of the Rpa34.5/Rpa49 domains on elongation speed in more detail. 199 When in vitro promoter-dependent transcription reactions were carried out at lower 200 temperature, the influence of the Rpa49CT and Rpa34.5/Rpa49NT domains on the 201 transcription rate was better detectable (Fig. 3A). As previously described (Pilsl et al., 202 2016a), addition of either the heterodimer Rpa34.5/Rpa49 or Rpa49CT to the initiation 203 inactive Rpa49 Pol I supported transcription initiation (Fig. 3 A, compare lanes 9-12 204 and 17 to 20 with 13 to 16). Addition of the heterodimer Rpa34.5/Rpa49 resulted in a bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

205 similar elongation speed as WT Pol I (Fig. 3A, compare lanes 1-4 with 5-8/ Suppl. Fig. 3). 206 Interestingly, addition of Rpa49CT increased the elongation speed in promoter- 207 dependent transcription at 0oC, (Fig. 3A, compare lanes 17-21 with 9-12). Elongation 208 speed of Rpa49 Pol I was also investigated in promoter-independent transcription 209 assays using tailed templates. In these reactions the presence of Rpa49CT also resulted 210 in increased elongation speed in the presence of 200M NTPs, when compared with WT 211 polymerase. In contrast, addition of the Rpa34.5/Rpa49NT dimerization domain did not 212 significantly change elongation speed of Rpa49 Pol I (Fig. 3B). These experiments 213 indicate that the complete heterodimer is necessary to restore WT processivity of 214 Rpa49 Pol I, and that the presence of Rpa49CT leads to an increased elongation speed. 215 216 Wrong nucleotides can be incorporated in the nascent RNA chain if the 217 Rpa34.5/Rpa49NT dimerization domain or the C-terminal part of Rpa12.2 is 218 absent 219 The pattern of radiolabelled transcripts observed before chase in tailed template 220 reactions with Rpa49 Pol I, indicated that the enzyme was not efficiently arrested after 221 the G-less cassette (Fig. 3B, first lane). This could be due to misincorporation of NTPs. To 222 investigate a general role of the Rpa49/Rpa34.5 heterodimer in controlling the correct 223 incorporation of nucleotides, we analysed how the Rpa34/Rpa49 subdomains 224 influenced transcription fidelity at the G-less cassette using promoter dependent 225 transcription. WT Pol I and Rpa49 Pol I in the presence of Rpa34/Rpa49 domains were 226 used for promoter-dependent in vitro transcription using templates with G-less 227 cassettes in the absence of GTP. The reaction was stopped after 5, 15 and 30 minutes 228 and supernatants (su) were separated from the immobilized template (p) to detect 229 possible differences in the stability of the arrested ternary complexes. Whereas WT Pol I 230 and Rpa49 Pol I in the presence of Rpa34.5/Rpa49 stopped precisely at the end of the 231 G-less cassette (Fig. 4A lanes 1- 2 and lanes 3 - 6), Rpa49 Pol I together with Rpa49CT 232 significantly extended the stalled transcript in the absence of GTP (Fig. 4A, compare 233 lanes 7-12 with lanes 1-6). Addition of the purified dimerization module to Rpa49 and 234 Rpa49CT polymerases reduced extension of the transcripts (Fig. 4A, lanes 13 -18), 235 arguing that the presence of Rpa34.5/Rpa49NT prevents elongation of the arrested 236 transcripts. The absence of the dimerization module did not significantly impair the 237 stability of the arrested ternary complex, since the ratio of immobilized transcripts bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

238 versus transcripts in the supernatant (su) was comparable in all reactions (Fig. 4A, 239 lower panel). Furthermore, washing the arrested complexes with increasing salt 240 concentrations (up to 1 M NaCl) did not result in disassembly of the complexes (data not 241 shown). This suggests that stalled complexes are stable, even in the absence of the 242 dimerization module. 243 244 Next, we wanted to see whether the Rpa49CT domain can also promote nucleotide 245 misincorporation of Rpa12.2 Pol I, lacking Rpa12.2 as well as the Rpa34.5/Rpa49 246 heterodimer. Therefore, we compared elongation of arrested transcripts in reactions 247 containing , WT Pol I, Rpa12.2CT Pol I, Rpa12.2 Pol I and Rpa12.2 Pol I in the 248 presence of Rpa49CT. Addition of Rpa49CT to Rpa12.2 Pol I supported RNA synthesis 249 in promoter-dependent transcription (Fig. 4B, compare lane 7 with lane 5) underlining 250 the important role of this domain in initiation (Beckouet et al., 2008, Pilsl et al., 2016a). 251 Furthermore, in contrast to Rpa12.2 Pol I, an increased number of readthrough 252 elongated transcripts after the G-less cassette were observed suggesting that the 253 Rpa49CT tWH in the absence of the Rpa34.5/Rpa49NT dimerization domain promoted 254 misincorporation of nucleotides. Interestingly, readthrough elongated transcripts were 255 also detected using Rpa12.2CT Pol I in transcription reactions (Fig. 4B compare lanes 3 256 and 1), which suggested that the C-terminus of Rpa12.2 is also required to prevent NTP 257 misincorporation. This is in accordance with a recently published finding (Sanz-Murillo 258 et al., 2018). Apparently, wrong nucleotides can be incorporated by different Pol I 259 mutants, but only in the presence of Rpa12.2 removal of misincorporated nucleotides 260 can occur. 261 It was recently reported that Rpa12.2C Pol I is not able to extend arrested and 262 backtracked transcripts which result in a displaced RNA 3´ end from the active enzyme 263 (Lisica et al., 2016). Accordingly, arrested and backtracked transcripts might accumulate 264 after the G-less cassette in such mutants. Extension of arrested and backtracked 265 transcripts should require an RNA 3´ cleavage activity which was reported to depend on 266 the C-terminal domain of subunit Rpa12.2 (aa 79 to aa 125) (Kuhn et al., 2007, Lisica et 267 al., 2016) and on the dimerization module of Rpa34/Rpa49 (Geiger et al., 2010). In fact, 268 if CTP, UTP and GTP, but no ATP were provided for chain elongation after washing the 269 arrested ternary complexes, Rpa12.2C Pol I could only poorly extend the nascent RNA, 270 while misincorporating nucleotides (Fig. 4B, compare lane 4 with lane 3). In contrast, bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

271 WT Pol I could further extend most of the radiolabelled RNAs until the position where 272 the first ATP should be incorporated (Figure 4B, compare lanes 1 with 2, see sequence of 273 the sense strand of the DNA template on the top). Interestingly, most transcripts 274 arrested after the G-less cassette could also be extended if Rpa49CT was added to 275 Rpa12 Pol I underlining that the presence of the dimerization domain is important to 276 keep the elongating polymerase in the arrested state if the cleavage supporting activity 277 of Rpa12.2 is lacking. On the other hand, the Rpa12C mutant which is lacking the RNA 278 cleavage supporting activity, but contains Rpa34.5/Rpa49 and the N-terminus of 279 Rpa12.2 could not resume efficient chain elongation. It was previously reported that 280 stalled Pol II transcripts due to the absence of one nucleotide can be extended with or 281 without RNA cleavage activity (Izban and Luse, 1993). Such elongation competent 282 ternary complexes which apparently produce mainly shallow backtracks (Izban and 283 Luse, 1993) (Lisica et al., 2016) could also arise in Pol I-dependent transcription at G- 284 less cassettes and could, thus, be extended even if Rpa12.2 is lacking. In contrast 285 recovery from deep backtrack depths requires the cleavage supporting activity of 286 Rpa12.2 (Lisica et al., 2016). It is possible that such deep backtrack depths are facilitated 287 in the presence of the dimerization module. Taken together, it is likely that the 288 dimerization module plays an important role to maintain stalled transcription 289 complexes, which can only resume elongation if the cleavage supporting activity of 290 Rpa12.2 is present. 291 292 293 Cooperation between the heterodimer and full length Rpa12.2 prevents NTP mis- 294 incorporation and is required to elongate stalled transcripts 295 To confirm that the dimerization domain influenced transcription fidelity and stalling of 296 elongation complexes by cooperating with Rpa12.2, transcription reactions using tailed 297 templates with G-less cassettes were performed (Fig. 5A). As stated above, transcription 298 reactions using tailed templates yielded always more transcripts with misincorporated 299 nucleotides (readthrough transcripts) than promoter-dependent transcription 300 reactions. As expected, addition of Rpa49CT to Rpa49 Pol I, Rpa12 Pol I and Rpa12C 301 Pol I stimulated readthrough transcription (Fig. 5A, compare lane 2 with 1, lane 6 with 5 302 and lane 10 with 9). Addition of the dimerization domain to Rpa49 reduced slightly 303 readthrough transcripts but did not significantly change the transcript pattern of the bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

304 other two mutant polymerases (Fig. 5A, compare lane 3 with 1, lane 7 with 5 and lane 11 305 with 9 and profile analysis aside). Addition of the dimerization domain to a Rpa49CT- 306 stimulated Rpa49 Pol I dependent reaction largely suppressed readthrough 307 transcription, confirming that cooperation of both domains of Rpa34.5/Rpa49 are 308 required for correct and efficient NTP incorporation (Fig. 5A, compare lane 4 and 2, and 309 the pattern profile aside). In contrast, addition of the dimerization domain to Rpa49CT- 310 stimulated transcription of either Rpa12 or Rpa12C resulted in not such pronounced 311 changes of the transcript pattern (compare lane 8 with 6 and lane 12 with 10 and 312 pattern profile aside). This indicates that the RNA cleavage supporting activity of 313 Rpa12.2 together with the dimerization domain or the complete heterodimer is required 314 to prevent NTP misincorporation at the G-less cassette. 315 316 Our previous analysis suggested that either WT Pol I or enzymes lacking the 317 dimerization domain could efficiently resume elongation of arrested transcripts after 318 the G-less cassette (see Fig. 1-4). As published for Pol I and II transcription (Izban and 319 Luse, 1993) (Lisica et al., 2016) and described above, different backtracking depths of 320 ternary elongation complexes can occur. Accordingly, polymerases can recover from 321 shallow backtracks by 1D diffusion whereas deeper backtracks require RNA cleavage 322 activity. Therefore, stalled complexes of Pol I mutants lacking the dimerization domain 323 might remain elongation-competent no matter whether the enzyme can cleave RNA or 324 not. In contrast, the presence of the dimerization domain could support a larger 325 backtrack depth which then requires RNA cleavage activity to restart elongation. To find 326 out whether the presence of the dimerization domain Rpa34.5/Rpa49NT is sufficient to 327 prevent chain elongation if RNA cleavage cannot take place, we asked which proportion 328 of arrested transcripts can be extended in the presence and absence of the dimerization 329 module Rpa34.5/Rpa49NT when Rpa12.2 is missing. Pulse experiments were 330 performed using tailed templates in the presence of 32P-CTP, ATP and UTP, ternary 331 complexes were washed and after addition of nucleotides the amount of transcripts at 332 the G-less cassette which were not extended were analysed after 30 and 180 seconds 333 (Fig. 5B/ Suppl Fig. 4A). As already shown in Fig. 2 and 4, a significant proportion of 334 Rpa12.2 Pol I was able to extend stalled transcripts. A similar effect was observed for 335 Rpa49 Pol I substituted with Rpa49CT. In contrast, addition of either the complete 336 heterodimer Rpa34.5/Rpa49 or the dimerization domain Rpa34.5/Rpa49NT revealed bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

337 that a major population of the transcripts remained arrested. The same was true for the 338 Rpa12C mutant. Promoter-dependent transcription reactions using the same 339 experimental approach showed comparable results with the exception that the influence 340 of Rpa34.5/Rpa49NT could not be tested in promoter-dependent assays (Suppl. Fig. 4B). 341 In summary, these experiments suggest that the dimerization domain is important to 342 establish a transcriptional state, in which Rpa12.2 dependent cleavage is required for 343 efficient extension of arrested transcripts. On the other hand, Pol I mutants lacking the 344 heterodimer or only the dimerization domain generated much less non-extendable 345 transcripts at the G-less cassette no matter whether the RNA cleaving activity of Rpa12.2 346 was available or not. Probably, these mutants bypass the backtracking/cleavage process 347 of the wildtype enzyme when the enzyme is stalled during elongation. 348 349 350 Pol I and Pol III which contain lobe-binding modules but not Pol II transcribe 351 through an assembled 352 In contrast to Pol I and Pol III the lb-module which is formed by subunits 353 RPA34.5/Rpa49 and Rpc37/Rpc53, respectively, is absent in Pol II. It was previously 354 published that purified Pol III can transcribe through a nucleosome (Studitsky et al., 355 1997) whereas Pol II is inhibited (Izban and Luse, 1991) without inclusion of additional 356 factors (Bondarenko et al., 2006). Stimulation of Pol II transcription through 357 nucleosomes was achieved in the presence of TFIIS and the elongation factor FACT 358 (Bondarenko et al., 2006) and when TFIIS and TFIIF were combined (Luse et al., 2011). 359 In mammalian Pol I and Pol III transcription FACT was described to facilitate chromatin 360 transcription (Birch et al., 2009). We wanted to know, if the three purified nuclear core 361 RNA polymerases from yeast without additional factors differ in their ability to 362 transcribe nucleosomal templates. A single nucleosome was assembled by salt dialysis 363 on a tailed template containing a 601 nucleosome positioning sequence (see Suppl. Fig. 5 364 and Material and Methods). This set-up allowed us to analyse the efficiency of affinity 365 purified Pol I to synthesize RNA from nucleosomal templates in comparison to the same 366 nucleosome-free templates without inclusion of initiation factors. To quantify the 367 efficiency of RNA synthesis through an assembled nucleosome, transcription reactions 368 were always performed in the presence of a nucleosome-free either shorter or longer 369 reference template (Fig. 6 and Fig. 7). Reactions were stopped after 30 min. Pol I, Pol II 370 and Pol III were purified according to a previously published protocol which allowed the bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

371 identical one-step affinity purification procedure using the same buffer conditions. The 372 purified enzymes were free of detectable cross-contamination of transcription factors or 373 RNA synthesizing enzymes (Merkl et al., 2014). Transcription reactions were performed 374 each with 7.5 nM of the respective polymerases and identical buffer conditions (50 mM 375 KoAc), which had been tested to allow transcription by all three polymerases. All three 376 polymerases were able to transcribe reference templates and the 601 sequence 377 containing templates without nucleosomes quite efficiently (Fig. 6, lanes 2, 4, 5, 7, 9, 10, 378 12, 14, 15). Differences in signal intensities between the generated RNAs were due to 379 the buffer conditions which were not optimal for all enzymes. The presence of a 380 nucleosome on the DNA template reduced the efficiency of transcription. However, the 381 level of inhibition differed significantly between the three polymerases. Whereas a 382 major fraction of Pol I and Pol III could transcribe the nucleosomal 601 sequence (Fig. 383 6A, lanes 1, 3, 11, 13), full length transcripts from nucleosomal templates were almost 384 not detectable in Pol II-dependent reactions (lanes 6 and 8). Upon longer exposure times 385 an additional transcript became visible in lanes 6 and 8 which length corresponded to 386 the size from the start site to the 5´end of the positioned nucleosome (Suppl Fig. 6). 387 Apparently, abortive or arrested transcripts were generated when the enzymes 388 encountered a nucleosome. This indicated that at least a minor fraction of Pol II initiated 389 transcription from the tailed template but was arrested or displaced upon collision with 390 a nucleosome. Quantification revealed about 40% reduction of RNA synthesis by Pol I 391 and Pol III in the presence of a nucleosome and almost complete loss of Pol II-derived 392 transcripts (Fig. 6). These results suggest that Pol I and Pol III which both contain 393 additional heterodimeric subcomplexes (Rpa34.5/Rpa49 and Rpc37/Rpc53, 394 respectively) in comparison to Pol II, have intrinsic activities to transcribe through a 395 nucleosomal barrier. 396 397 Pol I transcription of nucleosomal templates in vitro is compromised in the 398 absence of the Pol I-specific heterodimer Rpa34.5/Rpa49 and when the C-terminal 399 part of Rpa12.2 is missing 400 In Pol II-dependent transcription TFIIF together with the cleavage stimulating factor 401 TFIIS promotes elongation (Schweikhard et al., 2014) and RNA synthesis through 402 nucleosomes (Luse et al., 2011). Since Rpa34.5/49 and Rpa12.2 share structural 403 features with TFIIF and TFIIS, one could hypothesize that these subunits might facilitate 404 transcription through a nucleosome. Therefore, we investigated whether absence of bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

405 Rpa12.2 with and without the Rpa34.5/49 heterodimer had an impact on transcription 406 of a nucleosomal template. Consistent with a role of these subunits in transcription 407 through a nucleosome, both, Rpa12.2 Pol I andRpa49 Pol I could not as efficiently 408 transcribe nucleosomal templates as the WT enzyme (Fig. 7A, lanes 3, 4 and 5, 6). 409 Addition of increasing amounts of the purified heterodimer restored the ability of 410 Rpa49 Pol I to transcribe through a nucleosome (Fig. 7B, lanes 7-14, and 411 quantification), while the purified heterodimer alone contained no RNA synthesizing 412 activities (lanes 5 and 6). To determine the domains of the Rpa34.5/Rpa49 heterodimer 413 which are required to stimulate transcription through a nucleosome, either purified 414 dimerization domain Rpa34.5/49NT or the tandem winged helix-containing Rpa49CT 415 were added to a Rpa49 Pol I-dependent transcription reaction. Addition of the 416 complete heterodimer or Rpa49CT alone stimulated both, transcription of the reference 417 template, and transcription of the nucleosomal template. (The level of stimulation of the 418 reference template varied in dependency on the quality of the preparation of the 419 nucleosomal DNA matrix). The best recovery of transcriptional activity on the 420 nucleosomal template in reference to the nucleosome-free template was obtained 421 adding back either the entire heterodimer Rpa34.5/49 or Rpa49CT to the mutant 422 enzyme (Fig. 7C, lanes 3-5 and 9). Adding the dimerization domain Rpa34.5/Rpa49NT 423 alone to Rpa49 Pol I did not show significant enhancement of transcription (Fig. 7C, left 424 panel, compare lanes 3-5 and 6-8). RNA synthesis through nucleosomal templates using 425 promoter-dependent transcription assays showed comparable results (Table 1). 426 To find out whether the RNA cleavage-stimulating activity of Rpa12.2 contributes to 427 transcription through nucleosomes, transcription assays on tailed templates were 428 performed using WT Pol I and Rpa12CT. In the presence of nucleosomes transcription 429 using Rpa12CT was much stronger inhibited than with WT Pol I, suggesting that - in 430 addition to the heterodimer - RNA cleavage activity is also required for efficient passage 431 through a nucleosome. In summary, the dimerization module which is a constituent of 432 the Pol I lb-module, the C-terminal part of Rpa49 and the C-terminal part of Rpa12.2 433 seem to cooperate to support Pol I transcription of both, nucleosomal and nucleosome- 434 free templates. 435 436 437 438 bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

439 Discussion 440 441 Comparison of Rpa34.5/49 and Rpa12.2 with their Pol II counterparts 442 The functional properties of the heterodimeric Rpa34.5/Rpa49 complex and Rpa12.2 443 show significant similarities to the transcription factors TFIIE, TFIIF, and TFIIS, their 444 counterparts in Pol II dependent transcription (Kuhn et al., 2007, Geiger et al., 2010). 445 TFIIE and TFIIF have specific roles in initiation and elongation and are not components 446 of the core enzyme. Similar to Rpa49CT and, Rpa34.5/Rpa49NT, TFIIE and the 447 dimerization module of TFIIF bind each to opposite sides of Pol II, respectively, and 448 flank the promoter DNA. Both Pol II factors are parts of an intricate transcription 449 initiation network containing TFIIB, TBP and domains of the Pol II core enzyme which 450 are thought to encircle, retain and open the DNA (He et al., 2016, Plaschka et al., 2016). 451 The Tfa2 tWH domain of TFIIE corresponds to Rpa49Ct, but is only important for 452 transcription initiation. Tfa2 spans the polymerase cleft and is positioned close to the 453 site of DNA melting (Grunberg et al., 2012). Recent structural data revealed a similar 454 position for Rpa49CT which stabilizes the upstream DNA during transcription (Pilsl et 455 al., 2016a, Tafur et al., 2016, Jennebach et al., 2012). The Rpa49 linker, which is part of 456 Rpa49CT, connects the tWH domain of Rpa49 with the dimerization module, thereby 457 spanning the active site cleft and contacting the coiled coil (Tafur et al., 2016, Han et al., 458 2017). Based on the structures of different Pol I fractions, it was suggested that closing 459 of the Pol I clamp and establishing extensive contacts with the RNA and the upstream 460 DNA correlate with the stabilisation of the Rpa49CT linker region over the cleft. In 461 correlation to TFIIE function in initiation, this might cause the stabilisation of the tWH 462 domain of Rpa49CT during transcription initiation (Tafur et al., 2016, Han et al., 2017, 463 Engel et al., 2013). Contrary to TFIIE, Rpa49CT accompanies the elongating enzyme 464 (Beckouet et al., 2008). Therefore, clamp closing through Rpa49CT could also establish 465 an elongation promoting Pol I complex which is dedicated for the fast incorporation of 466 nucleotides. 467 468 TFIIF the counterpart of Rpa34.5/49NT was described as initiation and elongation 469 factor. This is in contrast to Rpa34.5/49NT which is neither required to stimulate 470 initiation in vitro (Pilsl et al., 2016a) nor for Pol I loading onto the promoter (Beckouet et 471 al., 2008). TFIIF predominantly associates with Pol II in promoter-proximal regions 472 (Mayer et al., 2010) (Krogan et al., 2002). In addition, it accompanies at least transiently bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

473 Pol II during RNA chain elongation (Rani et al., 2004, Mayer et al., 2010, Krogan et al., 474 2002, Cojocaru et al., 2008). It was shown, that TFIIF is involved in very early stages of 475 elongation (Yan et al., 1999, Cheng and Price, 2007, Ujvari et al., 2011) and that it 476 increases transcription rates in vitro (Renner et al., 2001, Izban and Luse, 1992, Zhang et 477 al., 2003), at least in part by suppressing pauses. Presumably, TFIIF promotes 478 transcription elongation together with the RNA cleavage stimulating factor TFIIS in a 479 synergistic manner (Schweikhard et al., 2014, Zhang et al., 2003, Zhang and Burton, 480 2004, Zhang et al., 2005), and the impact of both proteins is important to increase 481 transcription through nucleosomes in vitro (Luse et al., 2011). These elongation related 482 features resemble those of the Pol I dimerization module and its interaction with the 483 RNA cleavage stimulating factor Rpa12.2. 484 485 Functions of the heterodimeric Rpa34.5/Rpa49 complex and interplay with 486 Rpa12.2 487 The dimerization module Rpa34.5/49NT and the N-terminal part of Rpa12.2 form the 488 polymerase lobe-binding module which is not present in the Pol II enzyme. Our studies 489 underline that these subunits functionally cooperate. Efficient RNA chain elongation 490 was dependent on the Rpa34.5/Rpa49 complex. Apparently, this complex is important 491 for optimized Pol I processivity, elongation speed, pausing and RNA cleavage events. All 492 elongation features depended on the cooperation of both domains. In vitro transcription 493 indicated that Rpa49CT increased elongation speed, but lowered significantly Pol I 494 processivity and transcription fidelity, if the dimerization module Rpa34.5/Rpa49NT 495 was lacking. This suggested that the tandem winged helix (tWH) of Rpa49CT promotes 496 the core enzyme to incorporate nucleotides at higher rate, but at the cost of less 497 accuracy. Interaction of the tWH with the dimerization module reduced the speed of 498 RNA-synthesis and misincorporation of nucleotides, and, importantly, increased Pol I 499 processivity. 500 In the absence of the dimerization module, the presence of Rpa12.2 had neither 501 influence on speed, transcription fidelity, extension of arrested transcripts and 502 processivity (this manuscript), nor on RNA cleavage activity (Kuhn et al., 2007, Geiger et 503 al., 2010). This underlined that Rpa12.2 function depended on the neighbouring 504 dimerization domain and confirmed previous suggestions that the cleavage stimulatory 505 effect of the heterodimer is indirect and due to its proximity to Rpa12.2 (Jennebach et 506 al., 2012). Taken together, these findings suggest that the dimerization domain bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

507 containing lb-module is required to integrate Rpa12.2- and Rpa49CT-dependent 508 activities into the transcribing enzyme which finally ensures highly processive and 509 accurate transcription. It could be that a similar interplay is responsible for the reported 510 synergism between TFIIF with TFIIS which ensures Pol II proofreading (Thomas et al., 511 1998, Jeon and Agarwal, 1996, Schweikhard et al., 2014). 512 513 The interplay of the lobe binding subunits with stalled transcription complexes 514 If RNA polymerases encounter a barrier, the enzyme stalls, backtracks and arrests (Luse 515 et al., 2011, Lisica et al., 2016, Cheung and Cramer, 2011). Rapid relief of arrest requires 516 RNA cleavage stimulating proteins like TFIIS, Rpa12.2 or the Pol III subunit C11 and 517 cleavage of the 3´ end of the RNA which is disposed from the active center. In the 518 absence of RNA cleaving activity, NTPs were misincorporated in Pol I-dependent 519 transcription. If either the dimerization module or the C-terminus of Rpa12.2 was 520 lacking, transcription did not stop precisely at the G-less cassette. This resembles recent 521 findings that Rpa12C Pol I in contrast to WT Pol I can slowly incorporate nucleotides 522 opposite to a cyclobutene pyrimidine dimer (Sanz-Murillo et al., 2018). However, 523 although RNA cleavage activity is largely reduced in the absence of the dimerization 524 domain (Rpa49, Rpa12.2)(Kuhn et al., 2007), a significant fraction of these enzymes 525 can resume RNA chain elongation after ternary complexes were paused at G-less 526 cassettes (Fig. 1-4 ). It is possible that extension of RNA occurs if i) the enzyme 527 randomly diffuses forward to realign the RNA 3´end or ii) complexes do not enter the 528 deep backtracked state which requires RNA cleavage for elongation when they 529 encounter the G-less cassette arrest or other obstacles as it was previously reported for 530 Pol II transcription in vitro (Chang and Luse, 1997). The presence of Rpa49CT could 531 enforce such a non-arrested stage. Interestingly, Pol I transcription is not efficiently 532 terminated in rpa12 strains (Prescott et al., 2004), which could also be due to non- 533 arrested complexes at the termination protein Nsi1 (Reiter et al., 2012). The 534 dimerization module resides on the polymerase lobe in close vicinity to subunit Rpa12.2, 535 which is required for RNA cleavage of backtracked Pol I. If lack of the dimerization 536 module destabilizes Rpa12.2 such that it cannot stably access the active center, it is 537 conceivable that such an enzyme i) is not able to support cleavage reactions and ii) may 538 not enter a locked state with a 3´ RNA displaced from the active center. One 539 consequence would be a reduced transcription fidelity. On the other hand, it is possible 540 that the dimerization domain supports the generation of deep backtracked bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

541 intermediates in specific situations which can only resume elongation if RNA cleavage 542 can take place. It will be interesting to see if incorporation of a wrong nucleotide can 543 facilitate such a Rpa34.5/Rpa49NT driven backtrack. Very recently a manuscript was 544 submitted in which it was suggested that the association of the Rpa34.5/Rpa49 is 545 regulated by the C-terminal domain of Rpa12.2 (Tafur et al., 2018). If (transient) 546 dissociation of the dimerization domain by Rpa12.2 is important to restart RNA 547 synthesis, it is conceivable that a Pol I mutant lacking the Rpa12.2 C-terminus but 548 including the dimerization domain has problems to enter the elongation mode. 549 550 How to transcribe through a nucleosome? 551 Transcription through nucleosomes is a special challenge for eukaryotic RNA 552 polymerases since intrinsic stalling and backtracking is more pronounced in front of a 553 physical barrier like a nucleosome. Furthermore, the nucleosomal DNA recoils on the 554 octamer, locking the enzyme in the arrested state (Gaykalova et al., 2015). Rapid relief 555 of arrest requires TFIIS and cleavage of the 3´ end of the RNA which 556 is disposed from the active center of Pol II. TFIIF was reported to support TFIIS- 557 mediated in vitro transcription through nucleosomes (Luse et al., 2011). Both factors 558 were not associated to Pol II, when Pol II was affinity-purified using our stringent 559 conditions. Thus, Pol II was not able to transcribe through nucleosomes. In contrast Pol I 560 and Pol III which have the counterparts of TFIIF and TFIIS, the subunits Rpa34.5/Rpa49, 561 Rpa12.2, and Rpc37/Rpc53, Rpc11, respectively, tightly associated, were able to pass 562 through nucleosomes. For Pol I, efficient nucleosomal transcription depended on the 563 presence of the heterodimeric Rpa34.5/Rpa49 complex. Apparently, both modules 564 Rpa34.5/Rpa49NT and Rpa49CT as well as the C-terminal part of Rpa12.2 contributed 565 to the ability to transcribe through nucleosomes, suggesting that both cleavage activity 566 and Pol I binding to DNA (mediated by Rpa49CT) support passage through nucleosomes. 567 In the absence of the dimerization module, addition of Rpa49CT alone improved Pol I 568 movement through nucleosomes. As mentioned above, it is possible that such an enzyme 569 may not enter a locked state with a 3´ RNA displaced from the active center, suggesting 570 that elevated speed generated by Rpa49CT compensate for the missing backtracking and 571 RNA cleavage cycle. Whether or not the nucleosome is evicted by Pol I or the DNA is 572 transiently uncoiled during elongation remains to be determined. So far, the sensitivity 573 of our assays applied was not sufficient to allow clear-cut conclusions. bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

574 For passage through nucleosomes uncoiling of the template from the histone octamer 575 while maintaining the polymerase in the transcriptionally competent state are the most 576 important features. Histone chaperone FACT or elongation factors Spt4/5 or Paf1c are 577 examples of Pol II factors that facilitate DNA unwrapping from H2A/H2B dimers to 578 partially disassemble nucleosomes (Belotserkovskaya et al., 2003, Hsieh et al., 2013, 579 Crickard et al., 2017, Kim et al., 2010). The same factors were also reported either to 580 support Pol I elongation (Zhang et al., 2009, Zhang et al., 2010, Schneider et al., 2006) or 581 passage through nucleosomes (Birch et al., 2009). None of these factors was present in 582 the tailed template assays performed in this study. But, it is possible that a combinatory 583 effort between Pol I subunits and elongation factors further support transcription 584 through nucleosomes. 585 586 In vitro and in vivo phenotypes 587 In contrast to Pol II genes, Pol I transcribed genes are heavily packed with elongating 588 polymerases which do not leave much space for assembled nucleosomes (Merz et al., 589 2008, Conconi et al., 1989). It was previously shown that Pol I transcription is required 590 to achieve an open chromatin state in which the amount of DNA-bound nucleosomes is 591 strongly reduced (Merz et al., 2008, Wittner et al., 2011). According to our in vitro 592 studies the Pol I heterodimer Rpa34.5/Rpa49 and the Rpa12.2 C-terminus are involved 593 in this process. On the other hand mutant strains which are deleted in the heterodimer 594 or RPA12.2 are viable (Liljelund et al., 1992, Nogi et al., 1993) (Gadal et al., 1997). This 595 suggests that these mutant polymerases can still open the closed chromatin state (Albert 596 et al., 2011) and that other factors of the Pol I transcription machinery contribute to the 597 opening process. Such candidates could be the above mentioned FACT (Birch et al., 598 2009) or Paf1c (Zhang et al., 2010). Future structural and functional investigations are 599 necessary to elucidate mechanisms how stalled Pol I complexes can bypass transcription 600 obstacles in vivo with and without removal of a displaced RNA 3´end. Taken together our 601 studies underline that the specific subunit composition of Pol I adds to the functional 602 specialisation of the enzyme to transcribe the rRNA gene as efficiently as possible. bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

603 Materials and Methods 604 605 Yeast strains, plasmids, oligonucleotides and construction of transcription 606 templates 607 Oligonucleotides, plasmids and yeast strains used in this work are listed in suppl. Tables 608 1, 2 and 3. Molecular biological methods and transformation of yeast cells were 609 performed according to standard protocols (Sambrook et al., 1989, Burke et al., 2000, 610 Schiestl and Gietz, 1989). The generation of transcription templates is described in the 611 supporting information. Plasmid sequences are available upon request. For the 612 experiments in this study, yeast cells were grown at the indicated temperatures in either 613 YPD (2% w/v) peptone, 1% (w/v) yeast extract, 2% glucose). 614 Purification of proteins 615 Wild-type RNA polymerases I, II and III (Pol I, II and III) were purified from yeast strains 616 y2423 (Pol I), y2424 (Pol II), y2425 Pol III, (suppl. Table 1) via the protein A affinity tag 617 according to (Merkl et al., 2014). Purification of mutant Pol I y2670 (Rpa49), y2679 618 (Rpa12) and y2679 transformed with plasmid 1839 (Rpa12CT) was according to 619 (Pilsl et al., 2016a). Recombinant Rpa34/Rpa49, the dimerization domain 620 Rpa34.5/Rpa49NT (Rpa34/(Rpa49 aa 1-110) and Rpa49CT (aa 110-426) were purified 621 from E.coli according the protocol of (Pilsl et al., 2016a). The absolute efficiency of 622 single protein fractions in transcriptional activity varied slightly from preparation to 623 preparation. Peculiar attention concerned the expression and purification of Rpa49 Pol 624 I (y2670). To avoid the observed generation of suppressors this strain was first 625 cultivated in galactose, which allowed the expression of Rpa49 and then shifted to 626 glucose for 24 hours to shut off Rpa49 expression. Although depletion of subunits A49 627 and A34.5 was monitored by Coomassie-stained SDS PAGE and western blotting the 628 transcriptional activity varied slightly in different preparations. This could be due to 629 different levels of remaining Rpa49 or upcoming suppressors. Therefore, statistical 630 evaluation using biological replicates (which means different protein preparations from 631 independently cultivated strains) was not applicable. Recombinant Rpa34/Rpa49 and 632 its purified subdomains tend to loose activity when they are stored. Reliable results 633 were obtained using freshly prepared protein fractions. 634 Transcription elongation assays 635 Promoter-dependent elongation assay. Pulse-labelling of G-less transcripts on promoter- 636 containing templates and subsequent analysis were performed according to bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

637 (Tschochner, 1996, Pilsl et al., 2016a) with some modifications. If elongation should be 638 measured during 5 time points including the starting point at time point 0, the following 639 assay was performed. 1.5 ml reaction tubes (Sarstedt safety seal) were placed on ice. 5 x 640 0.5 to 1 l template (50 – to 100 ng DNA) (templates 2148 or 2313) immobilized to 641 magnetic beads was added, which corresponds to a final concentration of 5 – 10 nM per 642 transcription reaction (25 l reaction volume) . 5 x 1-2 l CF (0.5 to 1 pmol/l; final 643 concentration 20 - 40 nM) and 5 x 1-3 l Pol I-Rrn3 (final concentration 4 -12 nM) were 644 added to the tube. Where indicated, domains of the Rpa34.5/49 heterodimer were 645 added before transcription was started (final concentration 50 nM). 20 mM HEPES/KOH 646 pH 7.8 were added to a final volume of 5 x 12.5 l. Transcription was started adding 5 x

647 12.5 l transcription buffer 2 x without GTP (40 mM HEPES/KOH pH 7.8, 20 mM MgCl2, 648 10 mM EGTA, 5 mMDTT, 300 mM potassium acetate, 0.4 mM ATP, , 0.4 mM UTP, 0.02 649 mM CTP, 0.3 Ci 32P-CTP. The samples were incubated at 24oC between 5 and 30 min at 650 400 rpm in a thermomixer. After placement on ice, the supernatant was separated from 651 the magnetic beads and 2-3 washing steps using washbuffer W1 (20 mM HEPES/KOH

652 pH 7.8, 10 mM MgCl2, 5 mM EGTA, 2.5 mMDTT, 150 mM potassium acetate, 0.02 mM 653 ATP, 0.02 mM UTP, 0.02 mM CTP followed). The magnetic beads were resuspended in 654 150l washbuffer W1. 25 l were removed and added to 200 l Proteinase K buffer (0.5 655 mg/ml Proteinase K in 0.3 M NaCl, 10 mM Tris/HCl pH 7.5, 5 mM EDTA, 0.6% SDS) (time 656 point 0). To the remaining 125l either 12.5 l or 1.25l 10xNTPs (ATP, GTP, UTP, CTP 657 concentration each 2 mM) was added and immediately mixed. 25 or 27 l aliquots were 658 removed at the time points indicated and added to 200 l Proteinase K buffer. The 659 samples were incubated at 30oC for 15 min at 400 rpm in a thermomixer. 700 l Ethanol 660 p.a. were added and mixed. Nucleic acids were precipitated at -20oC over night or for 30 661 min at -80oC. The samples were centrifuged for 10 min at 12.000g and the supernatant 662 was removed. The precipitate was washed with 0.15 ml 70% ethanol. After 663 centrifugation, the supernatant was removed and the pellets were dried at 95oC for 2 664 minutes. RNA in the pellet was dissolved in 12 l 80% formamide, 0.1 TBE, 0,02% 665 bromophenol blue and 0.02% xylene cyanol. Samples were heated for 2 min under 666 vigorous shaking at 95oC and briefly centrifuged. After loading on a 6% polyacrylamide 667 gel containing 7 M urea and 1 x TBE RNAs were separated applying 25 watts for 30 - 40 668 min. The gel was dried after 10 min rinsing in water for 30 min at 80oC using a vacuum 669 dryer. Radiolabelled transcripts are visualised using a PhosphoImager. For bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

670 quantification, signal intensities were calculated using Multi Gauge (Fuji).

671 Tailed template elongation assay. The transcription were performed as described as 672 above on PCR-derived templates containing biotinylated oligo downstream of the 673 promoter in reverse direction and an oligo containing a Nb.BsmI (NEB) nicking site which 674 allows non-specific initiation of all RNA polymerases without the addition of initiation 675 factors.

676 Transcription through nucleosomes

677 Preparation of nucleosomal templates 678 Chromatin was reconstituted on templates for in vitro transcription containing one or 679 multiple 601 nucleosome positioning sequences (Lowary & Widom 1998). Dialysis 680 chambers were prepared from siliconized microreaction tubes (Eppendorf) by clipping 681 off the conical part and perforation of the cap with a red-hot metal rod (ø 0.5 cm). The 682 dialysis membrane (molecular weight cutoff 6.8kDa) was pre-wet in high salt buffer and 683 fixed between tube and cap. The dialysis chambers were put in a floater in a bucket 684 containing 300 ml high salt buffer, air bubbles were removed and the mixture of 685 histones and DNA was applied to the chambers. Dialysis from 2 M NaCl to 0.23 M NaCl 686 was performed over night at RT with constant stirring and at a low salt buffer flow rate 687 of 200 ml/h (3 l total). Upon completion, the assembly solution was transferred to a 688 siliconized tube, its volume was measured and chromatin was stored at 4°C. To 689 determine the assembly success, 5 μl of the reaction were supplemented with 1μl 690 loading buffer and analyzed on a 6% polyacrylamide, 0.4xTBE gel (Suppl Fig. 3). Gels 691 were stained in 0.4x TBE containing ethidium bromide for 15min, washed in 0.4x TBE 692 for 15 min and analysed on a FLA3000 imager (Fuji). 693 To determine optimal assembly conditions, histones were titrated to the DNA: ratio 0.4 : 1 0.6 : 1 0.8 : 1 1 : 1 histones : DNA high salt buffer 44μl 42.75μl 41.5μl 40.25μl BSA (10mg/ml) 1μl 1μl 1μl 1μl DNA template 2.5μl 2.5μl 2.5μl 2.5μl (2μg/μl) chicken histones 2.5μl 3.75μl 5μl 6.25μl (0.8mg/ml) bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

total volume 50μl 50μl 50μl 50μl 694 In most reaction series the ratios 0.6:1 or 0.8: 1 were used. Transcription reactions were 695 performed as described (Pilsl et al., 2016a). Each reaction contained 50- 100 ng of a 696 nucleosome-containing (2316) and a 50 - 100 ng naked template (1573). Templates of 697 approppriate sizes were generated by PCR reactions of different sizes using oligo 2115 698 and oligos 4231, 4232, 4233 or 4234. 699 700 701 Acknowledgements 702 703 We are grateful to members of the chair Biochemistry III for critical discussion and to 704 Elisabeth Silberhorn for technical assistance. This work was supported through grants 705 of the Deutsche Forschungsgemeinschaft (SFB 960). There are no competing interests. 706 bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

707 References 708 ALBERT, B., LEGER-SILVESTRE, I., NORMAND, C., OSTERMAIER, M. K., PEREZ- 709 FERNANDEZ, J., PANOV, K. I., ZOMERDIJK, J. C., SCHULTZ, P. & GADAL, O. 2011. 710 RNA polymerase I-specific subunits promote polymerase clustering to enhance 711 the rRNA gene transcription cycle. J Cell Biol, 192, 277-93. 712 APPLING, F. D., SCULL, C. E., LUCIUS, A. L. & SCHNEIDER, D. A. 2018. The A12.2 Subunit Is 713 an Intrinsic Destabilizer of the RNA Polymerase I Elongation Complex. Biophys J, 714 114, 2507-2515. 715 BECKOUET, F., LABARRE-MARIOTTE, S., ALBERT, B., IMAZAWA, Y., WERNER, M., GADAL, 716 O., NOGI, Y. & THURIAUX, P. 2008. Two RNA polymerase I subunits control the 717 binding and release of Rrn3 during transcription. Mol Cell Biol, 28, 1596-605. 718 BELOTSERKOVSKAYA, R., OH, S., BONDARENKO, V. A., ORPHANIDES, G., STUDITSKY, V. 719 M. & REINBERG, D. 2003. FACT facilitates transcription-dependent nucleosome 720 alteration. Science, 301, 1090-3. 721 BIRCH, J. L., TAN, B. C., PANOV, K. I., PANOVA, T. B., ANDERSEN, J. S., OWEN-HUGHES, T. 722 A., RUSSELL, J., LEE, S. C. & ZOMERDIJK, J. C. 2009. FACT facilitates chromatin 723 transcription by RNA polymerases I and III. Embo J, 28, 854-65. 724 BONDARENKO, V. A., STEELE, L. M., UJVARI, A., GAYKALOVA, D. A., KULAEVA, O. I., 725 POLIKANOV, Y. S., LUSE, D. S. & STUDITSKY, V. M. 2006. Nucleosomes can form a 726 polar barrier to transcript elongation by RNA polymerase II. Mol Cell, 24, 469-79. 727 BURKE, D., DAWSON, D. & STEARNS, T. 2000. Methods in Yeast Genetics, Cold Spring 728 Harbor. 729 CHANG, C. H. & LUSE, D. S. 1997. The H3/H4 tetramer blocks transcript elongation by 730 RNA polymerase II in vitro. J Biol Chem, 272, 23427-34. 731 CHENG, B. & PRICE, D. H. 2007. Properties of RNA polymerase II elongation complexes 732 before and after the P-TEFb-mediated transition into productive elongation. J Biol 733 Chem, 282, 21901-12. 734 CHEUNG, A. C. & CRAMER, P. 2011. Structural basis of RNA polymerase II backtracking, 735 arrest and reactivation. Nature, 471, 249-53. 736 COJOCARU, M., JERONIMO, C., FORGET, D., BOUCHARD, A., BERGERON, D., COTE, P., 737 POIRIER, G. G., GREENBLATT, J. & COULOMBE, B. 2008. Genomic location of the 738 human RNA polymerase II general machinery: evidence for a role of TFIIF and 739 Rpb7 at both early and late stages of transcription. Biochem J, 409, 139-47. 740 CONCONI, A., WIDMER, R. M., KOLLER, T. & SOGO, J. M. 1989. Two different chromatin 741 structures coexist in ribosomal RNA genes throughout the cell cycle. Cell, 57, 753- 742 61. 743 CRICKARD, J. B., LEE, J., LEE, T. H. & REESE, J. C. 2017. The elongation factor Spt4/5 744 regulates RNA polymerase II transcription through the nucleosome. Nucleic Acids 745 Res, 45, 6362-6374. 746 ENGEL, C., PLITZKO, J. & CRAMER, P. 2016. RNA polymerase I-Rrn3 complex at 4.8 A 747 resolution. Nat Commun, 7, 12129. 748 ENGEL, C., SAINSBURY, S., CHEUNG, A. C., KOSTREWA, D. & CRAMER, P. 2013. RNA 749 polymerase I structure and transcription regulation. Nature, 502, 650-5. 750 FERNANDEZ-TORNERO, C., MORENO-MORCILLO, M., RASHID, U. J., TAYLOR, N. M., RUIZ, 751 F. M., GRUENE, T., LEGRAND, P., STEUERWALD, U. & MULLER, C. W. 2013. Crystal 752 structure of the 14-subunit RNA polymerase I. Nature, 502, 644-9. 753 GADAL, O., MARIOTTE-LABARRE, S., CHEDIN, S., QUEMENEUR, E., CARLES, C., 754 SENTENAC, A. & THURIAUX, P. 1997. A34.5, a nonessential component of yeast 755 RNA polymerase I, cooperates with subunit A14 and DNA I to 756 produce a functional rRNA synthesis machine. Mol Cell Biol, 17, 1787-95. bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

757 GAYKALOVA, D. A., KULAEVA, O. I., VOLOKH, O., SHAYTAN, A. K., HSIEH, F. K., 758 KIRPICHNIKOV, M. P., SOKOLOVA, O. S. & STUDITSKY, V. M. 2015. Structural 759 analysis of nucleosomal barrier to transcription. Proc Natl Acad Sci U S A, 112, 760 E5787-95. 761 GEIGER, S. R., LORENZEN, K., SCHREIECK, A., HANECKER, P., KOSTREWA, D., HECK, A. J. 762 & CRAMER, P. 2010. RNA polymerase I contains a TFIIF-related DNA-binding 763 subcomplex. Mol Cell, 39, 583-94. 764 GRUNBERG, S., WARFIELD, L. & HAHN, S. 2012. Architecture of the RNA polymerase II 765 preinitiation complex and mechanism of ATP-dependent promoter opening. Nat 766 Struct Mol Biol, 19, 788-96. 767 HAN, Y., YAN, C., NGUYEN, T. H. D., JACKOBEL, A. J., IVANOV, I., KNUTSON, B. A. & HE, Y. 768 2017. Structural mechanism of ATP-independent transcription initiation by RNA 769 polymerase I. Elife, 6. 770 HE, Y., YAN, C., FANG, J., INOUYE, C., TJIAN, R., IVANOV, I. & NOGALES, E. 2016. Near- 771 atomic resolution visualization of human transcription promoter opening. 772 Nature, 533, 359-65. 773 HOFFMANN, N. A., JAKOBI, A. J., MORENO-MORCILLO, M., GLATT, S., KOSINSKI, J., 774 HAGEN, W. J., SACHSE, C. & MULLER, C. W. 2015. Molecular structures of 775 unbound and transcribing RNA polymerase III. Nature, 528, 231-6. 776 HSIEH, F. K., KULAEVA, O. I., PATEL, S. S., DYER, P. N., LUGER, K., REINBERG, D. & 777 STUDITSKY, V. M. 2013. Histone chaperone FACT action during transcription 778 through chromatin by RNA polymerase II. Proc Natl Acad Sci U S A, 110, 7654-9. 779 IZBAN, M. G. & LUSE, D. S. 1991. Transcription on nucleosomal templates by RNA 780 polymerase II in vitro: inhibition of elongation with enhancement of sequence- 781 specific pausing. Genes Dev, 5, 683-96. 782 IZBAN, M. G. & LUSE, D. S. 1992. Factor-stimulated RNA polymerase II transcribes at 783 physiological elongation rates on naked DNA but very poorly on chromatin 784 templates. J-Biol-Chem, 267, 13647-13655. 785 IZBAN, M. G. & LUSE, D. S. 1993. The increment of SII-facilitated transcript cleavage 786 varies dramatically between elongation competent and incompetent RNA 787 polymerase II ternary complexes. J-Biol-Chem, 268, 12874-12885. 788 JENNEBACH, S., HERZOG, F., AEBERSOLD, R. & CRAMER, P. 2012. Crosslinking-MS 789 analysis reveals RNA polymerase I domain architecture and basis of rRNA 790 cleavage. Nucleic Acids Res, 40, 5591-601. 791 JEON, C. & AGARWAL, K. 1996. Fidelity of RNA polymerase II transcription controlled by 792 elongation factor TFIIS. Proc Natl Acad Sci U S A, 93, 13677-82. 793 KHATTER, H., VORLANDER, M. K. & MULLER, C. W. 2017. RNA polymerase I and III: 794 similar yet unique. Curr Opin Struct Biol, 47, 88-94. 795 KIM, J., GUERMAH, M. & ROEDER, R. G. 2010. The human PAF1 complex acts in 796 chromatin transcription elongation both independently and cooperatively with 797 SII/TFIIS. Cell, 140, 491-503. 798 KROGAN, N. J., KIM, M., AHN, S. H., ZHONG, G., KOBOR, M. S., CAGNEY, G., EMILI, A., 799 SHILATIFARD, A., BURATOWSKI, S. & GREENBLATT, J. F. 2002. RNA polymerase 800 II elongation factors of : a targeted proteomics 801 approach. Mol Cell Biol, 22, 6979-92. 802 KUHN, C. D., GEIGER, S. R., BAUMLI, S., GARTMANN, M., GERBER, J., JENNEBACH, S., 803 MIELKE, T., TSCHOCHNER, H., BECKMANN, R. & CRAMER, P. 2007. Functional 804 architecture of RNA polymerase I. Cell, 131, 1260-72. bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

805 LILJELUND, P., MARIOTTE, S., BUHLER, J. M. & SENTENAC, A. 1992. Characterization and 806 mutagenesis of the gene encoding the A49 subunit of RNA polymerase A in 807 Saccharomyces cerevisiae. Proc-Natl-Acad-Sci-U-S-A, 89, 9302-5 issn: 0027-8424. 808 LISICA, A., ENGEL, C., JAHNEL, M., ROLDAN, E., GALBURT, E. A., CRAMER, P. & GRILL, S. 809 W. 2016. Mechanisms of backtrack recovery by RNA polymerases I and II. Proc 810 Natl Acad Sci U S A. 811 LOWARY, P. T. & WIDOM, J. 1998. New DNA sequence rules for high affinity binding to 812 histone octamer and sequence-directed nucleosome positioning. J Mol Biol, 276, 813 19-42. 814 LUSE, D. S., SPANGLER, L. C. & UJVARI, A. 2011. Efficient and rapid nucleosome traversal 815 by RNA polymerase II depends on a combination of transcript elongation factors. 816 J Biol Chem, 286, 6040-8. 817 MAYER, A., LIDSCHREIBER, M., SIEBERT, M., LEIKE, K., SODING, J. & CRAMER, P. 2010. 818 Uniform transitions of the general RNA polymerase II transcription complex. Nat 819 Struct Mol Biol, 17, 1272-8. 820 MERKL, P., PEREZ-FERNANDEZ, J., PILSL, M., REITER, A., WILLIAMS, L., GERBER, J., 821 BOHM, M., DEUTZMANN, R., GRIESENBECK, J., MILKEREIT, P. & TSCHOCHNER, H. 822 2014. Binding of the nsi1 to its cognate DNA site is sufficient to 823 terminate RNA polymerase I transcription in vitro and to induce termination in 824 vivo. Mol Cell Biol, 34, 3817-27. 825 MERZ, K., HONDELE, M., GOETZE, H., GMELCH, K., STOECKL, U. & GRIESENBECK, J. 2008. 826 Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized 827 chromatin associated with the high-mobility group protein Hmo1 and are largely 828 devoid of histone molecules. Genes Dev, 22, 1190-204. 829 NOGI, Y., YANO, R., DODD, J., CARLES, C. & NOMURA, M. 1993. Gene RRN4 in 830 Saccharomyces cerevisiae encodes the A12.2 subunit of RNA polymerase I and is 831 essential only at high temperatures. Mol. Cell. Biol., 13, 114-122. 832 OKUDA, M., WATANABE, Y., OKAMURA, H., HANAOKA, F., OHKUMA, Y. & NISHIMURA, Y. 833 2000. Structure of the central core domain of TFIIEbeta with a novel double- 834 stranded DNA-binding surface. EMBO J, 19, 1346-56. 835 PILSL, M., CRUCIFIX, C., PAPAI, G., KRUPP, F., STEINBAUER, R., GRIESENBECK, J., 836 MILKEREIT, P., TSCHOCHNER, H. & SCHULTZ, P. 2016a. Structure of the 837 initiation-competent RNA polymerase I and its implication for transcription. Nat 838 Commun, 7, 12126. 839 PILSL, M., MERKL, P. E., MILKEREIT, P., GRIESENBECK, J. & TSCHOCHNER, H. 2016b. 840 Analysis of S. cerevisiae RNA Polymerase I Transcription In Vitro. Methods Mol 841 Biol, 1455, 99-108. 842 PLASCHKA, C., HANTSCHE, M., DIENEMANN, C., BURZINSKI, C., PLITZKO, J. & CRAMER, P. 843 2016. Transcription initiation complex structures elucidate DNA opening. Nature, 844 533, 353-8. 845 PRESCOTT, E. M., OSHEIM, Y. N., JONES, H. S., ALEN, C. M., ROAN, J. G., REEDER, R. H., 846 BEYER, A. L. & PROUDFOOT, N. J. 2004. Transcriptional termination by RNA 847 polymerase I requires the small subunit Rpa12p. Proc Natl Acad Sci U S A, 101, 848 6068-73. 849 RANI, P. G., RANISH, J. A. & HAHN, S. 2004. RNA polymerase II (Pol II)-TFIIF and Pol II- 850 complexes: the major stable Pol II complexes and their activity in 851 transcription initiation and reinitiation. Mol Cell Biol, 24, 1709-20. 852 REITER, A., HAMPERL, S., SEITZ, H., MERKL, P., PEREZ-FERNANDEZ, J., WILLIAMS, L., 853 GERBER, J., NEMETH, A., LEGER, I., GADAL, O., MILKEREIT, P., GRIESENBECK, J. & bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

854 TSCHOCHNER, H. 2012. The Reb1-homologue Ydr026c/Nsi1 is required for 855 efficient RNA polymerase I termination in yeast. EMBO J. 856 RENNER, D. B., YAMAGUCHI, Y., WADA, T., HANDA, H. & PRICE, D. H. 2001. A highly 857 purified RNA polymerase II elongation control system. J Biol Chem, 276, 42601-9. 858 RHODES, D. & LASKEY, R. A. 1989. Assembly of nucleosomes and chromatin in vitro. 859 Methods Enzymol, 170, 575-85. 860 SADIAN, Y., TAFUR, L., KOSINSKI, J., JAKOBI, A. J., WETZEL, R., BUCZAK, K., HAGEN, W. J., 861 BECK, M., SACHSE, C. & MULLER, C. W. 2017. Structural insights into 862 transcription initiation by yeast RNA polymerase I. EMBO J, 36, 2698-2709. 863 SAMBROOK, J., FRITSCH, E. F. & MANIATIS, T. 1989. Molecular Cloning: A Laboratory 864 Manual., New York, Cold Spring Harbour Laboratory Press. 865 SANZ-MURILLO, M., XU, J., BELOGUROV, G. A., CALVO, O., GIL-CARTON, D., MORENO- 866 MORCILLO, M., WANG, D. & FERNANDEZ-TORNERO, C. 2018. Structural basis of 867 RNA polymerase I stalling at UV light-induced DNA damage. Proc Natl Acad Sci U 868 S A. 869 SCHIESTL, R. H. & GIETZ, R. D. 1989. High efficiency transformation of intact yeast cells 870 using single stranded nucleic acids as a carrier. Curr Genet, 16, 339-46. 871 SCHNEIDER, D. A., FRENCH, S. L., OSHEIM, Y. N., BAILEY, A. O., VU, L., DODD, J., YATES, J. 872 R., BEYER, A. L. & NOMURA, M. 2006. RNA polymerase II elongation factors Spt4p 873 and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA 874 processing. Proc Natl Acad Sci U S A, 103, 12707-12. 875 SCHWEIKHARD, V., MENG, C., MURAKAMI, K., KAPLAN, C. D., KORNBERG, R. D. & BLOCK, 876 S. M. 2014. Transcription factors TFIIF and TFIIS promote transcript elongation 877 by RNA polymerase II by synergistic and independent mechanisms. Proc Natl 878 Acad Sci U S A, 111, 6642-7. 879 STUDITSKY, V. M., KASSAVETIS, G. A., GEIDUSCHEK, E. P. & FELSENFELD, G. 1997. 880 Mechanism of transcription through the nucleosome by eukaryotic RNA 881 polymerase. Science, 278, 1960-3. 882 TAFUR, L., SADIAN, Y., HOFFMANN, N. A., JAKOBI, A. J., WETZEL, R., HAGEN, W. J. H., 883 SACHSE, C. & MULLER, C. W. 2016. Molecular Structures of Transcribing RNA 884 Polymerase I. Mol Cell, 64, 1135-1143. 885 TAFUR, L., SADIAN, Y., WETZEL, R., WEIS, F. & MULLER, C. W. 2018. Structural 886 rearrangement of TFIIS and TFIIE/TFIIF- like subunits in RNA polymerase I 887 transcription complexes. bioRxiv. 888 THOMAS, M. J., PLATAS, A. A. & HAWLEY, D. K. 1998. Transcriptional fidelity and 889 proofreading by RNA polymerase II. Cell, 93, 627-37. 890 TSCHOCHNER, H. 1996. A novel RNA polymerase-I dependent RNase activity that 891 shortens nascent transcripts from the 3´end. Proc. Natl. Acad. Sci. U.S.A., 93, 892 12914-12919. 893 UJVARI, A., PAL, M. & LUSE, D. S. 2011. The functions of TFIIF during initiation and 894 transcript elongation are differentially affected by phosphorylation by casein 895 2. J Biol Chem, 286, 23160-7. 896 VAN MULLEM, V., LANDRIEUX, E., VANDENHAUTE, J. & THURIAUX, P. 2002. Rpa12p, a 897 conserved RNA polymerase I subunit with two functional domains. Mol Microbiol, 898 43, 1105-13. 899 WITTNER, M., HAMPERL, S., STOCKL, U., SEUFERT, W., TSCHOCHNER, H., MILKEREIT, P. 900 & GRIESENBECK, J. 2011. Establishment and maintenance of alternative 901 chromatin states at a multicopy gene locus. Cell, 145, 543-54. bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

902 YAN, Q., MORELAND, R. J., CONAWAY, J. W. & CONAWAY, R. C. 1999. Dual roles for 903 transcription factor IIF in promoter escape by RNA polymerase II. J Biol Chem, 904 274, 35668-75. 905 ZHANG, C. & BURTON, Z. F. 2004. Transcription factors IIF and IIS and nucleoside 906 triphosphate substrates as dynamic probes of the human RNA polymerase II 907 mechanism. J Mol Biol, 342, 1085-99. 908 ZHANG, C., YAN, H. & BURTON, Z. F. 2003. Combinatorial control of human RNA 909 polymerase II (RNAP II) pausing and transcript cleavage by transcription factor 910 IIF, hepatitis delta antigen, and stimulatory factor II. J Biol Chem, 278, 50101-11. 911 ZHANG, C., ZOBECK, K. L. & BURTON, Z. F. 2005. Human RNA polymerase II elongation in 912 slow motion: role of the TFIIF RAP74 alpha1 helix in nucleoside triphosphate- 913 driven translocation. Mol Cell Biol, 25, 3583-95. 914 ZHANG, Y., SIKES, M. L., BEYER, A. L. & SCHNEIDER, D. A. 2009. The Paf1 complex is 915 required for efficient transcription elongation by RNA polymerase I. Proc Natl 916 Acad Sci U S A, 106, 2153-8. 917 ZHANG, Y., SMITH, A. D. T., RENFROW, M. B. & SCHNEIDER, D. A. 2010. The RNA 918 polymerase-associated factor 1 complex (Paf1C) directly increases the elongation 919 rate of RNA polymerase I and is required for efficient regulation of rRNA 920 synthesis. J Biol Chem, 285, 14152-9. 921 bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

922 Figure legends 923 924 Fig. 1 Depletion of the heterodimeric Rpa34.5/49 complex results in reduced Pol I 925 processivity 926 A) Experimental design. Promoter-dependent transcription was performed for 10min 927 on templates attached to magnetic beads using 70nM Rrn3, 20nM CF and 4nM Pol I and 928 in the presence of 200 M ATP, 200 M UTP, 10 M CTP (32P-CTP) but no GTP. 929 Transcription was arrested after 169 nt when the first GTP should be incorporated. 930 Arrested ternary complexes were washed twice with excess transcription buffer without 931 NTPs and radioactivity and were chased adding 200 M NTPs including GTP. At the time 932 points indicated 25 l aliquots were withdrawn and injected in 200 l 0.6% SDS, 0.3 M 933 NaCl, 5 mM EDTA, 0.5 mg/ml Proteinase K, which immediately stopped rRNA synthesis. 934 B) Promoter-dependent elongation assays were performed on template PIP_G-extend 935 2kb which produces a 1759 nt long run off transcript after the elongation block using 4 936 nM WT or 20 nM Rpa49 Pol I. Pulse labelling was for 5 min. Chase conditions were 200 937 M NTPs and 24oC. Radiolabelled transcripts were separated on a denaturing 938 polyacrylamide/urea gel and detected using a PhosphorImager. The arrows indicate 939 that at time point 15´ RNA extension of the WT enzyme was slightly advanced. 940 C) Comparative analysis of Pol I processivity between WT and Rpa49 enzyme of the 941 experiment shown in (B). Percentage of fully extended and arrested transcripts in 942 reference to the pulse labelled arrested transcript (time point 0) at the respective time 943 points. (blue and green: fully extended transcripts of WT and Rpa49; orange and 944 purple: arrested transcript of WT and Rpa49, respectively). The experiment is 945 representative for multiple replicates n>4. 946 947 Fig.2 Reduction of Pol I processivity appears in different mutant enzymes when 948 arrested transcripts should be extended at a G-less cassette 949 A) Promoter-dependent elongation assays were performed as described in Fig. 1, with 950 the exception that the initial pulse was performed for 20 min and that the separation 951 distance of the transcripts on the gel was longer. Assays were performed using 4nM Pol 952 I, Rpa12 and Rpa49 and 12 nM Rpa12. Each experiment is representative for at least 953 three replicates (see also Suppl Fig 2.). B) Percentage of fully extended transcripts in 954 reference to the pulse labelled arrested transcript (time point 0) at the respective time bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

955 points. C) Percentage of arrested transcripts in reference to the pulse labelled arrested 956 transcript (time point 0) at the respective time points. 957 958 959 Fig. 3 Pol I containing Rpa49CT but lacking the dimerization module 960 Rpa34.5/49NT accelerates transcription 961 A) Promoter-dependent elongation assays were performed as described in Fig. 1. 962 Combinations of different Rpa34.5/Rpa49 fractions were added to Rpa49 (4nM) before 963 transcription was started. Either 50 nM purified recombinant heterodimer 964 Rpa34.5/Rpa49 was used or the dimerization domain Rpa34.5/Rpa49NT lacking the 965 aminoacids 181 to 426 of Rpa49 or the C-terminal part of Rpa49 (Rpa49CT) which 966 corresponds to a protein fragment from aminoacid 111 to 426 of Rpa49. Pulse labelling 967 was for 5 min. Under this rather short labelling conditions most transcripts stopped at 968 the end of the G-less cassette. Chase conditions were 200 M NTPs and 0oC. 969 Radiolabelled transcripts were separated on a denaturing polyacrylamide/urea gel and 970 detected using a PhosphorImager. Elongation was faster in the presence of Rpa49CT. 971 The original gel section is shown in Suppl. Fig. 3. Longer exposed gel sections of lanes 972 13-16 and 17-20 are shown on the right. Experiments were repeated at least twice (see 973 also Fig. 3B). 974 975 B) Tailed template transcription to analyse effects of the dimerization domain and 976 Rpa49CT on transcription elongation. Left panel, experimental design was similar as in 977 Fig. 1 with the exception that transcription was initiated at a 3´ overhang of the 978 immobilized template which allowed RNA synthesis without addition of initiation 979 factors. Rpa34.5/Rpa49 or Rpa49CT (each 50 nM) were added to Rpa49 and analysed 980 as in A. Elongation was stopped after 15, 30 and 60 seconds, respectively. 981 982 Fig. 4 The heterodimer Rpa34.5/Rpa49 and the C-terminal part of Rpa12 are both 983 required for correct incorporation of nucleotides, but influence re-extension of 984 arrested transcripts differently 985 A) The dimerization module of Rpa34.5/Rpa49 is required to prevent Pol I from mis- 986 incorporation of nucleotides when transcription is arrested at the G-less cassette. Upper 987 panel Promoter-dependent transcription assays on immobilized templates containing a 988 G-less casette were performed in the presence of ATP, UTP and 32P-CTP using the bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

989 indicated Pol I fractions (5 nM). After the indicated time points supernatants (su) were 990 separated from pellets (p) and analyzed on a 6% polyacrylamide gel containing 6 M 991 urea. Lower panel Ternary stalled transcription complexes are rather stable. 992 Quantitation of the ratio pellet/supernatant of arrested transcripts synthesized on 993 immobilized templates from four different experiments. (dimer: Rpa34.5/Rpa49; CT: 994 Rpa49CT; CT + NT: Rpa49CT + Rpa34.5/Rpa49NT). (Experiment is representative for 995 two replicates). 996 B) Pulse labelling and extension of stalled transcripts of Pol I mutants lacking either the 997 C-terminal part of Rpa12.2 (Rpa12CT), or the entire Rpa12 and Rpa34.5/Rpa49 998 (Rpa12), or Rpa12.2 and the dimerization module (Rpa12 + Rpa49CT). Promoter- 999 dependent transcription assays on immobilized templates containing a G-less cassette 1000 were performed as in Fig. 1. After washing the ternary complexes with a buffer 1001 containing ATP, UTP and CTP (end concentration 200 M), an aliquot was removed. 1002 GTP, UTP and CTP, but no ATP were added to the remaining fraction. Transcription 1003 elongation was stopped after 3 min. The lower panel shows a longer exposure of the 1004 same gel to detect Rpa12-derived transcripts. Transcripts were analyzed on a 6% 1005 polyacrylamide gel containing 6 M urea. The DNA sequence of the end of the G-less 1006 cassette is indicated at the top. Arrows point to the first GTP and the first ATP after the 1007 G-less cassette. (Experiment is representative for two replicates). 1008 1009 1010 Fig. 5 Cooperation between Rpa12 and the heterodimer Rpa34.5/Rpa49 promotes 1011 correct incorporation of nucleotides and re-extension of arrested transcripts 1012 A) The dimerization domain supports transcription fidelity only if the RNA cleaving 1013 activity of Rpa12.2 is present. Transcription reactions were performed using 5 nM 1014 mutant enzymes in the presence or absence of Rpa34.5/Rpa49 domains as indicated 1015 (each 40nM). Pulse labelling on tailed templates containing a G-less cassettes occurred 1016 in the presence of 32P-CTP, ATP and UTP. Transcripts were analyzed on a 6% 1017 polyacrylamide gel containing 6M urea. Superimposed transcript profiles of the 4 single 1018 reactions are depicted near each section. The profiles were normalized to the band 1019 representing correct arrested transcripts. Note that Rpa34.5/Rpa49NT reduced mis- 1020 incorporation of nucleotides, when it was added to Rpa49 + Rpa49CT, which contains 1021 Rpa12.2. In contrast, reduction of NTP mis-incorporation was less effective after bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1022 addition of RPA34.5/Rpa49NT to mutants lacking either Rpa12.2 or Rpa12.2CT . 1023 Experiments were conducted at least in duplicate. 1024 1025 B) The dimerization domain without Rpa12.2 produces stalled transcripts at the G-less 1026 cassette which cannot be extended. Transcription complexes containing 32P-labelled 1027 RNA were generated on tailed templates using the Pol I mutants and protein fractions as 1028 indicated and stalled at the G-less cassette (10 min pulse labelling without GTP). 1029 Complexes were washed once, NTPs (200 M end concentration) were added and the 1030 reactions were stopped after 30 and 180 seconds respectively. Transcripts were 1031 analyzed on a 6% polyacrylamide gel containing 6 M urea. (Note, the radiolabeled band 1032 on top of all lanes is due to non-specific labelling). Quantification of the arrested 1033 transcripts (in brackets) is depicted at the diagram below. (Experiment is representative 1034 for several replicates n>3, see also Suppl. Fig. 4) 1035 1036 Fig 6. Purified Pol I and Pol III transcribe through a nucleosome on tailed 1037 templates more efficiently than Pol II. 1038 Nucleosomes were assembled on tailed templates containing a 601 nucleosome 1039 positioning sequence (601) (derived from plasmid 1253) as described in material and 1040 methods (see also Suppl Fig.5). Transcription reactions were performed in the presence 1041 of a nucleosome-containing and a nucleosome-free reference template. The cartoon on 1042 the left indicates the length of the transcripts derived from the nucleosomal and 1043 reference template. 1044 In vitro transcription assays were performed as described using 7.5 nM of purified RNA 1045 polymerases, identical buffer conditions and 10 nM of template 601 with or without 1046 nucleosomes and the reference template containing neither nucleosomes nor the 601 1047 sequence. The cartoon on the left indicates the length and the anticipated position of the 1048 nucleosome of the transcribed templates. 1049 Lower panel Quantification of the experiment. Signal intensities of 601 full length 1050 transcripts (lanes 1/2, 6/7 and 11/12) were normalized to reference full length 1051 transcript signal intensities in the respective lanes. Then, the ratio of the normalized 601 1052 full length transcript signal intensities in presence and absence of the nucleosome was 1053 calculated. Experiments were conducted at least in duplicate and mean values were 1054 plotted including error bars of the standard deviation. Quantification of the putative Pol 1055 II transcript after 24h exposition did not result in a signal above background level. bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1056 Length of reference transcript 240 nt 1057 Length of the 601 sequence 468 nt 1058 1059 1060 bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1061 Fig. 7 Efficient transcription through nucleosomes requires Rpa34.5/Rpa49 and 1062 Rpa12.2. 1063 A) Transcription reactions were performed on tailed templates using 20 nM Rpa49 or 1064 Rpa12 as in Fig. 6. The cartoon on the left indicates the length of the transcripts 1065 derived from the nucleosomal and reference template. Transcripts were analyzed on a 1066 6% polyacrylamide gel containing 6 M urea. Experiments were conducted in duplicate. 1067 B) The heterodimeric subunit Rpa34.5/Rpa49 of Pol I is required for efficient 1068 transcription through a nucleosome in tailed template assays. Upper panel. 1069 Transcription reactions were performed using 15 nM Pol I lacking subunits Rpa49 and 1070 Rpa34.5 (Rpa49), lanes 1 and 2). Increasing amounts of purified recombinant 1071 Rpa34.5/Rpa49 heterodimer were added to Rpa49-Pol I in reactions analysed in lanes 1072 5 to 12. Reactions analysed in lanes 3 and 4 were performed using 150 nM 1073 Rpa34.5/Rpa49 heterodimer, but no polymerase. (601 short indicates the position of a 1074 stalled transcript at the 5´end of a nucleosome; see also Suppl Fig. 4). 1075 Right panel. Quantification from two independent experiments using the same 1076 preparation of Rpa49. 1077 C) Effects of Rpa49 domains on transcription of a nucleosomal tailed template. Add-back 1078 experiments to Rpa49 were performed using 25, 50 and 75 nM of either purified 1079 recombinant heterodimeric subunits Rpa49CT, Rpa34.5/Rpa49NT, or 50 nM 1080 Rpa34.5/Rpa49. Concentration of Rpa49 Pol I was 10 nM. In contrast to the previous 1081 tailed template transcriptions the length of the templates were switched. Template 1082 concentration was 7.5 nM. The transcripts from the 601 and reference template were 1083 240 nt and 468 nt, respectively. This template combination gave less non-specific 1084 transcripts in the range between 250 nt and 500 nt in the presence of Rpa49CT. The 1085 stimulation of transcription through nucleosomes in reference to the stimulation of 1086 transcription on the nucleosome-free DNA is shown underneath. The same preparation 1087 of WT Pol I and Rpa49 was used. 1088 D) Efficient transcription through nucleosomes requires the C-terminal domain of 1089 Rpa12.2. Tailed template transcription was performed as in C, using 10 nM WT Pol I or 1090 Rpa12.2C. Quantification was performed as in Fig. 6. 1091 1092 1093 bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1094 Supplementary figure legends 1095 1096 Suppl Fig. 1 Separation of affinity-purified, ΔRpa49 Pol I ΔRpa12.2 Pol I, Rpa12C and 1097 Rpa34.5/49 domain-containing fractions on SDS PAGE 1098 A) 6 μg purified WT Pol I, ΔRPA49, ΔRPA12.2 and RPA12C were separated by SDS 1099 PAGE using a 4-12% gradient gel which was stained with SimplyBlue SafeStain 1100 (ThermoFisher) or with Silver. ΔRPA49 Pol I was affinity-purified from strain yJPF162- 1101 1a (2670) using a ProtA-TEV-tag fused to subunit A135 after 24 h depletion of subunit 1102 A49 in glucose containing medium (see Materials and Methods). Molecular weight 1103 standards (M) and Pol I subunits are indicated. B) The depletion of subunits Rpa49 and 1104 Rpa12.2 was confirmed in the affinity purified enzyme fraction by Western blotting 1105 using antibodies against Rpa49, Rpa12.2, which also recognizes Rpa14, and the two 1106 largest subunits. After blotting the membrane was cut into three pieces and equally 1107 treated with the different antibodies. Flag-tagged Rpa49 was separated as reference. 1108 (Note that Rpa49 is neither detected in ΔRPA49 nor in ΔRPA12.2 the band above Rpa49 1109 is due to nonspecific IgG interaction). Subunit RPA12.2 is only completely depleted in 1110 strain ΔRPA12.2 (Y2679) whereas the associated N-terminal part of Rpa12.2 is 1111 detectable in the Rpa12C preparation. C) Separation of recombinant A49/A34.5 1112 domains on SDS PAGE. comparable protein amounts were separated on a 15 % SDS gel 1113 and Coomassie stained. Lane 1: Rpa49 (amino acids 110-415), 2: Rpa34.5/49 (1-186), 3: 1114 A49/A34,5 full length; Expression and purification strategy see Material and Methods 1115 and (Pilsl et al., 2016a). Fractions were used for in vitro transcription. 1116 1117 Suppl. Fig. 2 1118 Reduction of Pol I processivity in different mutant enzymes when elongation at the G- 1119 less cassette occurs with 20 M NTPs 1120 Promoter-dependent elongation assays were performed as described in Fig. 2 with the 1121 1122 performed using 4 nM Pol I, Rpa12CT and Rpa49 (50 nM Rpa49CT) and 12 nM 1123 Rpa12. Percentage of fully extended and arrested transcripts in reference to the pulse 1124 labelled arrested transcript (time point 0) at the respective time points are depicted 1125 below each gel section. 1126 1127 bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1128 1129 Suppl. Fig. 3 1130 Original gel section of Fig 3A, lanes 1-8 1131 1132 1133 Suppl. Fig. 4 1134 A) The dimerization domain without Rpa12.2 produces stalled transcripts at the G-less 1135 cassette which cannot be extended. Same experiment as in Fig. 5B, however using 1136 different batches of Rpa12.2 and heterodimer fractions. Concentration of heterodimer 1137 fractions was 25 nM instead of 50 nM. The strong band at the top of the gel is non- 1138 specific and comigrates with fully extended transcripts. Percentage of the arrested 1139 transcripts is shown below the single lanes. 1140 B) Production of non- extendable arrested transcripts at the G-less cassette depends on 1141 the presence of the heterodimer and the absence of Rpa12.2 . Transcription complexes 1142 containing 32P-labelled RNA were generated using promoter-dependent transcription 1143 using the Pol I mutants and protein fractions as indicated and stalled at the G-less 1144 cassette (10 min pulse labelling without GTP). Complexes were washed twice, NTPs 1145 (200 M end concentration) were added and the reactions were stopped after 30 and 1146 180 seconds respectively. Transcripts were analyzed on a 6% polyacrylamide gel 1147 containing 6 M urea. Percentage of the arrested transcripts is shown below the single 1148 lanes. 1149 1150 1151 1152 Suppl. Fig. 5 1153 Purified core histones H2A, H2B, H3 and H4 from chicken erythrocytes were assembled 1154 according to (Rhodes and Laskey, 1989) using the salt gradient dialysis technique. A) 1155 Schematic outline. B) 12% PAGE (Coomassie stain) of purified chicken histones. C) A 1156 typical assembly reaction (50 l) contained 5 g DNA carrying the 601 nucleosome 1157 positioning sequence (Lowary and Widom, 1998), varying amounts of histone octamer, 1158 200 ng BSA/ml, and 250 ng competitor DNA in high salt buffer (10 mM Tris, pH 7.6, 2 M 1159 NaCl, 1 mM EDTA, 0.05% NP-40, 2 mM -mercaptoethanol). The salt was continuously 1160 reduced to 200 mM NaCl during 16–20 h. The quality of the assembly reaction was bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1161 analysed on a 5% PAA gel in 0.4 x TBE followed by ethidium bromide staining. Different 1162 ratios of DNA to nucleosomes were analysed by electromobility shift assays to reveal 1163 optimal conditions for nucleosome assembly (left panel). Protection assay: 0.5 mg of 1164 naked and nucleosome associated template were incubated with and without restriction 1165 enzyme Nb.BsmI (NEB). Note that the 601 containing sequence was not cleaved in the 1166 presence of a nucleosome. 1167 1168 Suppl. Fig 6. A shorter transcript is detected if Pol II transcription is arrested by a 1169 nucleosome on tailed templates 1170 Longe exposure of lane 6 of Fig. 6 indicated an additional RNA which corresponded to 1171 the length of an arrested transcript. 1172 bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Fig. 1

A B WT (4 nM) ΔRpa49 (20nM)

sec chase 0 5 15 45 120 0 5 15 45 120 full length transcript

200 µM/ 24oC

arrested transcript

C 1 2 3 4 5 6 7 8 9 10 1.2 transcripts 100 1 WT 200 mM NTP WT chased chase 0.8 80 arrested WT 200 mM NTP WT arrested 0.6 60 arrested D49 200 mM NTP ΔRpa49 chased 0.4 40 chase

extended or 0.2 20 D49 200 mM NTP ΔRpa49 arrested arrested

fully 0 0 50 100 150 sec units bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

ΔRpa49 WT Pol I Rpa12 Rpa12 C Fig. 2 Δ Δ + Rpa49CT A 0 15 30 45 60 75 0 15 30 45 60 0 15 30 45 60 0 15 30 45 60 chase sec fully extended transcripts

chase 200µM NTPs 24oC arrested transcripts CCCC

Pol I casePe

less G- promoter

arrested transcripts

B fully extended transcripts C 120 120

WT 200 mM NTP WT 100 WT 200 mM NTP WT 100 arrested

transcripts 80

80 D A12 200 uM NTP ΔRpa12 D A12 200 uM ΔRpa12 transcripts

NTP arrested 60 chase 60 A12NT 200 uM NTP A12NT 200 uM ΔRpa12ΔC 40 ΔRpa12ΔC 40

extended NTP arrested

chase

arrested ΔRpa49 + 20 ΔRpa49 + 20 D49+CT 200 uM

fully D49+CT 200 uM NTP Rpa49CT NTParrested Rpa49CT 0 units 0

units 0 20 40 60 80 0 20 40 60 80 bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A Fig. 3 ΔRpa49 + ΔRpa49 + ΔRpa49 + WT Rpa34.5/Rpa49 Rpa34.5/Rpa49 ΔRpa49 Rpa49CT ΔRpa49 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 sec chase full length transcript chase 200µM NTPs 0oC

arrested transcript CCCC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 13 14 15 16 21 22 23 24 Pol I casePe G- less ΔRpa49 + ΔRpa49 + ΔRpa49 promoter Rpa49CT Rpa34.5/Rpa49NT 0 15 30 60 0 15 30 60 0 15 30 60 sec chase B

200 µM/ 24oC bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Fig. 4

A B G-less cassePe 5´ ATACCAAACCAATTCAACACAAGGGTGAAAAGAGTT

ΔRpa12 4nM WT Rpa12ΔCT ΔRpa12 + Rpa49CT

30 5 30 5 15 30 5 15 30 minutes pulse p su p su p su p su p su p su p su p su p su pulse Rme -GTP p s wash

+GTP,CTP UTP arrested transcripts at Pol I

CCCC

G-less cassePe CCCC Pol I

CCCC casePe casePe

Pol I less less

casePe 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 2 3 4 5 6 7 8

G- pulse with G-

less 100 32 1 G- P-CTP, UTP, ATP promoter 0.8 promoter 0.6 50

0.4 transcripts

percentage immobilized 0.2

0 0 WT dimer CT CT+NT + ΔRpa49 bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Fig. 5 A ΔRpa49 ΔRpa12 Rpa12ΔC + + + + + + Rpa34.5/49NT + + + + + + Rpa49CT

pulse CCCC -GTP

Pol I - case)e 1 2 3 4 5 6 7 8 9 10 11 12 + Rpa49CT less + Rpa34.5/49NT G- tailed + Rpa34.5/49NT template + Rpa49CT 3´ ΔRpa12 ΔRpa12 + Rpa34.5 + Rpa34.5/ /49NT ΔRpa12 Rpa12ΔC ΔRpa49 B 49NT + Rpa49CT +Rpa49CT 0 30 180 0 30 180 0 30 180 0 30 180 0 30 180 sec

1 Pol I

pulse -GTP

wash arrested transcripts +NTPs CCCC CCCC

Pol I case)e case)e

160

ΔRpa12 140 less less

G- Datenreihe1 G- 120 ΔRpa12+ Rpa34.5/Rpa49NT 100 Datenreihe2

transcripts ΔRpa12+ Rpa34.5/Rpa49 80 Datenreihe3 3´ 3´ tailed 60 Datenreihe4 Rpa12ΔC template 40 arrested 20 Rpa12DC ΔRpa49+ Rpa49CT 0 0 0 3030 180180 sec bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Fig. 6

nucleosome

tailed template bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Fig. 7 A

1 2 3 4 5 6 tailed template

B length transcripts ra>o of normalized full

tailed template

C D

1- 2- 2.5- 3.6- 1.2- 0.7- 0.7- 1.7- fold normalized s>mula>on of nucleosomal transcript bioRxiv preprint doi: https://doi.org/10.1101/433375; this version posted October 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

normalized stimulation of transcription through nucleosomes ΔRpa49 1 + Rpa34.5/49 2.7 +/- 0.6 + Rpa34.5/49 NT 1.2 +/- 0.1 + Rpa49 CT 2.0 +/- 0.3

Table 1. Addition of the complete heterodimer RPA34.5/49 or Rpa49CT to ΔRpa49 results in stimulation of promoter-dependent transcription on nucleosomal templates. Signal intensities of 601 full length transcripts were normalized to reference full length transcript signal intensities in the respective lanes. Then, the ratio of the normalized 601 full length transcript signal intensities in presence and absence of the nucleosome was calculated. The value for the ΔRpa49 enzyme was set to 1 and the stimulation adding the different Rpa34.5/49 domains (150nM) was calculated. Mean values of each two experiments are shown.

Suppl. Fig. 1

A B C C D M D . Rpa49 Pol I WT Rpa49 M DRpa12 Rpa12DC Rpa49 Rpa12 rec WT D D Rpa12

Rpa190 M Rpa190 Rpa190 Rpa49CTRpa34.5/49NTRpa34.5/49 Rpa135 Rpa135 Rpa135 Rpa190 Rpa135

Rpa49 Rpa43 Rpa43 AC40 Rpa49 Rpa49 AC40 Rpa34.5 Rpa43 AC40 ABC27 Rpa34.5 ABC27 AC19 TEV ABC27 AC19 ABC23 AC19 ABC23 Rpa14 ABC23 Rpa14 Rpa14 Rpa14 Rpa12.2 Rpa12 ABC10a/b ABC10a/b Rpa12NT 1 2 3 Coomassie/silver-stained gels Westernblot Coomassie-stained gel DRpa49 WT Pol I DRpa12 Rpa12DCT Suppl. Fig. 2 + Rpa49CT 0 15 30 45 60 90 0 15 30 45 60 0 15 30 45 60 0 15 30 45 60 chase sec

chase 20µM NTPs 24oC CCCC Pol I Pol casette less - G

promoter 100 80 60 40 20

0 0 20 40 60 90 0 20 40 60 0 20 40 60 0 20 40 60 sec

fully elongated transcripts arrested transcripts Suppl. Fig. 3 A DRpa49 + WT (4nM) Rpa34.5/Rpa49 0 20 40 60 0 20 40 60 sec chase full length transcript chase 200µM NTPs 0oC CCCC 1 2 3 4 5 6 7 8 Pol I Pol casette less - G

promoter Suppl. Fig. 4

A B

Rpa12 + DRpa12 + Rpa12 + D D DRpa12 Rpa34.5/Rpa49 Rpa12DC + Rpa49CT DRpa12 Rpa34.5/Rpa49NT Rpa34.5/Rpa49NT 0 30 180 0 30 180 0 30 180 sec chase 0 30 60 0 30 60 0 30 60 sec chase Pol I Pol pulse I Pol -GTP

pulse wash -GTP

+NTPs wash arrested arrested transcripts +NTPs CCCC

transcripts CCCC CCCC CCCC 100 60 49 100 90 93 100 115 87 percentage arrested Pol I Pol Pol I Pol

transcripts casette casette 100 14 5 100 40 27 100 65 60 casette casette less less percentage arrested - less - less - - G G G G transcripts ´ ´ 3 tailed 3 template promoter Suppl. Fig. 5

Suppl. Fig. 5

A B

kDa C 70 2M NaCl 55 45 35 25

H3 601 200mM NaCl H2B 15 H2A H4

C ratio histones : DNA naked nucleosome - 0.4 0.6 0.8 1 + - + - BsiWI bp nucleosomal template 601 5000 naked vector backbone bp 2000 1500 1000 1000 700 500 500 naked template 601 400 400 300 300 200

200 100 Suppl. Fig. 6 Supplementary

Table 1: Strains

Strain Name Parental strain Genotype Reference

BY4741 A135- MATa, his3-1 leu2-0 met15-0 ura3- (Hierlmeier y2423 TEV-ProtA BY4741 0 rpa135::RPA135-TEV-ProtA- et al., 2013) kanMX6

yJPF162-1a (Pilsl et al., Y2670 BY4741 his31; leu20; met15-0; ura3-0; RPA135-TEV-ProtA::kanMX6 2016) HIS3MX::GAL::HA-RPA49

MATa, his3-1, leu2-0, lys2-, ura3-0 (Merkl et Y2679 yJPF165-1a BY4741 RPA135-TEV-ProtA::kanMX6 al., 2014) HIS3MX::GAL::HA-RPA12

y2423 BY4741 A135- BY4741 MATa, his3-1 leuy2-0 met15-0 (Hierlmeier TEV-ProtA ura3-0 rpa135::RPA135-TEV- et al., 2013) ProtA-kanMX6 y2424 BY4741 Rpb2- BY4741 MATa, his3-1 leu2-0 met15-0 ura3- (Hierlmeier TEV-ProtA 0 rpb2::RPB2-TEV-ProtA-kanMX6 et al., 2013)

y2425 BY4741 C128- BY4741 MATa, his3-1 leu2-0 met15-0 ura3- this study TEV-ProtA 0 c128::C128-TEV-ProtA-kanMX6

To obtain the yeast mutant expressing Rpa12.2DCT plasmid p1839 was transformed in strain y2679. Cultivation was in glucose-containing medium.

Table 2: Plasmids

EcoRI/EcoRV digested amplicon from Merkl et al., 1247 pRS316 SIRT G-less with 2107/2108 into pUC19PIP g-601 KasI, blunted/EcoRI digested pUC19 601 2014)

contains Pol I promoter template with rDNA terminator including T1 T-rich element for in vitro transcription Merkl et al., 1961 pUC 19 PIP g- TER SacI/BamHI cut terminator region 2014) amplified with primers #3050/3051 was cloned into SacI/BamHI cut vector #1247

amplicon of primers #2479 and #2480 pUC 19 PIP g- TER from pT11 phosphorylated and cloned into Merkl et al., 2148 elongated SacI cut #1961 2014)

Merkl et al., 2313 pUC19 PIP g- elong extended g-less cassette to enhance 601 labelling in the elongation assay; 2014) for promotor-dependent transcription SacI-fragment (3780 bp) from YepSIRT (Musters et al., 1989) containing ETS1 (Pilsl et al., 435 pMAX1 including Pol I promoter and 520 bp at the 2016) C-terminus of 25S rDNA cloned in pBluescript (6731 bp)

tailed template DNA containing 601 Merkl et al., 1253 pUC 19 tail G-601 binding sequence for unspecific in vitro 2014) transcription

tailed template DNA without 601 binding Merkl et al., 1573 pUC19 tail G- sequence for unspecific in vitro 2014) transcription

2316 pUC19 tail g- elong extended g-less cassette to enhance this study 601 labelling in the elongation assay; for promotor-independent Trx

pCM182-LEU2- 1839 plasmid expressing the N-terminus of this study rpa12delC Rpa12 (aa 1- aa 85)

Table 3: Oligonucleotides

4228 PIP_Reb1_fwd ttacccggggcacctgtc amplification of rDNA promoter including Reb1 BS

4018 3´r bio 601 1k biotin_cctacagcgtgagctatgagaaag amplification of biotinylated 1000nt template

4220 3´r bio 601 2k biotin_ cgaggtcgaggggcacctgtcac amplification of biotinylated 2000nt template 4231 601 rev 263 nt gcctgcaggtcgactctag amplification of 263 nt long RNA coding DNA

4232 601 rev 263 nt biotin_ gcctgcaggtcgactctag amplification of 263 nt long RNA coding biotinylated DNA

4233 601 rev 191nt atccccttggcggttaaaacg amplification of 191 nt long RNA coding DNA

4234 601 rev 448nt ctggcacgacaggtttccc amplification of 448 nt long RNA coding DNA

4220 tail fwd ATCACCTTACCCTATACTTACTCG competitor for tailed competitor rev template transcription

2115 5´for_tail_primer CGCTAGGCGCCGATATCACCTTACCC Forward primer for tailed TATACTTACTCGCATTCCCTACAAT template TCTACTTCATACCAAACCAATTC

4176 #pMax rev 320 nt ttactattgcggtaacattcat amplification of trx template pMAX1 with#2973

4177 #pMax rev 480 nt atattgtgtggagcaaagaaat amplification of trx template pMAX1 with#2973

2973 35S rRNA UE fwd agcttaaattgaagttttctc amplification of trx template pMAX1