1

RESEARCH ARTICLE 1

2

Critical Role of Transcript Cleavage in Arabidopsis RNA Polymerase II 3

Transcriptional Elongation 4

5 6

Wojciech Antosz1, Jules Deforges

2, Kevin Begcy

3, Astrid Bruckmann

4, Yves Poirier

2, Thomas 7

Dresselhaus1 and Klaus D. Grasser

1,58 9

10 1 Department of Cell Biology & Plant Biochemistry, Biochemistry Centre, University of Regensburg, 11 Germany 12 2 Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland 13 3 Environmental Horticulture Department, University of Florida, Gainesville, FL 32611-0670, USA 14 4 Department for Biochemistry I, Biochemistry Centre, University of Regensburg, Germany 15 5 Corresponding Author: Klaus.Grasser@ur.de 16

17 Short title: Transcript cleavage by RNA polymerase II 18

19 One-sentence summary: Backtracking/arrest of RNA polymerase II commonly occurs during 20 transcript elongation and reactivation of the enzyme is a prerequisite for plant growth. 21

22 The author responsible for distribution of materials integral to the findings presented in this article in 23 accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Klaus D. 24 Grasser (Klaus.Grasser@ur.de) 25

26 27

ABSTRACT 28

Transcript elongation factors associate with elongating RNA polymerase II (RNAPII) to 29

control the efficiency of mRNA synthesis and consequently modulate plant growth and 30

development. Encountering obstacles during transcription such as nucleosomes or particular 31

DNA sequences may cause backtracking and transcriptional arrest of RNAPII. The elongation 32

factor TFIIS stimulates the intrinsic transcript cleavage activity of the polymerase, which is 33

required for efficient rescue of backtracked/arrested RNAPII. A TFIIS mutant variant 34

(TFIISmut) lacks the stimulatory activity to promote RNA cleavage, but instead efficiently 35

inhibits unstimulated transcript cleavage by RNAPII. We could not recover viable 36

Arabidopsis thaliana tfIIs plants constitutively expressing TFIISmut. Induced, transient 37

expression of TFIISmut in tfIIs plants provoked severe growth defects, transcriptomic 38

changes and massive, transcription-related redistribution of elongating RNAPII within 39

transcribed regions towards the transcriptional start site. The predominant site of RNAPII 40

accumulation overlapped with the +1 nucleosome, suggesting that upon inhibition of RNA 41

cleavage activity, RNAPII arrest prevalently occurs at this position. In the presence of 42

TFIISmut, the amount of RNAPII was reduced, which could be reverted by inhibiting the 43

proteasome, indicating proteasomal degradation of arrested RNAPII. Our findings suggest 44

that polymerase backtracking/arrest frequently occurs in plant cells and RNAPII-reactivation 45

is essential for correct transcriptional output and proper growth/development. 46

47

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Plant Cell Advance Publication. Published on March 9, 2020, doi:10.1105/tpc.19.00891

©2020 American Society of Plant Biologists. All Rights Reserved

2

INTRODUCTION 49

In eukaryotes, the abundance of functional mRNAs is precisely controlled in a spatially and 50

temporally defined manner. Besides post-transcriptional mechanisms (e.g. control of splicing 51

or mRNA stability) synthesis of pre-mRNAs by RNA polymerase II (RNAPII) is accurately 52

regulated. For a considerable time, it was assumed that the transcription of protein-coding 53

genes is controlled exclusively during transcriptional initiation. However, the elongation stage 54

of RNAPII transcription also turned out to be highly dynamic and tightly regulated. As a 55

distinguishing feature, the carboxy-terminal domain of the large subunit of RNAPII (RNAPII-56

CTD) is modified during subsequent steps of transcript synthesis. Thus, residues within 57

conserved heptapeptide repeats (e.g. S2, S5) of the RNAPII-CTD are differentially 58

phosphorylated in the course of transcriptional elongation (Hajheidari et al., 2013; Harlen and 59

Churchman, 2017). 60

The importance of regulating the progression of RNAPII is also reflected by existence 61

of a variety of transcript elongation factors (TEFs). Although transcript elongation is 62

generally processive, it represents a rather discontinuous process, involving pausing, 63

backtracking and even transcriptional arrest, requiring the action of certain TEFs to stimulate 64

RNAPII progression. TEFs also enable efficient transcript synthesis in the chromatin context, 65

since nucleosomes represent major obstacles to transcriptional elongation. Consequently, 66

TEFs act as histone chaperones, modify histones within transcribed regions, or regulate 67

catalytic properties of RNAPII to ensure that elongation occurs efficiently (Chen et al., 2018; 68

Selth et al., 2010; Sims et al., 2004). 69

Among the modulators of RNAPII activity is TFIIS that facilitates RNAPII 70

transcription through blocks to elongation (Fish and Kane, 2002; Wind and Reines, 2000). 71

TFIIS deeply inserts a highly conserved, β-hairpin of its C-terminal domain into the RNAPII 72

complex approaching the polymerase active site to reactivate arrested RNAPII (Kettenberger 73

et al., 2003). In addition to its RNA polymerisation activity, RNAPII has a relatively weak 74

intrinsic RNA nuclease activity. Structural studies demonstrated that reactivation of arrested 75

RNAPII is accomplished by TFIIS-induced extensive conformational changes in the 76

elongation complex (Kettenberger et al., 2003). Consequently, the backtracked/arrested RNA 77

is mobilised and two invariant acidic side chains positioned at the tip of the TFIIS hairpin 78

complement the enzyme active site. Thereby, TFIIS stimulates the weak intrinsic nuclease 79

activity of RNAPII, resulting in cleavage of the backtracked/arrested RNA and generating a 80

new RNA 3´ end at the active site that allows transcription to resume (Cheung and Cramer, 81

3

2011; Wang et al., 2009). Consistent with these structural studies, earlier in vitro transcription 82

analyses demonstrated that the TFIIS-promoted RNA cleavage stimulated transcript 83

elongation by RNAPII (Izban and Luse, 1992; Jeon et al., 1994). 84

In view of the importance of TFIIS for transcript elongation in vitro, it was surprising 85

that yeast TFIIS (Dst1) is dispensable for cells grown under normal conditions suggesting that 86

the slower, unstimulated RNA cleavage activity of RNAPII is sufficient for transcription 87

when yeast cells are grown at standard conditions (Fish and Kane, 2002; Svejstrup, 2007; 88

Wind and Reines, 2000). Alternatively, the apparently non-essential function of TFIIS in 89

yeast could imply that transcription in vivo is a smoother process than in vitro, so that 90

transcript cleavage and escape from arrest are not important. 91

Expression of a mutant version of TFIIS in wild type yeast cells caused severe 92

dominant-negative growth defects and expression in ∆dst1 cells even proved to be lethal 93

(Sigurdsson et al., 2010). In this mutant version (termed TFIISmut), the two conserved acidic 94

residues at the tip of the hairpin were changed to alanine residues, which resulted in a loss of 95

the transcript cleavage stimulatory activity. Beyond that, this mutation efficiently inhibited the 96

intrinsic RNAPII transcript cleavage reaction (Imashimizu et al., 2013; Jeon et al., 1994; 97

Kettenberger et al., 2004; Sigurdsson et al., 2010). The experiments in yeast further suggested 98

that transcriptional elongation problems frequently occur in vivo and require reactivation of 99

backtracked/arrested RNAPII (Sigurdsson et al., 2010). 100

Several TEFs including TFIIS were found to associate with elongating RNAPII in 101

plant cells, forming the transcript elongation complex (TEC) (Antosz et al., 2017). Moreover, 102

various studies particularly in the Arabidopsis thaliana model have demonstrated that 103

different TEFs are critical for proper plant growth and development (Van Lijsebettens and 104

Grasser, 2014). Hence, RNAPII-associated TEFs (e.g. FACT, PAF1C, SPT4/SPT5, 105

Elongator) were found to modulate diverse processes such as hormone signalling, circadian 106

clock and developmental transitions (Dürr et al., 2014; He et al., 2004; Lolas et al., 2010; Ma 107

et al., 2018; Nelissen et al., 2010; Oh et al., 2004). However, similar to the situation in yeast, 108

TFIIS seems to be non-essential in Arabidopsis under normal growth conditions, as mutant 109

plants deficient in TFIIS or plants over-expressing TFIIS show basically wild type appearance 110

(Grasser et al., 2009; Mortensen and Grasser, 2014). 111

Interestingly, despite their apparent wild type phenotype tfIIs mutants exhibit defects 112

in seed dormancy (Grasser et al., 2009; Liu et al., 2011), which are caused by reduced 113

transcript levels of DELAY OF GERMINATION1 (Liu et al., 2011; Mortensen and Grasser, 114

4

2014). This gene is expressed seed-specifically and is a known QTL for seed dormancy in 115

Arabidopsis (Bentsink et al., 2006). Expression of the TFIISmut variant in Arabidopsis wild 116

type plants resulted in a range of distinct developmental defects including leaf serration and 117

reduced stem elongation. Viable transformants could not be recovered when it was attempted 118

to express TFIISmut in tfIIs mutant background (Dolata et al., 2015). This finding indicates 119

that in Arabidopsis the endonucleolytic RNA cleavage activity of RNAPII is required for 120

viability as it was observed in yeast (Sigurdsson et al., 2010). 121

To further study the role of the intrinsic nuclease activity of RNAPII in Arabidopsis in 122

the absence of endogenous TFIIS, we established an inducible expression system of TFIISmut 123

in the tfIIs mutant background. Induction of TFIISmut expression initially caused marked 124

growth defects and ultimately lethality. Using this inducible system, we investigated the 125

effects of transient expression of TFIISmut and transcript cleavage inhibition on plant growth 126

and cell proliferation as well as on gene expression pattern and genome-wide RNAPII 127

distribution. 128

129

RESULTS 130

Induced expression of TFIISmut severely impairs growth and cell proliferation of tfIIs 131

plants 132

To study the effect of TFIISmut expression in plants, the two invariant acidic residues (D359, 133

E360 in Arabidopsis TFIIS, Figure 1A,B) positioned at the tip of the highly conserved hairpin 134

of the C-terminal domain were changed to alanine. To that end the genomic TFIIS sequence 135

was mutagenised and expressed under control of the native promoter in tfIIs plants. Consistent 136

with a previous study (Dolata et al., 2015), we were unable to obtain viable, homozygous tfIIs 137

plants, harbouring the TFIISmut construct. This confirmed that the intrinsic nuclease activity 138

of RNAPII is essential in Arabidopsis. 139

To further analyse the endonucleolytic RNA cleavage by RNAPII, we employed 140

transient expression of TFIISmut (and for comparison TFIIS) based on a β-estradiol inducible 141

system (Brand et al., 2006) schematically presented in Supplemental Figure 1A. The 142

sequences encoding TFIISmut/TFIIS were N-terminally fused to GFP to visualise expression 143

of the proteins. tfIIs plants were transformed with these constructs and three independent 144

transgenic lines each were further characterised by PCR-based genotyping (Supplemental 145

Figure 1B). Analysis of leaves and roots of these plants at different times after addition of β-146

estradiol (or mock treated with ethanol) by confocal fluorescence microscopy revealed 147

5

comparable induction kinetics for GFP-TFIISmut and GFP-TFIIS (Supplemental Figure 2). 148

Nuclear GFP fluorescence was detectable after 3h of induction. After longer induction periods 149

(6h, 24h) the fluorescence signal was observed in a larger number of cells with increasing 150

intensity. These analyses showed that both GFP-TFIISmut and GFP-TFIIS are efficiently 151

expressed upon β-estradiol induction. 152

To examine the effect of induced TFIISmut expression on plant growth, seeds of three 153

independent lines each expressing inducibly GFP-TFIISmut or GFP-TFIIS were sown on 154

Murashige and Skoog (MS) medium supplemented with β-estradiol or mock treated. Images 155

taken seven days after stratification (7 DAS) demonstrated that GFP-TFIISmut plants were 156

severely affected in growth. Many seeds were unable to establish seedling growth at all, 157

whereas the growth of controls is indistinguishable from that of Col-0 wild type plants (Figure 158

1C). These observations confirm the deleterious effects of the expression of GFP-TFIISmut in 159

the tfIIs background on plant growth, whereas plants expressing TFIIS (Col-0; tfIIs expressing 160

GFP-TFIIS) or plants lacking TFIIS (tfIIs) grow comparably. 161

To further evaluate the effects of temporary TFIISmut expression on plants, we 162

measured the root growth of seedlings. Seedlings (8 days after stratification, 8 DAS) were 163

transferred onto medium supplemented with β-estradiol or ethanol, and growth of the primary 164

root was scored at different times after induction. In comparison to non-induced plants, a clear 165

inhibition of root growth was observed at different times after induction with GFP-TFIISmut 166

plants, but not with GFP-TFIIS (Figure 2A). Quantification of absolute and relative root 167

elongation (Figure 2B,C) demonstrated that root growth of GFP-TFIISmut plants was 168

distinctly decreased. 169

Employing the cell cycle marker pCYCB1;1-GFP that allows detection of cells at the 170

G2-M phase of the cell cycle (Ubeda-Tomás et al., 2009), we examined whether reduced cell 171

proliferation might be a cause of decreased root growth. To allow visualisation of the cell 172

cycle marker, the pCYCB1;1-GFP construct was introduced into plants that harbour inducible 173

expression cassettes of TFIISmut/TFIIS fused to a GS-tag ((Bürckstümmer et al., 2006); see 174

below for details). When compared with GS-TFIIS, β-estradiol induced expression of GS-175

TFIISmut resulted in a significantly reduced number of mitotic cells (Figure 2D,E), indicating 176

that decreased cell proliferation contributed to reduced root growth. 177

178

Like TFIIS, TFIISmut interacts with the TEC, but exhibits reduced nuclear mobility 179

6

Using protein affinity purification in combination with mass spectrometry (AP-MS), we 180

examined the association of TFIISmut and TFIIS with the RNAPII-TEC and compared the 181

composition of the complex. Both proteins fused to an N-terminal GS-tag were expressed 182

under control of the β-estradiol inducible system in Arabidopsis PSB-D suspension cultured 183

cells and affinity-purified using IgG-chromatography. This method was successfully 184

employed before to isolate various nuclear protein complexes (Antosz et al., 2017; Dürr et al., 185

2014; Nelissen et al., 2010). Both fusion proteins were efficiently isolated from protein 186

extracts that showed a comparable band pattern as analysed by SDS-PAGE (Supplemental 187

Figure 3A). Co-purified proteins were examined initially using immunoblotting with different 188

RNAPII-specific antibodies (Supplemental Figure 3B). Both GS-TFIISmut and GS-TFIIS co-189

purified with RNAPII, as observed before for Arabidopsis TFIIS (Antosz et al., 2017). The 190

S2P modified version co-purifies ~3-fold more prominently with GS-TFIISmut than with GS-191

TFIIS. Further analysis of the co-purified proteins by AP-MS revealed that in addition to 192

TFIIS and RNAPII subunits, various RNAPII-associated TEFs were detected (Table 1). 193

Among the identified TEFs were characteristic TEC-components such as PAF1C, FACT and 194

SPT4/SPT5. These results indicated that both TFIIS and TFIISmut interact with the RNAPII-195

TEC that exhibits a comparable composition. 196

In view of the TFIISmut-induced inhibition of RNAPII-mediated transcript cleavage 197

that results in transcriptional arrest, elongation complexes containing TFIISmut may display 198

reduced nuclear dynamics. To examine whether TFIIS and TFIISmut associate with 199

complexes of different mobility, we studied their dynamics in Arabidopsis cells. GFP-200

TFIISmut and GFP-TFIIS were expressed in PSB-D cells and analysed by fluorescence 201

recovery after photobleaching (FRAP). FRAP measurements demonstrated that GFP-202

TFIISmut displayed a clearly lower mobility in nuclei (Figure 3A). Further quantification of 203

the data revealed that GFP-TFIISmut re-distributed more slowly after photobleaching than 204

GFP-TFIIS resulting in an increased half-time of the protein (t1/2 = 0.74 s vs. t1/2 = 0.26 s), 205

while the mobile fraction (Mf = 69.7% vs. Mf = 97.7%) of GFP-TFIISmut is reduced relative 206

to that of GFP-TFIIS (Figure 3B,C). Taken together our results suggest that TFIISmut 207

associates properly with the RNAPII-TEC, but it exhibits a reduced nuclear mobility, which 208

may be due to RNAPII stalling upon association with TFIISmut. 209

210

Marked changes in the transcriptome induced by the expression of TFIISmut 211

7

To explore whether the presence of TFIISmut (relative to that of TFIIS) results in an altered 212

transcriptome, changes in mRNA abundance upon induced expression of TFIISmut vs. TFIIS 213

were analysed by RNAseq. cDNAs prepared from polyadenylated mRNAs of total RNA 214

samples of 9-DAS GFP-TFIISmut and GFP-TFIIS seedlings either after 24h of β-estradiol 215

induction or mock treated were analysed by next-generation sequencing. Differentially 216

expressed genes (DEGs; log2FC > 1, P < 0.001) were identified between the different plant 217

lines and depending on induction conditions. Modest transcriptomic changes (< 200 DEGs) 218

were observed between the mock controls and upon induction of GFP-TFIIS, whereas 219

induction of GFP-TFIISmut resulted in more severe (> 1.100 DEGs) transcriptomic 220

alterations (Figure 4A). Among the DEGs dependent on GFP-TFIISmut expression, up-221

regulation of genes was more prominent than down-regulation. Visualisation of the 222

transcriptomic changes using a heat map representing all transcribed genes illustrates the 223

relative similarity between the mock treated samples (and GFP-TFIIS induction), whereas the 224

induction of GFP-TFIISmut yields a very different transcriptomic pattern (Figure 4B,C). Gene 225

ontology analysis of DEGs indicated that stress-responsive genes are prominently up-226

regulated, while genes involved in cell metabolism are preferentially down-regulated 227

(Supplemental Table 1), which may reflect the fact that TFIISmut expression provokes plant 228

stress responses. 229

230

Expression of TFIISmut results in massive redistribution of RNAPII over transcribed 231

regions 232

Next, we examined the recruitment of GFP-TFIISmut and GFP-TFIIS to transcribed regions. 233

Using chromatin immunoprecipitation (ChIP) with GFP-specific antibodies their association 234

with two relatively long genes (At3g02260, At1g49090) was analysed, whose expression 235

(according to the RNAseq data) was unaffected by the applied induction conditions. The 236

association with different regions of the genes was quantified by qPCR. GFP-TFIISmut and 237

GFP-TFIIS associated with sites around the transcription start site (TSS) and within the 238

transcribed region, but not with non-transcribed regions down-stream of the polyadenylation 239

site (pA) (Supplemental Figure 4). Moreover, within the transcribed regions an increased 240

association of GFP-TFIISmut relative to that of GFP-TFIIS was observed that may imply 241

perturbed transcriptional elongation in the presence of GFP-TFIISmut. This might be evoked 242

by the accumulation of backtracked/arrested elongation complexes (see below) and is 243

consistent with the observed reduced mobility of TFIISmut-containing complexes. 244

8

The genomic distribution of elongating RNAPII has not been thoroughly studied in 245

plants. Therefore, the association of RNAPII was analysed genome-wide by ChIPseq using 246

antibodies specific for the phosphorylated forms of the RNAPII-CTD, modified at positions 2 247

and 5 (S2P and S5P). The role of TFIISmut was examined by analysing tfIIs plants induced to 248

express GFP-TFIISmut after a 24 h β-estradiol treatment or mock treated for the same time. 249

Analysis of the ChIPseq data revealed that in the mock treated samples RNAPII occupancy 250

(S2P and S5P) correlated with gene expression levels as determined by RNAseq (Figure 251

5A,B) illustrating the basic accordance of both datasets. Generally, we observed RNAPII 252

accumulation over the transcribed regions of active genes. In the mock control, the 253

distribution of RNAPII-S2P showed an enrichment from the 5´ to the 3´ end with maximal 254

accumulation around the pA (Figure 5A). Compared to that RNAPII-S5P was more uniformly 255

distributed over the transcribed region (Figure 5B). Genes that were found to be associated 256

with decreased amounts of RNAPII upon GFP-TFIISmut expression tended to show reduced 257

transcript levels, while increased RNAPII association generally was reflected by elevated 258

transcript levels (Supplemental Figures 5,6), illustrating the expected connection between 259

RNAPII occupancy and transcriptional outcome. Additionally, the induced expression of 260

GFP-TFIISmut was accompanied by a clear redistribution of both RNAPII-S2P and –S5P 261

towards the TSS (Figure 5C,D). We also noticed that RNAPII redistribution differed between 262

long and short genes. For RNAPII-S2P, the redistribution towards the TSS is clearly more 263

pronounced for long genes (TSS-pA >2 kb) than for short genes (TSS-pA < 1 kb), while the 264

differences are less marked for RNAPII-S5P (Supplemental Figure 7). 265

Further analysis of the global RNAPII distribution around the TSS upon GFP-266

TFIISmut expression demonstrated that the peak particularly of RNAPII-S2P overlaps with 267

that of the nucleosome (+1) downstream of the TSS (Figure 6A,B). For this comparison, we 268

reanalysed data determined using the MNase-seq approach (Li et al., 2014). The likely 269

stalling of RNAPII at this position downstream of the TSS is also observed for S5P-modified 270

RNAPII, albeit to a lesser extent (Figure 6A,B). In view of the changes in the RNAPII profile 271

upon expression of TFIISmut, we quantified the accumulation of RNAPII at the TSS. We 272

calculated the promoter proximal enrichment of RNAPII (PPEP) for RNAPII-S2P (PPEP-273

S2P) and RNAPII-S5P (PPEP-S5P) as a measure for the association of RNAPII just 274

downstream of the TSS (0 – 500 bp) relative to the rest of the transcribed region. Of ~17000 275

genes 731 and 659 genes upon TFIISmut expression showed significantly increased PPEP-276

S2P and PPEP-S5P, respectively (z-score < -2). Genes with significantly decreased PPEP-S2P 277

9

were not detected, whereas only two genes showed decreased PPEP-S5P (z-score > 2). 278

Examples of individual genes illustrate the accordance of massive RNAPII redistribution and 279

calculated PPEP values (Figure 6C). To examine whether increased PPEP values correlate 280

with gene expression levels, analysed genes were subdivided into four classes of expression 281

according to above described RNAseq data. Increased PPEP-S2P and PPEP-S5P were 282

observed with genes expressed at high, medium and low levels, but not with non-expressed 283

genes (Figure 7). The magnitude of PPEP changes essentially conforms with the expression 284

level of the gene classes. These analyses demonstrate that upon induced expression of GFP-285

TFIISmut RNAPII accumulates close to the TSS, as though in the absence of its transcript 286

cleavage activity RNAPII would undergo frequently transcriptional arrest during early 287

elongation. 288

289

Proteasomal degradation of arrested RNAPII 290

As a last resort arrested yeast RNAPII is specifically targeted for proteasomal degradation 291

(Somesh et al., 2005). We therefore examined whether a similar mechanism may exist in 292

plants. Protein extracts of plants induced to express GFP-TFIIS and GFP-TFIISmut and 293

corresponding controls were analysed by immunoblotting using an antibody directed against 294

NRPB1, the large subunit of RNAPII. To suppress protein synthesis, the plants were 295

additionally treated with cycloheximide. Immunoblot analyses revealed that the amount of 296

NRPB1 upon expression of GFP-TFIISmut was reduced by ~50% relative to control samples, 297

while this effect was not observed upon expression of GFP-TFIIS (Figure 8A). To examine 298

whether the decrease of NRPB1 levels was mediated by the proteasome, GFP-TFIISmut 299

plants were additionally treated with the proteasome inhibitor MG132. In the presence of 300

MG132, the amount of NRPB1 was not reduced (Figure 8A,B) and remained at the level of 301

the controls. These findings indicated that similar to the situation in yeast, expression of 302

TFIISmut resulted in increased transcriptional arrest of RNAPII in Arabidopsis and the 303

arrested polymerases were removed by proteasome-mediated degradation. 304

305

306

DISCUSSION 307

Transcript elongation by RNAPII is a discontinuous process and transcription problems 308

leading to backtracking/arrest of the enzyme occur relatively frequently. There are various 309

causes disturbing transcriptional elongation including certain DNA sequences that are more 310

10

difficult to transcribe than others, packaging of the DNA into nucleosomes/chromatin, other 311

DNA-binding proteins or topological constraints (Gómez-Herreros et al., 2012; Palangat and 312

Larson, 2012; Svejstrup, 2007). While RNAPI and RNAPIII have a strong intrinsic RNA 313

cleavage activity to reverse transcriptional arrest, the cleavage activity of RNAPII requires 314

assistance by TFIIS to enable rapid backtrack recovery (Lisica et al., 2016; Ruan et al., 2011). 315

In light of this it seems surprising that TFIIS is not essential for the growth of yeast cells 316

(Archambault et al., 1992; Nakanishi et al., 1992) and that the growth of Arabidopsis tfIIs 317

mutant plants under standard conditions is largely indistinguishable from that of wild type 318

plants (Grasser et al., 2009). 319

The TFIIS acidic hairpin is highly conserved (cf. Fig. 1A) and the inhibitory effect of 320

mutation of the two invariant acidic hairpin residues on the RNA cleavage activity of RNAPII 321

has been demonstrated comprehensively (Imashimizu et al., 2013; Jeon et al., 1994; 322

Kettenberger et al., 2004; Sigurdsson et al., 2010). Thus, it may be expected that plant acidic 323

hairpin mutant TFIISmut comparably inhibits transcript cleavage by RNAPII. Consistently, 324

expression of TFIISmut causes various growth and developmental defects in wild type 325

Arabidopsis plants (Dolata et al., 2015). The observation that viable tfIIs plants constitutively 326

expressing TFIISmut cannot be recovered suggests that the RNA cleavage activity of RNAPII 327

is critical for viability in plants, as it is in yeast (Sigurdsson et al., 2010). In wild type plants, 328

endogenous TFIIS competes with TFIISmut for RNAPII binding, resulting in partial 329

suppression of transcript cleavage activity, whereas in tfIIs plants in the absence of 330

endogenous TFIIS, TFIISmut efficiently inhibits cleavage activity. Inducible expression of 331

TFIISmut in tfIIs plants confirmed the detrimental effects on Arabidopsis growth and 332

viability. 333

Comparative characterisation of protein interactions of TFIISmut and TFIIS revealed 334

that both proteins interact preferentially with the elongating RNAPII as components of the 335

TEC. Notably, FRAP measurements demonstrated that in Arabidopsis nuclei TFIISmut 336

displayed a lower mobility (fluorescence recovery, mobile fraction) than TFIIS. This is 337

consistent with the more prominent association of TFIISmut with arrested RNAPII. 338

Transcriptome analyses demonstrated that induced expression of TFIISmut resulted in 339

substantial changes in mRNA abundance. Expression of TFIIS caused only minor 340

transcriptomic changes, as expected from the mild phenotype of tfIIs plants and their 341

moderate differences in mRNA abundance relative to wild type plants (Grasser et al., 2009). 342

In the presence of TFIISmut, we observed a preferential down-regulation of genes related to 343

11

cell metabolism as well as an up-regulation of stress responsive genes. Both effects may be a 344

consequence of the fact that transient TFIISmut expression results in frequent transcriptional 345

arrest, provoking severe plant stress symptoms along with corresponding transcriptional 346

reprogramming (Baena-González and Sheen, 2008; Cohen and Leach, 2019; Less et al., 347

2011), eventually compromising cell viability. 348

In plants, genome-wide distribution of transcribing RNAPII is not well investigated, 349

but global run-on sequencing (GRO-seq) and native elongating transcript sequencing (NET-350

seq) analyses indicated certain differences relative to yeast and mammals (Hetzel et al., 2016; 351

Zhu et al., 2018). Thus, RNAPII was found to display characteristic accumulation in 352

proximity to pAs. Our ChIPseq experiments using antibodies directed against elongating 353

RNAPII (CTD S2P, S5P) generally showed an enrichment over the transcribed region of 354

active genes consistent with earlier analyses of individual loci by ChIP-qPCR (Ding et al., 355

2011; Dürr et al., 2014) and association of RNAPII is in good agreement with transcript levels 356

of the corresponding genes. For RNAPII-S2P an accumulation over the transcribed region of 357

genes towards the pA was observed that is consistent with the above-mentioned enrichment of 358

RNAPII at pAs (Hetzel et al., 2016; Zhu et al., 2018). In general, the distribution pattern of 359

RNAPII-S2P along transcribed regions is similar to that observed in yeast and mammals 360

(Hajheidari et al., 2013; Harlen and Churchman, 2017; Jeronimo et al., 2016). On the other 361

hand, Arabidopsis RNAPII-S5P was relatively evenly distributed over transcribed regions, 362

whereas in yeast and mammals RNAPII-S5P peaks at the TSS and levels decline towards the 363

pA (Hajheidari et al., 2013; Harlen and Churchman, 2017; Jeronimo et al., 2016). 364

Induced expression of TFIISmut resulted in a clear redistribution of elongating 365

RNAPII. Regarding the distribution of both RNAPII-S2P and –S5P, we observed a substantial 366

shift within transcribed regions towards the TSS (PPEP). In yeast, the density of RNAPII 367

became progressively lower within the transcribed region upon TFIISmut expression, 368

suggesting that TFIISmut inhibits transcript elongation (Sigurdsson et al., 2010). Recent 369

studies demonstrated that co-expression of mouse TFIISmut along with the endogenous TFIIS 370

in human cells impaired the ability of 5´ paused RNAPII to resume elongation due to 371

inhibition of the RNA cleavage activity (Sheridan et al., 2019). In addition, it was found that 372

the expression of TFIIS in human cells resulted in slower elongation rates of RNAPII and in 373

relative depletion of polymerases towards the end of genes (Zatreanu et al., 2019). The 374

importance of TFIIS for release of stalled RNAPII to proceed elongation was also 375

demonstrated in Drosophila (Adelman et al., 2005). Neither the distribution of elongating 376

12

RNAPII mapped in the absence of TFIIS in this study, nor GRO-seq analyses (Hetzel et al., 377

2016) indicated RNAPII pausing during early elongation in plants in the manner of that 378

exemplified by mammals. Promoter-proximal pausing of RNAPII was recently reported at a 379

subset of Arabidopsis genes based on GRO-seq and NET-seq experiments (Zhu et al., 2018). 380

However, in this study a rather broad peak of RNAPII accumulation was detected around 381

+300 down-stream of the TSS, whereas the well-characterised promoter-proximal pausing in 382

mammals occurs in a narrow region between +25 and +50 (Core and Adelman, 2019). 383

Therefore, it remains to be clarified whether the accumulation of plant RNAPII down-stream 384

of the TSS indeed represents RNAPII pausing comparable to that described in mammals or 385

whether it represents a slowing-down of transcriptional elongation possibly due to obstacles 386

to transcription (see also below). Consistent with the lack of compelling evidence for RNAPII 387

pausing in Saccharomyces cerevisiae, Caenorhabditis elegans and plants, orthologues of the 388

subunits of the important pausing regulatory complex NELF are not encoded in genomes of 389

organisms including yeast, worms and plants. This implies a functional connection between 390

NELF and promoter-proximal RNAPII pausing and its regulated escape into productive 391

elongation (Core and Adelman, 2019; Van Lijsebettens and Grasser, 2014). 392

The observed TFIISmut-induced PPEP in Arabidopsis distinctly overlaps with the 393

position of the +1 nucleosome at the 5´ end of the transcribed region. The +1 nucleosome is 394

most tightly positioned and often contains histone variants such as H2A.Z, features that are 395

apparently also relevant in plants (Jarillo and Piñeiro, 2015; Jiang and Pugh, 2009; Struhl and 396

Segal, 2013). Nucleosomes in general represent obstacles to transcript elongation by RNAPII 397

(Kulaeva et al., 2013; Teves et al., 2014) and passage of the +1 nucleosome appears to be 398

particularly critical for the efficiency of transcription (Nock et al., 2012; Weber et al., 2014). 399

This could be related to the elongation rate of RNAPII, which is slowest near promoters 400

(Danko et al., 2013). Various studies have demonstrated that TFIIS can partially relieve the 401

nucleosomal barrier to transcript elongation (Ishibashi et al., 2014; Kireeva et al., 2005; Nock 402

et al., 2012; Ujvári et al., 2008). The stimulatory effect of TFIIS on chromatin transcription 403

likely is related to the fact that RNAPII frequently backtracks upon encountering the 404

nucleosome barrier, which can lead to nucleosome-induced RNAPII arrest (Kireeva et al., 405

2005; Weber et al., 2014). Therefore, the accumulation of RNAPII upon expression of 406

Arabidopsis TFIISmut at the position of the +1 nucleosome may reflect that the enzyme in the 407

absence of its RNA cleavage activity cannot efficiently pass the nucleosomal barrier resulting 408

in a significant fraction of arrested RNAPII. Since permanently stalled RNAPII complexes 409

13

obstruct transcription of affected genes, in yeast and mammals arrested RNAPII becomes 410

ubiquitinated and degraded by the proteasome (Wilson et al., 2013). The induced expression 411

of TFIISmut resulted in reduced levels of RNAPII that can be reverted by the proteasome 412

inhibitor MG132, suggesting that the proteasomal degradation pathway of arrested RNAPII is 413

essentially conserved in plants. However, expression of TFIISmut efficiently inhibits the 414

RNA cleavage activity of RNAPII, resulting in prevailing transcriptional arrest. 415

Consequently, the degradation machinery is not capable anymore to sufficiently remove 416

arrested RNAPII complexes, causing profound transcription problems that may impair plant 417

growth and eventually compromise viability. In conclusion, our analyses indicate that 418

backtracking of RNAPII frequently occurs in plant cells during transcriptional elongation on 419

chromatin templates and they underscore the importance of reactivating backtracked/arrested 420

RNAPII for efficient transcript elongation as a prerequisite for correct transcriptional output 421

and proper plant growth/development. 422

423

424

METHODS 425

Plasmid constructions 426

The required gene or cDNA sequences were amplified by PCR with KAPA DNA polymerase 427

(PeqLab) using Arabidopsis thaliana genomic DNA or cDNA as template and the primers 428

(providing also the required restriction enzyme cleavage sites) listed in Supplemental Table 2. 429

The PCR fragments were inserted into suitable plasmids using standard methods or Gateway 430

cloning technology. All plasmid constructions were checked by DNA sequencing, and details 431

of the plasmids generated in this work are summarised in Supplemental Table 3. 432

433

Plant material, transformation and documentation 434

Arabidopsis thaliana (Col-0) was grown at 21°C under long-day (LD) conditions (16h 435

photoperiod per day, 100 µmol m-2

s-1

using Philips Master Eco white fluorescent tubes) on 436

MS medium (Antosz et al., 2017; Dürr et al., 2014). The T-DNA insertion line tfIIs-1 was 437

previously reported (Grasser et al., 2009). After sowing, seeds were stratified in darkness for 438

48h at 4°C prior to incubation in a plant incubator (Percival Scientific). Agrobacterium-439

mediated transformation of Col-0 or tfIIs plants and characterisation of obtained transgenic 440

lines was performed as previously described (Antosch et al., 2015; Dürr et al., 2014). Plant 441

phenotypes including root growth in the presence of β-estradiol were documented as 442

14

described (Dürr et al., 2014; Lolas et al., 2010). For other experiments, plants were initially 443

grown vertically on MS medium with 1% phyto agar and then transferred onto MS medium 444

supplemented with 2 µM β-estradiol (Sigma Aldrich). Additionally, transferred plants were 445

covered with sterile metal grids, submerged in liquid MS medium supplemented with 2 µM β-446

estradiol and vacuum-infiltrated for 10 min. Excess liquid MS medium was subsequently 447

removed and plants were incubated for the desired induction time. 448

449

PCR-based genotyping and quantitative PCR (qPCR) 450

To distinguish between plants being wild type, heterozygous, or homozygous for the T-DNA 451

insertions, genomic DNA was isolated from leaves. Genomic DNA was used for PCR 452

analysis with Taq DNA polymerase (PeqLab) and primers specific for DNA insertions and 453

target genes (Supplemental Table 2). DNA precipitated during ChIP assay was analysed by 454

qPCR with locus-specific primers (Supplemental Table 2). Five microlitres of precipitated 455

DNA was used for amplification by PCR using KAPA SYBR FAST Universal reagents 456

(PeqLab) and a Mastercycler ep realplex2 (Eppendorf). Data was analysed after normalisation 457

to the Input as previously described (Dürr et al., 2014). 458

459

Antibodies 460

For ChIP and/or immunoblotting the following primary antibodies were used: α-RNAPII-461

CTD-S2P (Abcam, ab5095), α-RNAPII-CTD-S5P (Abcam, ab1791), α-RNAPII-CTD 462

(Abcam, ab817), GFP-Trap (ChromoTeq), α-GS (Sigma Aldrich) and α-UAP56 (Kammel et 463

al., 2013). Antibodies were used at dilutions of 1:2000 (immunoblots) and 1:100 (ChIP). 464

465

Affinity-purification and mass spectrometry (AP-MS) analysis of proteins 466

Arabidopsis suspension cultured PSB-D cells were maintained and transformed as previously 467

described (van Leene et al., 2015). Isolation of GS-tagged proteins, purification and mass 468

spectrometric analyses were essentially performed as previously described (Antosz et al., 469

2017). In brief, after sonification of 15 g cells in extraction buffer the unspecific endonuclease 470

Benzonase (50 u/ml extract) was added to degrade DNA and RNA. GS-tagged proteins were 471

affinity-purified using IgG-coupled magnetic beads and eluted proteins were analysed by 472

SDS-PAGE and digested with trypsin. Peptides were separated by reversed-phase 473

chromatography and the LC-system was coupled on-line to a maXis plus UHR-QTOF System 474

(Bruker Daltonics) via a nanoflow electrospray source (Bruker Daltonics). Data-dependent 475

15

acquisition of MS/MS spectra and processing of the data was performed as previously 476

described (Antosz et al., 2017). Experimental background of contaminating proteins that were 477

isolated with the unfused GS-tag or that co-purify non-specifically independent of the used 478

bait protein was subtracted. 479

480

Plant protein extraction and immunoblotting 481

Whole protein extracts from PSD-B cultures were prepared using the procedure described 482

above, whereas the whole protein extracts from Arabidopsis seedlings were prepared as 483

described (Tsugama et al., 2011). After separation by SDS-PAGE, proteins were electro-484

transferred onto PVDF membrane and detected by chemiluminescence (Antosch et al., 2015; 485

Antosz et al., 2017). 486

487

Light microscopy and FRAP 488

For cell imaging, roots (5 - 10 DAS) and fragments of Arabidopsis leaves (5 DAS) were 489

mounted in H2O on objective slides with cover slips. Confocal laser scanning microscopy 490

(CLSM) was performed using a SP8 microscope (Leica) equipped with 40x/1.3 Oil or 63x/1.3 491

glycerol objectives as previously described (Dürr et al., 2014; Pfab et al., 2018). GFP was 492

excited using an Argon laser at 488 nm and mCherry/propidium iodide were exited using a 493

DPSS laser at 561 nm. GFP emission was detected with a hybrid detector at 500 - 550 nm. 494

For fluorescence recovery after photobleaching (FRAP) PSB-D cells were used and a 495

rectangle ROI area of 9 µm2 was defined for bleaching performed at 100% laser power with 496

eight bleaching pulse iterations. 50 pre-bleach and 100 post-bleach images were taken with 497

1% laser power. The raw measurements were processed using the easyFRAP software with 498

double full-scale normalisation (Rapsomaniki et al., 2012). Obtained normalised values were 499

used to determine half life time and mobile fraction. All pictures were generated using X-Las 500

software (Leica). 501

502

Transcript profiling by RNAseq 503

Total RNA was isolated from 10 DAS seedlings grown vertically on sterile plates with MS 504

medium with or without 24 h β-estradiol induction using the RNeasy R Mini Plant kit 505

(Qiagen). cDNA libraries were prepared using the TruSeq stranded mRNA sample 506

preparation kit (Illumina) and quality was assessed using a Bioanalyzer 2100 (Agilent 507

16

Technologies). Sequencing was carried out using a HiSeq 1000 (Illumina) instrument. Raw 508

reads were trimmed using Trimmomatic (Bolger et al., 2014) with a minimum average quality 509

score greater than 30 and a minimum length of 70 bp. Processed reads were mapped to the 510

Arabidopsis reference transcriptome assembly (TAIR10) using TopHat 2.1.1 (Kim et al., 511

2013) and Bowtie 2.3.4. (Langmead and Salzberg, 2012), allowing up to two base mismatches 512

per read. Reads mapped to multiple locations were discarded. For the analysis of differentially 513

expressed genes (DEGs), data was transformed logarithmically and normalised using a 514

quantile normalisation method. DEGs were determined by DEseq2 (Love et al., 2014) with a 515

threshold |log2 FPKM| > 2 and an adjusted p value < 0.001 using the Benjamini and 516

Hochberg’s method (Benjamini and Hochberg, 1995) for multiple testing. DEG analysis and 517

heat maps were generated using R environment 3.2.2. Genes with Fragments Per Kilobase of 518

transcript per Million (FPKM) ≥ 5 were considered transcriptionally active. 519

520

Chromatin immunoprecipitation (ChIP): ChIP-qPCR and ChIPseq 521

Seedlings (10 DAS) grown on MS medium were treated with 2 µM β-estradiol or ethanol 522

(mock) for 24 before harvesting. Following crosslinking with 1% formaldehyde, plant 523

material was further processed and used for immunoprecipitation as previously described 524

(Antosz et al., 2017; Dürr et al., 2014). For ChIP-qPCR, 5µl precipitated DNA (diluted 1:200 525

for input and 1:10 for GFP-trap and the control without antibody) was analysed by qPCR with 526

locus-specific primers (Supplemental Table 2). 527

For ChIPseq, libraries were generated using the DNA SMART ChIP-Seq kit 528

(Clontech) and quality was assessed using an Agilent Technologies 2100 Bioanalyzer. 529

Sequencing was performed with the Illumina HiSeq 1000. Raw reads were trimmed with 530

Trimmomatic and mapped to the Arabidopsis reference genome (TAIR10) using Bowtie2.3.4. 531

Nucleosome occupancy was determined from MNase-seq data (Li et al., 2014). 532

Data processing was performed using bamCoverage from the DeepTools suit to 533

calculate read density per nucleotide and three biological replicates were merged prior to the 534

calculation. Meta-analysis of relative RNAPII distribution over transcribed loci (TSS-pA) and 535

additional regions was performed using computeMatrix from the DeepTools suit (with 536

maxThreshold 500). RNAPII density was additionally determined around transcription start 537

site (TSS; [-2000;2000]) and polyadenylation site (pA; [-2000;2000]). HTSeq suit was used to 538

determine RNAPII read counts within gene bodies. Obtained raw values were normalised 539

using DEseq2 and averaged for three biological replicates. Fold change (FC) between studied 540

17

conditions was calculated and transformed logarithmically. Significantly different occupancy 541

was determined with a threshold |log2 FC| > 1 and p-value < 0.1 adjusted using the Benjamini 542

and Hochberg’s method for multiple testing. RNAPII-S2P and RNAPII-S5P profiles over 543

single genes were visualised using Integrative Genomics Browser (IGB) with standard 544

settings. 545

To determine promoter proximal enrichment of RNAPII (PPEP), RNAPII read counts 546

from three biological replicates were summed up and the number of tags was determined for 547

each gene in the gene body (+500 bp to pA) and in the promoter-proximal region (TSS to 548

+500 bp). Genes with ≥ 10 or more tags in the promoter-proximal region were further 549

analysed and the number of tags were calculated in the promoter proximal region relatively to 550

the gene body region. Obtained values were compared relatively between studied conditions 551

and transformed logarithmically resulting in log2 PPEP values for which Z-scores were 552

calculated (Juntawong et al., 2014). Changes in PPEP were considered significantly different 553

for the |z-score| > 2 between studied conditions. 554

555

556

Accession Numbers 557

Sequence data of TFIIS (At2g38560) and RNAPII-NRPB1 (At4g35800) can be found in the 558

GenBank/EMBL libraries. 559

RNA-seq and ChIP-seq data was deposited in the NCBI Gene Expression Omnibus under the 560

Accession numbers GSE129072 and GSE128316, respectively. 561

562

563

Supplemental Data 564

Supplemental Figure 1. β-estradiol induced expression of GFP-TFIIS and GFP-TFIISmut in 565

tfIIs plants. 566

Supplemental Figure 2. Analysis of the induction of GFP-TFIIS and GFP-TFIISmut by 567

CLSM. 568

Supplemental Figure 3. Association TFIIS and TFIISmut with RNAPII. 569

Supplemental Figure 4. TFIISmut accumulates within transcribed regions. 570

Supplemental Figure 5. RNAPII-S2P and RNAPII-S5P occupancy correlates with transcript 571

levels. 572

18

Supplemental Figure 6. Genome-wide correlation of RNAPII-S2P/-S5P occupancy and 573

transcript levels. 574

Supplemental Figure 7. Redistribution of RNAPII-S2P/-S5P upon TFIISmut expression in 575

relation to gene length. 576

Supplemental Table 1. Overrepresented GO terms among the differentially expressed genes 577

(DEGs) upon TFIISmut expression. 578

Supplemental Table 2. Oligonucleotide primers used in this study and construction of 579

plasmids. 580

Supplemental Table 3. Plasmids used in this study. 581

582

583

ACKNOWLEDGEMENTS 584

We thank Simon Obermeyer for TFIIS structure modelling, Simon Mortensen for 585

contributions during early stages of the project, Eduard Hochmuth for recording mass spectra 586

and Peter Doerner and Arp Schnittger for the pCYCB1;1-GFP marker line. This work was 587

supported by the German Research Foundation (DFG) through grant GR1159/14-2 to K.D.G. 588

and SFB960 to T.D., a grant from the Swiss National Foundation to Y.P. (Sinergia grant 589

CRSII3_154471), as well as the EC Marie Curie Research Training Network “Chromatin in 590

Plants – European Training and Mobility” (CHIP-ET, FP7-PEOPLE-2013-ITN607880) to 591

K.D.G.592

593

594

AUTHOR CONTRIBUTIONS 595

W.A. and K.D.G. designed the research and wrote the manuscript. W.A., J.D. and K.B. 596

performed the research. W.A., J.D., K.B., A.B., Y.P., T.D. and K.D.G. analysed the data. 597

A.B., Y.P., T.D. and K.D.G. provided the reagents and the tools for the analysis. All authors598

commented on the manuscript and contributed to the writing. 599

600

601

602

603

604

605

19

606

Table 1. Known TEC components co-purifying with GS-TFIIS and GS-TFIISmut

TFIIS1

TFIISmut1

Interactor2 AGI Complex

3748 / 3 2882 / 3 TFIIS AT2G38560 TFIIS

1487 / 3 1763 / 3 NRPB1 AT4G35800 Polymerase II

1378 / 3 1319 / 3 NRPB2 AT4G21710 Polymerase II

466 / 3 551 / 3 NRP(B/D/E)3a AT2G15430 Polymerase II

462 / 3 474 / 3 NRP(A/B/C/D)5 AT3G22320 Polymerase II

216 / 3 149 / 2 NRPB7 AT5G59180 Polymerase II

105 / 2 NRP(B/D/E)9a AT3G16980 Polymerase II

904 / 3 859 / 3 CDC73 AT3G22590 PAF1C

504 / 3 416 / 3 VIP4, LEO1 AT5G61150 PAF1C

553 / 3 408 / 3 ELF7, PAF1 AT1G79730 PAF1C

438 / 3 408 / 2 VIP3, SKI8 AT4G29830 PAF1C

327 / 3 245 / 3 VIP5, RTF1 AT1G61040 PAF1C

342 / 2 546 / 3 SPT6L AT1G65440 SPT6

364 / 3 649 / 3 SPT5-2 AT4G08350 SPT4/SPT5

435 / 2 544 / 3 SPT16 AT4G10710 FACT

211 / 2 261 / 3 SSRP1 AT3G28730 FACT

218 / 2 171 / 2 ELP1, ELO2 AT5G13680 Elongator

607

608

609

610

611

612

613

1

The numbers indicate the respective average MASCOT score and how many times the proteins were detected in

three independent APs. The proteins that were detected in less than two out of three AP are not listed.

2

TEC components previously identified as interactors of elongating Arabidopsis RNAPII (Antosz et al., 2017) are

listed here.

20

REFERENCES 614

Adelman,K., Marr,M.T., Werner,J., Saunders,A., Ni,Z., Andrulis,E.D., and Lis,J.T. (2005). 615

Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS. Mol. 616

Cell 17: 103-112. 617

Antosch,M., Schubert,V., Holzinger,P., Houben,A., and Grasser,K.D. (2015). Mitotic lifecycle of 618

chromosomal 3xHMG-box proteins and the role of their N-terminal domain in the association with 619

rDNA loci and proteolysis. New Phytol. 208: 1067-1077. 620

Antosz,W., Pfab,A., Ehrnsberger,H.F., Holzinger,P., Köllen,K., Mortensen,S.A., Bruckmann,A., 621

Schubert,T., Längst,G., Griesenbeck,J., Schubert,V., Grasser,M., and Grasser,K.D. (2017). 622

Composition of the Arabidopsis RNA polymerase II transcript elongation complex reveals the 623

interplay between elongation and mRNA processing factors. Plant Cell 29: 854-870. 624

Archambault,J., Lacroute,F., Ruet,A., and Friesen,J.D. (1992). Genetic interaction between 625

transcription elongation factor TFIIS and RNA polymerase II. Mol. Cell. Biol. 12: 4142-4152. 626

Baena-González,E. and Sheen,J. (2008). Convergent energy and stress signaling. Trends Plant Sci. 627

13: 474-482. 628

Benjamini,Y. and Hochberg,Y. (1995). Controlling the false discovery rate: a practical and powerful 629

approach to multiple testing. J. R. Statist. Soc. B 57: 289-300. 630

Bentsink,L., Jowett,J., Hanhart,C.J., and Koornneef,M. (2006). Cloning of DOG1, a quantitative 631

trait locus controlling seed dormancy in Arabidopsis. Proc. Natl. Acad. Sci. USA 103: 17042-632

17047. 633

Bolger,A.M., Lohse,M., and Usadel,B. (2014). Trimmomatic:a flexible trimmer for Illumina 634

sequence data. Bioinformatics 30: 2114-2120. 635

Brand,L., Hörler,M., Nüesch,E., Vassalli,S., Barrell,P., Yang,W., Jefferson,R.A., 636

Grossniklaus,U., and Curtis,M.D. (2006). A versatile and reliable two-component system for 637

tissue-specific gene induction in Arabidopsis. Plant Physiol. 141: 1194-1204. 638

Bürckstümmer,T., Bennett,K.L., Preradovic,A., Schütze,G., Hantschel,O., Superti-Furga,G., 639

and Bauch,A. (2006). An efficient tandem affinity purification procedure for interaction 640

proteomics in mammalian cells. Nat. Meth. 3: 1013-1019. 641

Chen,F.X., Smith,E.R., and Shilatifard,A. (2018). Born to run: control of transcription elongation 642

by RNA polymerase II. Nat. Rev. Mol. Cell Biol. 19: 464-478. 643

Cheung,A.C.M. and Cramer,P. (2011). Structural basis of RNA polymerase II backtracking, arrest 644

and reactivation. Nature 471: 249-253. 645

Cohen,S.P. and Leach,J.E. (2019). Abiotic and biotic stresses induce a core transcriptome response 646

in rice. Sci. Rep. 9: 6273. 647

Core,L. and Adelman,K. (2019). Promoter-proximal pausing of RNA polymerase II: a nexus of gene 648

regulation. Genes Dev. 33: 960-982. 649

21

Danko,C.G., Hah,N., Luo,X., Martins,A.L., Core,L., Lis,J.T., Siepel,A., and Kraus,W.L. (2013). 650

Signaling pathways differentially affect RNA polymerase II initiation, pausing, and elongation rate 651

in cells. Mol. Cell 50: 212-222. 652

Ding,Y., Avramova,Z., and Fromm,M. (2011). Two distinct roles of ARABIDOPSIS HOMOLOG 653

OF TRITHORAX1 (ATX1) at promoters and within transcribed regions of ATX1-regulated genes. 654

Plant Cell 23: 350-363. 655

Dolata,J., Guo,Y., Kolowerzo,A., Smolinski,D., Brzyzek,G., Jarmolowski,A., and Swiezewski,S. 656

(2015). NTR1 is required for transcription elongation checkpoints at alternative exons in 657

Arabidopsis. EMBO J. 34: 544-558. 658

Dürr,J., Lolas,I.B., Sørensen,B.B., Schubert,V., Houben,A., Melzer,M., Deutzmann,R., 659

Grasser,M., and Grasser,K.D. (2014). The transcript elongation factor SPT4/SPT5 is involved in 660

auxin-related gene expression in Arabidopsis. Nucleic Acids Res. 42: 4332-4347. 661

Fish,R.N. and Kane,C.M. (2002). Promoting elongation with transcript cleavage stimulatory factors. 662

Biochim. Biophys. Acta 1577: 287-307. 663

Gómez-Herreros,F., de Miguel-Jiménez,L., Millán-Zambrano,G., Peñate,X., Delgado-Ramos,L., 664

Muñoz-Centeno,M.C., and Chávez,S. (2012). One step back before moving forward: regulation 665

of transcription elongation by arrest and backtracking. FEBS Lett. 586: 2820-2825. 666

Grasser,M., Kane,C.M., Merkle,T., Melzer,M., Emmersen,J., and Grasser,K.D. (2009). 667

Transcript elongation factor TFIIS is involved in Arabidopsis seed dormancy. J. Mol. Biol. 386: 668

598-611. 669

Hajheidari,M., Koncz,C., and Eick,D. (2013). Emerging roles for RNA polymerase II CTD in 670

Arabidopsis. Trends Plant Sci. 18: 633-643. 671

Harlen,K.M. and Churchman,L.S. (2017). The code and beyond: transcription regulation by the 672

RNA polymerase II carboxy-terminal domain. Nat. Rev. Mol. Cell Biol. 18: 263-273. 673

He,Y., Doyle,M.R., and Amasino,R.M. (2004). PAF1-complex-mediated histone methylation of 674

FLOWERING LOCUS C chromatin is required for the vernalization-responsive, winter-annual 675

habit in Arabidopsis. Genes Dev. 18: 2774-2784. 676

Hetzel,J., Duttke,S.H., Benner,C., and Chory,J. (2016). Nascent RNA sequencing reveals distinct 677

features in plant transcription. Proc. Natl. Acad. Sci. USA 113: 12316-12321. 678

Imashimizu,M., Kireeva,M., Lubowska,L., Gotte,D., Parks,A.R., Strathern,J.N., and Kashlev,M. 679

(2013). Intrinsic translocation barrier as an initial step in pausing by RNA polymerase II. J. Mol. 680

Biol. 425: 697-712. 681

Ishibashi,T., Dangkulwanich,M., Coello,Y., Lionberger,T.A., Lubkowska,L., Ponticelli,A.S., 682

Kashlev,M., and Bustamante,C. (2014). Transcription factors IIS and IIF enhance transcription 683

efficiency by differentially modifying RNA polymerase pausing dynamics. Proc. Natl. Acad. Sci. 684

USA 111: 3419-3424. 685

22

Izban,M.G. and Luse,D.S. (1992). The RNA polymerase II ternary complex cleaves the nascent 686

transcript in a 3'-5' direction in the presence of elongation factor SII. Genes Dev. 6: 1342-1356. 687

Jarillo,J.A. and Piñeiro,M. (2015). H2A.Z mediates different aspects of chromatin function and 688

modulates flowering responses in Arabidopsis. Plant J. 83: 96-109. 689

Jeon,C., Yoon,H., and Agarwal,K. (1994). The transcription factor TFIIS zinc ribbon dipeptide Asp-690

Glu is critical for stimulation of elongation and RNA cleavage by RNA polymerase II. Proc. Natl. 691

Acad. Sci. USA 91: 9106-9110. 692

Jeronimo,C., Collin,P., and Robert,F. (2016). The RNA polymerase II CTD: the increasing 693

complexity of a low-complexity protein domain. J. Mol. Biol. 428: 2607-2622. 694

Jiang,C. and Pugh,B.F. (2009). Nucleosome positioning and gene regulation: advances through 695

genomics. Nat. Rev. Genet. 10: 161-172. 696

Juntawong,P., Girke,T., Bazin,J., and Bailey-Serres,J. (2014). Translational dynamics revealed by 697

genome-wide profiling of ribosome footprints in Arabidopsis. Proc. Natl. Acad. Sci. USA 111: 698

E203-E212. 699

Kammel,C., Thomaier,M., Sørensen,B.B., Schubert,T., Längst,G., Grasser,M., and Grasser,K.D. 700

(2013). Arabidopsis DEAD-box RNA helicase UAP56 interacts with both RNA and DNA as well 701

as with mRNA export factors. PLoS One 8: e60644. 702

Kettenberger,H., Armache,K.-J., and Cramer,P. (2003). Architecture of the RNA polymerase II-703

TFIIS complex and implications for mRNA cleavage. Cell 114: 347-357. 704

Kettenberger,H., Armache,K.-J., and Cramer,P. (2004). Complete RNA polymerase II elongation 705

complex structure and its interactions with NTP and TFIIS. Mol. Cell 16: 955-965. 706

Kim,D., Pertea,G., Trapnell,C., Pimentel,H., Kelley,R., and Salzberg,S.L. (2013). TopHat2: 707

accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. 708

Genome Biol. 14: R36. 709

Kireeva,M.L., Hancock,B., Cremona,G.H., Walter,W., Studitsky,V.M., and Kashlev,M. (2005). 710

Nature of nucleosomal barrier to RNA polymerase II. Mol. Cell 18: 108. 711

Kulaeva,O.I., Hsieh,F.-K., Chang,H.-W., Luse,D.S., and Studitsky,V.M. (2013). Mechanism of 712

transcription through a nucleosome by RNA polymerase II. Biochim. Biophys. Acta 1829: 76-83. 713

Langmead,B. and Salzberg,S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat. Meth. 9: 714

357-359.715

Less,H., Angelovici,R., Tzin,V., and Galili,G. (2011). Coordinated gene networks regulating 716

Arabidopsis plant metabolism in response to various stresses and nutritional cues. Plant Cell 23: 717

1264-1271. 718

Li,G., Liu,S., Wang,J., He,J., Huang,H., Zhang,Y., and Xu,L. (2014). ISWI proteins participate in 719

the genome-wide nucleosome distribution in Arabidopsis. Plant J. 78: 706-714. 720

23

Lisica,A., Engel,C., Jahnel,M., Roldán,É., Galburt,E.A., Cramer,P., and Grill,S.W. (2016). 721

Mechanisms of backtrack recovery by RNA polymerases I and II. Proc. Natl. Acad. Sci. USA 113: 722

2946-2951. 723

Liu,Y., Geyer,R., van Zanten,M., Carles,A., Li,Y., Hörold,A., van Nocker,S., and Soppe,W.J. 724

(2011). Identification of the Arabidopsis REDUCED DORMANCY 2 gene uncovers a role for the 725

polymerase associated factor 1 complex in seed dormancy. PLoS One 6: e22241. 726

Lolas,I.B., Himanen,K., Grønlund,J.T., Lynggaard,C., Houben,A., Melzer,M., Van 727

Lijsebettens,M., and Grasser,K.D. (2010). The transcript elongation factor FACT affects 728

Arabidopsis vegetative and reproductive development and genetically interacts with HUB1/2. Plant 729

J. 61: 686-697. 730

Love,M.L., Huber,W., and Anders,S. (2014). Moderated estimation of fold change and dispersion 731

for RNA-seq data with DESeq2. Genome Biol. 15: 550. 732

Ma,Y., Gil,S., Grasser,K.D., and Mas,P. (2018). Targeted recruitment of the basal transcriptional 733

machinery by LNK clock components controls the circadian rhythms of nascent RNAs in 734

Arabidopsis. Plant Cell 30: 907-924. 735

Mortensen,S.A. and Grasser,K.D. (2014). The seed dormancy defect of Arabidopsis mutants lacking 736

the transcript elongation factor TFIIS is caused by reduced expression of the DOG1 gene. FEBS 737

Lett. 588: 47-51. 738

Nakanishi,T., Nakano,A., Nomura,K., Sekimizu,K., and Natori,S. (1992). Purification, gene 739

cloning, and gene disruption of the transcription elongation factor S-II in Saccharomyces 740

cerevisiae. J. Biol. Chem. 267: 13200-13204. 741

Nelissen,H., DeGroeve,S., Fleury,D., Neyt,P., Bruno,L., Bitonti,M.B., Vandenbussche,F., Van 742

Der Straeten,D., Yamaguchi,T., Tsukaya,H., Witters,E., de Jaeger,G., Houben,A., and Van 743

Lijsebettens,M. (2010). Plant Elongator regulates auxin-related genes during RNA polymerase II-744

mediated transcription elongation. Proc. Natl. Acad. Sci. USA 107: 1678-1683. 745

Nock,A., Ascano,J.M., Barrero,M.J., and Malik,S. (2012). Mediator-regulated transcription through 746

the +1 nucleosome. Mol. Cell 48: 837-848. 747

Oh,S., Zhang,H., Ludwig,P., and van Nocker,S. (2004). A mechanism related to the yeast 748

transcriptional regulator Paf1c is required for expression of the Arabidopsis FLC/MAF MADS box 749

gene family. Plant Cell 16: 2940-2953. 750

Palangat,M. and Larson,D.R. (2012). Complexity of RNA polymerase II elongation dynamics. 751

Biochim. Biophys. Acta 1819: 667-672. 752

Pfab,A., Bruckmann,A., Nazet,J., Merkl,R., and Grasser,K.D. (2018). The adaptor protein ENY2 753

is a component of the deubiquitination module of the Arabidopsis SAGA transcriptional co-754

activator complex but not of the TREX-2 complex. J. Mol. Biol. 430: 1479-1494. 755

24

Rapsomaniki,M.A., Kotsantis,P., Symeonidou,I.E., Giakoumankis,N.N., Taraviras,S., and 756

Lygerou,Z. (2012). easyFRAP: an interactive, easy-to-use tool for qualitative and quantitative 757

analysis of FRAP data. Bioinformatics 28: 1800-1801. 758

Ruan,W., Lehmann,E., Thomm,M., Kostrewa,D., and Cramer,P. (2011). Evolution of two modes 759

of intrinsic RNA polymerase transcript cleavage. J. Biol. Chem. 286: 18701-18707. 760

Selth,L.A., Sigurdsson,S., and Svejstrup,J.Q. (2010). Transcript elongation by RNA Polymerase II. 761

Ann. Rev. Biochem. 79: 271-293. 762

Sheridan,R.M., Fong,N., D´Alessandro,A., and Bentley,D.L. (2019). Widespread backtracking by 763

RNA Pol II Is a major effector of gene activation, 5' pause release, termination, and transcription 764

elongation rate. Mol. Cell 73: 107-118. 765

Sigurdsson,S., Dirac-Svejstrup,A.B., and Svejstrup,J.Q. (2010). Evidence that transcript cleavage 766

is essential for RNA polymerase II transcription and cell viability. Mol. Cell 38: 202-210. 767

Sims,R.J., Belotserkovskaya,R., and Reinberg,D. (2004). Elongation by RNA polymerase II: the 768

short and the long of it. Genes Dev. 18: 2437-2468. 769

Somesh,B.P., Reid,J., Liu,W.F., Søgaard,T.M., Erdjument-Bromage,H., Tempst,P., and 770

Svejstrup,J.Q. (2005). Multiple mechanisms confining RNA polymerase II ubiquitylation to 771

polymerases undergoing transcriptional arrest. Cell 121: 913-923. 772

Struhl,K. and Segal,E. (2013). Determinants of nucleosome positioning. Nat. Struct. Mol. Biol. 20: 773

267-273.774

Svejstrup,J.Q. (2007). Contending with transcriptional arrest during RNAPII transcript elongation. 775

Trends Biochem. Sci. 32: 165-171. 776

Teves,S.S., Weber,C.M., and Henikoff,S. (2014). Transcribing through the nucleosome. Trends 777

Biochem. Sci. 39: 577-586. 778

Tsugama,D., Liu,S., and Takano,T. (2011). A rapid chemical method for lysing Arabidopsis cells 779

for protein analysis. Plant Meth. 7: 22. 780

Ubeda-Tomás,S., Federici,F., Casimiro,I., Beemster,G.T., Bhalerao,R., Swarup,R., Doerner,P., 781

Haseloff,J., and Bennett,M.J. (2009). Gibberellin signaling in the endodermis controls 782

Arabidopsis root meristem size. Curr. Biol. 19: 1194-1199. 783

Ujvári,A., Hsieh,F.-K., Luse,S.W., Studitsky,V.M., and Luse,D.S. (2008). Histone N-terminal tails 784

interfere with nucleosome traversal by RNA polymerase II. J. Biol. Chem. 283: 32236-32243. 785

van Leene,J., Eeckhout,D., Cannoot,B., De Winne,N., Persiau,G., van de Slijke,E., 786

Vercruysse,L., Dedecker,M., Vandepoele,K., Martens,L., Witters,E., Gevaert,K., and de 787

Jaeger,G. (2015). An improved toolbox to unravel the plant cellular machinery by tandem affinity 788

purification of Arabidopsis protein complexes. Nat. Protoc. 10: 169-187. 789

Van Lijsebettens,M. and Grasser,K.D. (2014). Transcript elongation factors: shaping transcriptomes 790

after transcript initiation. Trends Plant Sci. 19: 717-726. 791

25

Wang,D., Bushnell,D.A., Huang,X., Westover,K.D., Levitt,M., and Kornberg,R.D. (2009). 792

Structural basis of transcription: backtracked RNA Polymerase II at 3.4 angstrom resolution. 793

Science 324: 1203-1206. 794

Weber,C.M., Ramachandran,S., and Henikoff,S. (2014). Nucleosomes are context-specific, 795

H2A.Z-modulated barriers to RNA polymerase. Mol. Cell 53: 819-830. 796

Wilson,M.D., Harreman,M., and Svejstrup,J.Q. (2013). Ubiquitylation and degradation of 797

elongating RNA polymerase II: the last resort. Biochim. Biophys. Acta 1829: 151-157. 798

Wind,M. and Reines,D. (2000). Transcription elongation factor SII. BioEssays 22: 327-336. 799

Yang,J. and Zhang,Y. (2015). I-TASSER server: new development for protein structure and function 800

predictions. Nucleic Acids Res. 43: W174-W181. 801

Zatreanu,D., Han,Z., Mitter,R., Tumini,E., Williams,H., Gregersen,L., Dirac-Svejstrup,A.B., 802

Roma,S., Stewart,A., Aguilera,A., and Svejstrup,J.Q. (2019). Elongation Factor TFIIS Prevents 803

Transcription Stress and R-Loop Accumulation to Maintain Genome Stability. Mol. Cell 76: 57-69. 804

Zhu,J., Liu,M., Liu,X., and Dong,Z. (2018). RNA polymerase II activity revealed by GRO-seq and 805

pNET-seq in Arabidopsis. Nat. Plants 4: 1112-1123. 806

807

808

FIGURE LEGENDS 809

Figure 1. Induced expression of TFIISmut in tfIIs mutant background severely affects plant 810

growth. A, amino acid sequence alignment of the C-terminal part of TFIIS from Arabidopsis 811

(At), rice (Os), human (Hs) and the yeast S. cerevisiae (Sc). Asterisks indicate conserved 812

residues and the invariant acidic residues (D359, E360 indicated in red) that were mutated in 813

this work (and the corresponding residues were mutated in other studies (Imashimizu et al., 814

2013; Jeon et al., 1994; Kettenberger et al., 2004; Sigurdsson et al., 2010)). B, cartoon 815

representation of the computational structure model of domains II and III of Arabidopsis 816

TFIIS. The model (C-score: 0.82) was generated using the I-TASSER server (Yang and 817

Zhang, 2015). Domain II is indicated in green, the interdomain linker in orange, domain III in 818

yellow and the position of the two invariant acidic hairpin residues (changed to alanine 819

residues in TFIISmut) is highlighted in red and marked by an arrow. C, growth of Arabidopsis 820

plants expressing TFIISmut is severely impaired. 7-DAS plants of three independent plant 821

lines each expressing GFP-TFIISmut or GFP-TFIIS were grown in comparison to the Col-0 822

wild type on MS medium under inductive conditions (+ β-estradiol) or mock treated (ethanol). 823

824

26

Figure 2. Plants expressing TFIISmut exhibit reduced growth of the primary root and 825

decreased cell proliferation. A, inhibition of root growth upon expression of TFIISmut. 8-826

DAS plants were transferred onto MS plates containing β-estradiol to induce expression of 827

GFP-TFIISmut and GFP-TFIIS, or mock-treated. Images were taken at the indicated times. 828

White arrow (and black line) indicate the position of the root tip at the time of transfer (0h). 829

Root elongation was measured to determine absolute (B) and relative (C) elongation. B, dots 830

represent mean values ±SD and dotted lines represent linear regression (n = 6). C, bars 831

represent mean values ±SD and asterisks indicate the outcome of the Student´s t-Test: * 832

p<0.05, ** p<0.01, *** p<0.001. D, CLSM analysis of GFP fluorescence of root tips of 5-833

DAS plants induced (or mock-treated) for 24h to express GS-TFIIS or GS-TFIISmut and 834

harbouring the pCYCB1;1-GFP reporter. E, bars represent mean values ±SD of the number of 835

mitotic, GFP expressing cells (n = 5 plants per line/condition). Characters indicate the 836

outcome of a multi-comparison Tukey´s test (p<0.05). 837

838

Figure 3. Nuclear mobility of TFIISmut is markedly lower than that of TFIIS. FRAP analysis 839

of Arabidopsis PSB-D cells induced for 24h to express TFIISmut or TFIIS. A, mean 840

fluorescence recovery curves ±SD (full scale normalisation) representing 40 pre-bleach and 841

50 post-bleach time points. Calculated half-time (t1/2, B) and mobile fraction (Mf, C) 842

represented as box plots with * p<0.05 according to Student´s t-Test. 843

844

Figure 4. Expression of TFIISmut leads to major transcriptomic changes. A, schematic 845

representation of the number of DEGs (up/down) between the analysed plant lines/conditions. 846

B, heat map (n = 15836) of the transcriptomic changes between the analysed plant 847

lines/conditions. Only genes with FPKM ≥ 5 in at least one line/condition were considered 848

and the hierarchical clustering is indicated on the left. C, heat map legend with yellow-blue 849

gradient indicating the log2FPKM [-2; 2]. The magenta line indicates the number of values in 850

a given colour range. 851

852

Figure 5. The genomic occupancy of elongating RNAPII is related to the corresponding 853

transcript levels and the distribution within transcribed regions is altered upon expression of 854

27

TFIISmut. As indicated, gene-averaged profiles represent the distribution of RNAPII-S2P 855

(A,C) and RNAPII-S5P (B,D) over transcribed regions (TSS – pA) according to the ChIPseq 856

data of 10-DAS plants induced for 24h to express GFP-TFIISmut (C,D; dark magenta) or 857

mock treated (A,B; light magenta). Profiles represent genes with no (18754), low (3488), 858

medium (6975) or high (3488) transcript levels based on the FPKM values determined by 859

RNAseq. 860

861

Figure 6. In the presence of TFIIS elongating RNAPII accumulates downstream of the TSS at 862

a position overlapping with the +1 nucleosome. A, Gene-averaged profiles of the RNAPII-863

S2P and RNAPII-S5P distribution around the TSS according to ChIPseq data of 10-DAS 864

plants induced for 24h to express GFP-TFIISmut (dark magenta) or mock treated (light 865

magenta). Profiles represent 33574 and 33560 loci for RNAPII-S2P and RNAPII-S5P, 866

respectively. B, the black line represents the nucleosome occupancy of 14-DAS plants 867

calculated from MNase-seq data (Li et al., 2014) that was combined with the profiles of 868

RNAPII-S2P and RNAPII-S5P after induced GFP-TFIISmut expression. C, ChIP-seq profiles 869

of RNAPII-S2P and RNAPII-S5P at the At5g11090 and At2g20562 loci with increased PPEP 870

values upon β-estradiol vs. mock treatment. Plots were generated using IGB using 871

representative biological replicates. In gene models, thin bars represent UTRs, thick bars 872

represent exons and dotted lines represent introns. 873

874

875

Figure 7. TFIISmut induced changes in PPEP are related to transcript levels. (A) Scheme 876

depicting the promoter proximal region (0 to +500 bp, blue) and the rest of the transcribed 877

region to pA (orange), as well as calculation of PPEP. Changes in PPEP-S2P (B) and PPEP-878

S5 (C) upon TFIISmut expression vs. mock treatment (dark magenta vs. bright magenta, 879

respectively) were calculated for genes with no (18754), low (3488), medium (6975) or high 880

(3488) transcript levels based on FPKM values determined by RNAseq. PPEP-S2P and 881

PPEP-S5 values are visualised by box plots and asterisks indicate the outcome of the 882

Student´s t-Test: * p<0.05, *** p<0.001. 883

884

28

Figure 8. Upon TFIISmut expression the amount of the large subunit NRPB1 of RNAPII is 885

decreased by proteasomal degradation. A, 10-DAS plants inducibly expressing GFP-TFIIS or 886

GFP-TFIISmut were treated for 24h with the inducer β-estradiol (or mock-treated) and the 887

proteasome inhibitor MG132 as indicated. All plants were additionally co-treated with 888

cycloheximide. Total protein extracts of these plants were analysed by immunoblotting using 889

an antibody directed against the NRPB1-CTD and as loading control an antibody directed 890

against the RNA helicase UAP56. B, band intensities were quantified using ImageJ. Bars 891

represent mean values ±SD of three independent experiments. Data were statistically analysed 892

using one-way ANOVA and characters indicate the outcome of a multi-comparison Tukey´s 893

test (p<0.05). 894

Figure 1. Induced expression of TFIISmut in tfIIs mutant background severely affects plant growth. A, amino acid sequence

alignment of the C-terminal part of TFIIS from Arabidopsis (At), rice (Os), human (Hs) and the yeast S. cerevisiae (Sc).

Asterisks indicate conserved residues and the invariant acidic residues (D359, E360 indicated in red) that were mutated in

this work (and the corresponding residues were mutated in other studies (Imashimizu et al., 2013; Jeon et al., 1994;

Kettenberger et al., 2004; Sigurdsson et al., 2010)). B, cartoon representation of the computational structure model of

domains II and III of Arabidopsis TFIIS. The model (C-score: 0.82) was generated using the I-TASSER server (Yang and

Zhang, 2015). Domain II is indicated in green, the interdomain linker in orange, domain III in yellow and the position of the

two invariant acidic hairpin residues (changed to alanine residues in TFIISmut) is highlighted in red and marked by an arrow.

C, growth of Arabidopsis plants expressing TFIISmut is severely impaired. 7-DAS plants of three independent plant lines

each expressing GFP-TFIISmut or GFP-TFIIS were grown in comparison to the Col-0 wild type on MS medium under

inductive conditions (+ β-estradiol) or mock treated (ethanol).

Figure 2. Plants expressing TFIISmut exhibit reduced growth of the primary root and decreased cell proliferation. A, inhibition of root

growth upon expression of TFIISmut. 8-DAS plants were transferred onto MS plates containing β-estradiol to induce expression of

GFP-TFIISmut and GFP-TFIIS, or mock-treated. Images were taken at the indicated times. White arrow (and black line) indicate the

position of the root tip at the time of transfer (0h). Root elongation was measured to determine absolute (B) and relative (C) elongation.

B, dots represent mean values ±SD and dotted lines represent linear regression (n = 6). C, bars represent mean values ±SD and asterisks

indicate the outcome of the Student´s t-Test: * p<0.05, ** p<0.01, *** p<0.001. D, CLSM analysis of GFP fluorescence of root tips of

5-DAS plants induced (or mock-treated) for 24h to express GS-TFIIS or GS-TFIISmut and harbouring the pCYCB1;1-GFP reporter. E,

bars represent mean values ±SD of the number of mitotic, GFP expressing cells (n = 5 plants per line/condition). Characters indicate the

outcome of a multi-comparison Tukey´s test (p<0.05).

Figure 3. Nuclear mobility of TFIISmut is markedly lower than that of TFIIS. FRAP analysis of Arabidopsis PSB-D cells

induced for 24h to express TFIISmut or TFIIS. A, mean fluorescence recovery curves ±SD (full scale normalisation)

representing 40 pre-bleach and 50 post-bleach time points. Calculated half-time (t1/2, B) and mobile fraction (Mf, C)

represented as box plots with * p<0.05 according to Student´s t-Test.

A

GFP-TFIIS

(mock) GFP-TFIIS

(b-estradiol)

GFP-TFIISmut

(b-estradiol)

77/119

848/280

840/431 26/114

DEG

up/down

GFP-TFIISmut

(mock)

B

C

Figure 4. Expression of TFIISmut leads to major transcriptomic changes. A, schematic representation of the number of DEGs

(up/down) between the analysed plant lines/conditions. B, heat map (n = 15836) of the transcriptomic changes between the

analysed plant lines/conditions. Only genes with FPKM ≥ 5 in at least one line/condition were considered and the hierarchical

clustering is indicated on the left. C, heat map legend with yellow-blue gradient indicating the log2FPKM [-2; 2]. The

magenta line indicates the number of values in a given colour range.

Color Key and Histogram

Value

-2 -1 0 1 2

0

70

00

Co

un

t

mock b-estradiol

GFP-

TFIIS

GFP-

TFIISmut GFP-

TFIIS

GFP-

TFIISmut

Figure 5. The genomic occupancy of elongating RNAPII is related to the corresponding transcript levels and the

distribution within transcribed regions is altered upon expression of TFIISmut. As indicated, gene-averaged profiles

represent the distribution of RNAPII-S2P (A,C) and RNAPII-S5P (B.D) over transcribed regions (TSS – TES) according

to the ChIPseq data of 10-DAS plants induced for 24h to express GFP-TFIISmut (C,D; dark magenta) or mock treated

(A,B; light magenta). Profiles represent genes with no (18754), low (3488), medium (6975) or high (3488) transcript

levels based on the FPKM values determined by RNAseq.

Figure 6. In the presence of TFIIS elongating RNAPII accumulates downstream of the TSS at a position overlapping with the +1

nucleosome. A, Gene-averaged profiles of the RNAPII-S2P and RNAPII-S5P distribution around the TSS according to ChIPseq data

of 10-DAS plants induced for 24h to express GFP-TFIISmut (dark magenta) or mock treated (light magenta). Profiles represent

33574 and 33560 loci for RNAPII-S2P and RNAPII-S5P, respectively. B, the black line represents the nucleosome occupancy of 14-

DAS plants calculated from MNase-seq data (Li et al., 2014) that was combined with the profiles of RNAPII-S2P and RNAPII-S5P

after induced GFP-TFIISmut expression. C, ChIP-seq profiles of RNAPII-S2P and RNAPII-S5P at the At5g11090 and At2g20562

loci with increased PPEP values upon β-estradiol vs. mock treatment. Plots were generated using IGB using representative biological

replicates. In gene models, thin bars represent UTRs, thick bars represent exons and dotted lines represent introns.

Figure 7. TFIISmut induced changes in PPEP are related to transcript levels. (A) Scheme depicting the promoter proximal

region (0 to +500 bp, blue) and the rest of the transcribed region to pA (orange), as well as calculation of PPEP. Changes in

PPEP-S2P (B) and PPEP-S5 (C) upon TFIISmut expression vs. mock treatment (dark magenta vs. bright magenta,

respectively) were calculated for genes with no (18754), low (3488), medium (6975) or high (3488) transcript levels based on

FPKM values determined by RNAseq. PPEP-S2P and PPEP-S5 values are visualised by box plots and asterisks indicate the

outcome of the Student´s t-Test: * p<0.05, *** p<0.001.

Figure 8. Upon TFIISmut expression the amount of the large subunit NRPB1 of RNAPII is decreased by proteasomal

degradation. A, 10-DAS plants inducibly expressing GFP-TFIIS or GFP-TFIISmut were treated for 24h with the inducer β-

estradiol (or mock-treated) and the proteasome inhibitor MG132 as indicated. All plants were additionally co-treated with

cycloheximide. Total protein extracts of these plants were analysed by immunoblotting using an antibody directed against

the NRPB1-CTD and as loading control an antibody directed against the RNA helicase UAP56. B, band intensities were

quantified using ImageJ. Bars represent mean values ±SD of three independent experiments. Data were statistically analysed

using one-way ANOVA and characters indicate the outcome of a multi-comparison Tukey´s test (p<0.05).

Parsed CitationsAdelman,K., Marr,M.T., Werner,J., Saunders,A., Ni,Z., Andrulis,E.D., and Lis,J.T. (2005). Efficient release from promoter-proximal stallsites requires transcript cleavage factor TFIIS. Mol. Cell 17: 103-112.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Antosch,M., Schubert,V., Holzinger,P., Houben,A., and Grasser,K.D. (2015). Mitotic lifecycle of chromosomal 3xHMG-box proteins andthe role of their N-terminal domain in the association with rDNA loci and proteolysis. New Phytol. 208: 1067-1077.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Antosz,W., Pfab,A., Ehrnsberger,H.F., Holzinger,P., Köllen,K., Mortensen,S.A., Bruckmann,A., Schubert,T., Längst,G., Griesenbeck,J.,Schubert,V., Grasser,M., and Grasser,K.D. (2017). Composition of the Arabidopsis RNA polymerase II transcript elongation complexreveals the interplay between elongation and mRNA processing factors. Plant Cell 29: 854-870.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Archambault,J., Lacroute,F., Ruet,A., and Friesen,J.D. (1992). Genetic interaction between transcription elongation factor TFIIS andRNA polymerase II. Mol. Cell. Biol. 12: 4142-4152.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Baena-González,E. and Sheen,J. (2008). Convergent energy and stress signaling. Trends Plant Sci. 13: 474-482.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Benjamini,Y. and Hochberg,Y. (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R.Statist. Soc. B 57: 289-300.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bentsink,L., Jowett,J., Hanhart,C.J., and Koornneef,M. (2006). Cloning of DOG1, a quantitative trait locus controlling seed dormancy inArabidopsis. Proc. Natl. Acad. Sci. USA 103: 17042-17047.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bolger,A.M., Lohse,M., and Usadel,B. (2014). Trimmomatic:a flexible trimmer for Illumina sequence data. Bioinformatics 30: 2114-2120.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Brand,L., Hörler,M., Nüesch,E., Vassalli,S., Barrell,P., Yang,W., Jefferson,R.A., Grossniklaus,U., and Curtis,M.D. (2006). A versatile andreliable two-component system for tissue-specific gene induction in Arabidopsis. Plant Physiol. 141: 1194-1204.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bürckstümmer,T., Bennett,K.L., Preradovic,A., Schütze,G., Hantschel,O., Superti-Furga,G., and Bauch,A. (2006). An efficient tandemaffinity purification procedure for interaction proteomics in mammalian cells. Nat. Meth. 3: 1013-1019.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Chen,F.X., Smith,E.R., and Shilatifard,A. (2018). Born to run: control of transcription elongation by RNA polymerase II. Nat. Rev. Mol.Cell Biol. 19: 464-478.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Cheung,A.C.M. and Cramer,P. (2011). Structural basis of RNA polymerase II backtracking, arrest and reactivation. Nature 471: 249-253.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Cohen,S.P. and Leach,J.E. (2019). Abiotic and biotic stresses induce a core transcriptome response in rice. Sci. Rep. 9: 6273.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Core,L. and Adelman,K. (2019). Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation. Genes Dev. 33: 960-982.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Danko,C.G., Hah,N., Luo,X., Martins,A.L., Core,L., Lis,J.T., Siepel,A., and Kraus,W.L. (2013). Signaling pathways differentially affect RNApolymerase II initiation, pausing, and elongation rate in cells. Mol. Cell 50: 212-222.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ding,Y., Avramova,Z., and Fromm,M. (2011). Two distinct roles of ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) at promoters and

within transcribed regions of ATX1-regulated genes. Plant Cell 23: 350-363.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Dolata,J., Guo,Y., Kolowerzo,A., Smolinski,D., Brzyzek,G., Jarmolowski,A., and Swiezewski,S. (2015). NTR1 is required for transcriptionelongation checkpoints at alternative exons in Arabidopsis. EMBO J. 34: 544-558.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Dürr,J., Lolas,I.B., Sørensen,B.B., Schubert,V., Houben,A., Melzer,M., Deutzmann,R., Grasser,M., and Grasser,K.D. (2014). Thetranscript elongation factor SPT4/SPT5 is involved in auxin-related gene expression in Arabidopsis. Nucleic Acids Res. 42: 4332-4347.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Fish,R.N. and Kane,C.M. (2002). Promoting elongation with transcript cleavage stimulatory factors. Biochim. Biophys. Acta 1577: 287-307.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Gómez-Herreros,F., de Miguel-Jiménez,L., Millán-Zambrano,G., Peñate,X., Delgado-Ramos,L., Muñoz-Centeno,M.C., and Chávez,S.(2012). One step back before moving forward: regulation of transcription elongation by arrest and backtracking. FEBS Lett. 586: 2820-2825.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Grasser,M., Kane,C.M., Merkle,T., Melzer,M., Emmersen,J., and Grasser,K.D. (2009). Transcript elongation factor TFIIS is involved inArabidopsis seed dormancy. J. Mol. Biol. 386: 598-611.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Hajheidari,M., Koncz,C., and Eick,D. (2013). Emerging roles for RNA polymerase II CTD in Arabidopsis. Trends Plant Sci. 18: 633-643.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Harlen,K.M. and Churchman,L.S. (2017). The code and beyond: transcription regulation by the RNA polymerase II carboxy-terminaldomain. Nat. Rev. Mol. Cell Biol. 18: 263-273.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

He,Y., Doyle,M.R., and Amasino,R.M. (2004). PAF1-complex-mediated histone methylation of FLOWERING LOCUS C chromatin isrequired for the vernalization-responsive, winter-annual habit in Arabidopsis. Genes Dev. 18: 2774-2784.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Hetzel,J., Duttke,S.H., Benner,C., and Chory,J. (2016). Nascent RNA sequencing reveals distinct features in plant transcription. Proc.Natl. Acad. Sci. USA 113: 12316-12321.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Imashimizu,M., Kireeva,M., Lubowska,L., Gotte,D., Parks,A.R., Strathern,J.N., and Kashlev,M. (2013). Intrinsic translocation barrier asan initial step in pausing by RNA polymerase II. J. Mol. Biol. 425: 697-712.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ishibashi,T., Dangkulwanich,M., Coello,Y., Lionberger,T.A., Lubkowska,L., Ponticelli,A.S., Kashlev,M., and Bustamante,C. (2014).Transcription factors IIS and IIF enhance transcription efficiency by differentially modifying RNA polymerase pausing dynamics. Proc.Natl. Acad. Sci. USA 111: 3419-3424.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Izban,M.G. and Luse,D.S. (1992). The RNA polymerase II ternary complex cleaves the nascent transcript in a 3'-5' direction in thepresence of elongation factor SII. Genes Dev. 6: 1342-1356.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Jarillo,J.A. and Piñeiro,M. (2015). H2A.Z mediates different aspects of chromatin function and modulates flowering responses inArabidopsis. Plant J. 83: 96-109.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Jeon,C., Yoon,H., and Agarwal,K. (1994). The transcription factor TFIIS zinc ribbon dipeptide Asp-Glu is critical for stimulation ofelongation and RNA cleavage by RNA polymerase II. Proc. Natl. Acad. Sci. USA 91: 9106-9110.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Jeronimo,C., Collin,P., and Robert,F. (2016). The RNA polymerase II CTD: the increasing complexity of a low-complexity protein domain.J. Mol. Biol. 428: 2607-2622.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Jiang,C. and Pugh,B.F. (2009). Nucleosome positioning and gene regulation: advances through genomics. Nat. Rev. Genet. 10: 161-172.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Juntawong,P., Girke,T., Bazin,J., and Bailey-Serres,J. (2014). Translational dynamics revealed by genome-wide profiling of ribosomefootprints in Arabidopsis. Proc. Natl. Acad. Sci. USA 111: E203-E212.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kammel,C., Thomaier,M., Sørensen,B.B., Schubert,T., Längst,G., Grasser,M., and Grasser,K.D. (2013). Arabidopsis DEAD-box RNAhelicase UAP56 interacts with both RNA and DNA as well as with mRNA export factors. PLoS One 8: e60644.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kettenberger,H., Armache,K.-J., and Cramer,P. (2003). Architecture of the RNA polymerase II-TFIIS complex and implications for mRNAcleavage. Cell 114: 347-357.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kettenberger,H., Armache,K.-J., and Cramer,P. (2004). Complete RNA polymerase II elongation complex structure and its interactionswith NTP and TFIIS. Mol. Cell 16: 955-965.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kim,D., Pertea,G., Trapnell,C., Pimentel,H., Kelley,R., and Salzberg,S.L. (2013). TopHat2: accurate alignment of transcriptomes in thepresence of insertions, deletions and gene fusions. Genome Biol. 14: R36.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kireeva,M.L., Hancock,B., Cremona,G.H., Walter,W., Studitsky,V.M., and Kashlev,M. (2005). Nature of nucleosomal barrier to RNApolymerase II. Mol. Cell 18: 108.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kulaeva,O.I., Hsieh,F.-K., Chang,H.-W., Luse,D.S., and Studitsky,V.M. (2013). Mechanism of transcription through a nucleosome by RNApolymerase II. Biochim. Biophys. Acta 1829: 76-83.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Langmead,B. and Salzberg,S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat. Meth. 9: 357-359.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Less,H., Angelovici,R., Tzin,V., and Galili,G. (2011). Coordinated gene networks regulating Arabidopsis plant metabolism in response tovarious stresses and nutritional cues. Plant Cell 23: 1264-1271.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Li,G., Liu,S., Wang,J., He,J., Huang,H., Zhang,Y., and Xu,L. (2014). ISWI proteins participate in the genome-wide nucleosomedistribution in Arabidopsis. Plant J. 78: 706-714.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lisica,A., Engel,C., Jahnel,M., Roldán,É., Galburt,E.A., Cramer,P., and Grill,S.W. (2016). Mechanisms of backtrack recovery by RNApolymerases I and II. Proc. Natl. Acad. Sci. USA 113: 2946-2951.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Liu,Y., Geyer,R., van Zanten,M., Carles,A., Li,Y., Hörold,A., van Nocker,S., and Soppe,W.J. (2011). Identification of the ArabidopsisREDUCED DORMANCY 2 gene uncovers a role for the polymerase associated factor 1 complex in seed dormancy. PLoS One 6:e22241.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lolas,I.B., Himanen,K., Grønlund,J.T., Lynggaard,C., Houben,A., Melzer,M., Van Lijsebettens,M., and Grasser,K.D. (2010). Thetranscript elongation factor FACT affects Arabidopsis vegetative and reproductive development and genetically interacts with HUB1/2.Plant J. 61: 686-697.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Google Scholar: Author Only Title Only Author and Title

Love,M.L., Huber,W., and Anders,S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol. 15: 550.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ma,Y., Gil,S., Grasser,K.D., and Mas,P. (2018). Targeted recruitment of the basal transcriptional machinery by LNK clock componentscontrols the circadian rhythms of nascent RNAs in Arabidopsis. Plant Cell 30: 907-924.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Mortensen,S.A. and Grasser,K.D. (2014). The seed dormancy defect of Arabidopsis mutants lacking the transcript elongation factorTFIIS is caused by reduced expression of the DOG1 gene. FEBS Lett. 588: 47-51.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Nakanishi,T., Nakano,A., Nomura,K., Sekimizu,K., and Natori,S. (1992). Purification, gene cloning, and gene disruption of thetranscription elongation factor S-II in Saccharomyces cerevisiae. J. Biol. Chem. 267: 13200-13204.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Nelissen,H., DeGroeve,S., Fleury,D., Neyt,P., Bruno,L., Bitonti,M.B., Vandenbussche,F., Van Der Straeten,D., Yamaguchi,T., Tsukaya,H.,Witters,E., de Jaeger,G., Houben,A., and Van Lijsebettens,M. (2010). Plant Elongator regulates auxin-related genes during RNApolymerase II-mediated transcription elongation. Proc. Natl. Acad. Sci. USA 107: 1678-1683.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Nock,A., Ascano,J.M., Barrero,M.J., and Malik,S. (2012). Mediator-regulated transcription through the +1 nucleosome. Mol. Cell 48: 837-848.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Oh,S., Zhang,H., Ludwig,P., and van Nocker,S. (2004). A mechanism related to the yeast transcriptional regulator Paf1c is required forexpression of the Arabidopsis FLC/MAF MADS box gene family. Plant Cell 16: 2940-2953.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Palangat,M. and Larson,D.R. (2012). Complexity of RNA polymerase II elongation dynamics. Biochim. Biophys. Acta 1819: 667-672.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Pfab,A., Bruckmann,A., Nazet,J., Merkl,R., and Grasser,K.D. (2018). The adaptor protein ENY2 is a component of the deubiquitinationmodule of the Arabidopsis SAGA transcriptional co-activator complex but not of the TREX-2 complex. J. Mol. Biol. 430: 1479-1494.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Rapsomaniki,M.A., Kotsantis,P., Symeonidou,I.E., Giakoumankis,N.N., Taraviras,S., and Lygerou,Z. (2012). easyFRAP: an interactive,easy-to-use tool for qualitative and quantitative analysis of FRAP data. Bioinformatics 28: 1800-1801.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ruan,W., Lehmann,E., Thomm,M., Kostrewa,D., and Cramer,P. (2011). Evolution of two modes of intrinsic RNA polymerase transcriptcleavage. J. Biol. Chem. 286: 18701-18707.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Selth,L.A., Sigurdsson,S., and Svejstrup,J.Q. (2010). Transcript elongation by RNA Polymerase II. Ann. Rev. Biochem. 79: 271-293.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sheridan,R.M., Fong,N., D´Alessandro,A., and Bentley,D.L. (2019). Widespread backtracking by RNA Pol II Is a major effector of geneactivation, 5' pause release, termination, and transcription elongation rate. Mol. Cell 73: 107-118.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sigurdsson,S., Dirac-Svejstrup,A.B., and Svejstrup,J.Q. (2010). Evidence that transcript cleavage is essential for RNA polymerase IItranscription and cell viability. Mol. Cell 38: 202-210.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sims,R.J., Belotserkovskaya,R., and Reinberg,D. (2004). Elongation by RNA polymerase II: the short and the long of it. Genes Dev. 18:2437-2468.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Somesh,B.P., Reid,J., Liu,W.F., Søgaard,T.M., Erdjument-Bromage,H., Tempst,P., and Svejstrup,J.Q. (2005). Multiple mechanismsconfining RNA polymerase II ubiquitylation to polymerases undergoing transcriptional arrest. Cell 121: 913-923.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Struhl,K. and Segal,E. (2013). Determinants of nucleosome positioning. Nat. Struct. Mol. Biol. 20: 267-273.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Svejstrup,J.Q. (2007). Contending with transcriptional arrest during RNAPII transcript elongation. Trends Biochem. Sci. 32: 165-171.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Teves,S.S., Weber,C.M., and Henikoff,S. (2014). Transcribing through the nucleosome. Trends Biochem. Sci. 39: 577-586.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Tsugama,D., Liu,S., and Takano,T. (2011). A rapid chemical method for lysing Arabidopsis cells for protein analysis. Plant Meth. 7: 22.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ubeda-Tomás,S., Federici,F., Casimiro,I., Beemster,G.T., Bhalerao,R., Swarup,R., Doerner,P., Haseloff,J., and Bennett,M.J. (2009).Gibberellin signaling in the endodermis controls Arabidopsis root meristem size. Curr. Biol. 19: 1194-1199.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ujvári,A., Hsieh,F.-K., Luse,S.W., Studitsky,V.M., and Luse,D.S. (2008). Histone N-terminal tails interfere with nucleosome traversal byRNA polymerase II. J. Biol. Chem. 283: 32236-32243.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

van Leene,J., Eeckhout,D., Cannoot,B., De Winne,N., Persiau,G., van de Slijke,E., Vercruysse,L., Dedecker,M., Vandepoele,K.,Martens,L., Witters,E., Gevaert,K., and de Jaeger,G. (2015). An improved toolbox to unravel the plant cellular machinery by tandemaffinity purification of Arabidopsis protein complexes. Nat. Protoc. 10: 169-187.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Van Lijsebettens,M. and Grasser,K.D. (2014). Transcript elongation factors: shaping transcriptomes after transcript initiation. TrendsPlant Sci. 19: 717-726.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Wang,D., Bushnell,D.A., Huang,X., Westover,K.D., Levitt,M., and Kornberg,R.D. (2009). Structural basis of transcription: backtrackedRNA Polymerase II at 3.4 angstrom resolution. Science 324: 1203-1206.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Weber,C.M., Ramachandran,S., and Henikoff,S. (2014). Nucleosomes are context-specific, H2A.Z-modulated barriers to RNApolymerase. Mol. Cell 53: 819-830.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Wilson,M.D., Harreman,M., and Svejstrup,J.Q. (2013). Ubiquitylation and degradation of elongating RNA polymerase II: the last resort.Biochim. Biophys. Acta 1829: 151-157.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Wind,M. and Reines,D. (2000). Transcription elongation factor SII. BioEssays 22: 327-336.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Yang,J. and Zhang,Y. (2015). I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Res. 43:W174-W181.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zatreanu,D., Han,Z., Mitter,R., Tumini,E., Williams,H., Gregersen,L., Dirac-Svejstrup,A.B., Roma,S., Stewart,A., Aguilera,A., andSvejstrup,J.Q. (2019). Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability.Mol. Cell 76: 57-69.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zhu,J., Liu,M., Liu,X., and Dong,Z. (2018). RNA polymerase II activity revealed by GRO-seq and pNET-seq in Arabidopsis. Nat. Plants 4:1112-1123.

Pubmed: Author and Title

Google Scholar: Author Only Title Only Author and Title

DOI 10.1105/tpc.19.00891; originally published online March 9, 2020;Plant Cell

and Klaus D. GrasserWojciech Antosz, Jules Deforges, Kevin Begcy, Astrid Bruckmann, Yves Poirier, Thomas Dresselhaus

ElongationCritical Role of Transcript Cleavage in Arabidopsis RNA Polymerase II Transcriptional

 This information is current as of October 9, 2020

 

Supplemental Data /content/suppl/2020/03/09/tpc.19.00891.DC1.html

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