BRF1 120 MCB.00910 13.full

User Manual: BRF1- 120

Open the PDF directly: View PDF .
Page Count: 34

Mapping the protein interaction network for the TFIIB-related 1

factor Brf1 in the RNA polymerase III pre-initiation complex 2

Seok-Kooi Khoo1,2, Chih-Chien Wu2, Yu-Chun Lin2, Jin-Cheng Lee2, and 4

Hung-Ta Chen1,2# 5

1. Taiwan International Graduate Program, Graduate Institute of Life 7

Sciences, National Defense Medical Center, Taipei, Taiwan, R.O.C. 8

2. Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, R.O.C. 9

# Correspondence to HT Chen, Institute of Molecular Biology, Academia Sinica, 12

128 Sec. 2 Academia Rd., Taipei 115, Taiwan, R.O.C. 13

Phone: + 886 2 27824778, Fax: + 886 2 27826085 14

E-mail: htchen012@gate.sinica.edu.tw 15

Running Title: Brf1 protein network in the pre-initiation complex 17

Keywords: Brf1/ RNA Polymerase III/ transcription initiation/ Bdp1/ C34 18

MCB Accepts, published online ahead of print on 25 November 2013

Mol. Cell. Biol. doi:10.1128/MCB.00910-13

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

Abstract 21

The TFIIB-related factor Brf1 is essential for RNA polymerase (Pol) III 22

recruitment and open promoter formation in transcription initiation. We site-23

specifically incorporated non-natural amino acid cross-linker to Brf1 to map its 24

protein interaction targets in the pre-initiation complex (PIC). Our cross-linking 25

analysis in the N-terminal domain of Brf1 indicated a pattern of multiple protein 26

interactions reminiscent of TFIIB in the polymerase active site cleft. In addition to 27

the TFIIB-like protein interactions, the Brf1 cyclin repeats subdomain is in contact 28

with the Pol III-specific C34 subunit. With site-directed hydroxyl radical probing, 29

we further revealed the binding between Brf1 cyclin repeats and the highly 30

conserved region connecting C34 winged-helix domains 2 and 3. In contrast to 31

the N-terminal domain of Brf1, the C-terminal domain contains extensive binding 32

sites for TBP and Bdp1 to hold together the TFIIIB complex on the promoter. 33

Overall, the domain architecture of the PIC derived from our cross-linking data 34

explains how individual structural subdomains of Brf1 integrate the protein 35

network from the Pol III active center to the promoter for transcription initiation. 36

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

Introduction 38

Eukaryotic RNA polymerase (Pol) III transcribes precursor tRNAs, 5S 39

ribosomal RNA, small nuclear RNAs such as U6 and 7SK RNAs, and a number 40

of small nucleolar and microRNAs (1). In yeast (Saccharomyces cerevisiae), the 41

Pol III transcription apparatus consists of the 17-subunit Pol III and three other 42

transcription factors: the single-polypeptide TFIIIA, the three-subunit TFIIIB and 43

the six-subunit TFIIIC (2, 3). TFIIIA and TFIIIC function as the promoter 44

recognition factors, and TFIIIB is recruited to the promoter through TFIIIC. TFIIIB 45

is composed of the TFIIB-related factor Brf1, the TATA-box binding protein TBP, 46

and the SANT domain-containing subunit Bdp1. Previous biochemical studies 47

indicated that Brf1 and TBP cooperatively assemble onto DNA upstream of the 48

transcription start site, and Bdp1 binds to the Brf1-TBP-DNA complex mainly 49

through its SANT domain (4-10). The TFIIIB-DNA assembly is required for 50

subsequent Pol III recruitment and transcript initiation. Both Brf1 and Bdp1 have 51

been found to interact with Pol III and function in promoter opening (4, 11-14). 52

The N-terminal domain of yeast Brf1 (Brf1n; aa. 1-286) contains a zinc 53

ribbon fold (aa. 3-34) and a cyclin-fold repeat subdomain (aa. 83-282) (Figure 54

1A), both of which are homologous to those in the general transcription factor 55

TFIIB of the Pol II system. Based on biochemical and structural analyses, TFIIB 56

ribbon and cyclin-fold repeats are respectively positioned in the RNA exit tunnel 57

and on the wall domain of Pol II (15-20). In addition, the connecting region 58

between the TFIIB ribbon and cyclin repeat domain is structurally resolved to 59

contain B-reader and B-linker motifs interacting with the polymerase active center. 60

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

Based on sequence comparison, the connecting region in Brf1n, which we refer 61

to as N-linker, contains low sequence homology with TFIIB. However, this region 62

might also contribute to the binding of the polymerase active center as previous 63

genetic analyses revealed the involvement of ribbon and N-linker in open 64

complex formation (11, 13). 65

The C-terminal half of Brf1 (Brf1c) is Pol III-specific and is not conserved 66

among the TFIIB family, which, in addition to Brf1 and TFIIB, also includes Rrn7 67

(TAF1B in human) in the Pol I system (21-24). Yeast Brf1c (aa. 287-596) contains 68

three homologous sequence blocks, I (aa. 287-304), II (aa. 461-515) and III (aa. 69

570-596) (Figure 1A), that are conserved in S. cerevisiae, Schizosaccharomyces 70

pombe, Candida albicans, Kluyveromyces lactis and Homo sapiens (22, 25). 71

Brf1c exists mostly as a scaffold that holds together the three TFIIIB subunits (12, 72

26). In particular, structural analysis of the Brf1-TBP-DNA complex indicated that 73

homology block II is positioned along the convex and lateral surfaces of TBP, and 74

the block also interacts with Bdp1 (5, 6, 10, 22, 26-28). The homology blocks are 75

separated by two non-conserved connecting regions that we refer to as C-linkers 76

1 and 2 (Figure 1A). 77

Previous genetic and pairwise protein-protein interaction analyses have 78

identified Brf1 interacting partners. In addition to TBP and Bdp1 of the TFIIIB 79

complex, Brf1 interacts with the τ131 (Tfc4) subunit of TFIIIC and two of the Pol 80

III subunits, C34 and C17 (29-33). However, most of the previous studies 81

involved large protein fragments of Brf1, and a detailed and more precise 82

characterization of the Brf1 protein network is not yet available. In this study, we 83

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

site-specifically incorporated a non-natural photo-reactive amino acid p-benzoyl-84

L-phenylalanine (BPA) to the yeast Brf1 to map protein-protein interactions within 85

the Pol III pre-initiation complex (PIC). BPA incorporated in the amino acid 86

sequence of Brf1n revealed cross-linking with TBP and the C160 and C128 87

subunits of the Pol III active site cleft as well as two smaller subunits, C34 and 88

C17. The Brf1-C34 interaction was further analyzed by site-specific hydroxyl 89

radical analysis that revealed the connection between the Brf1 cyclin repeat 90

subdomain and a conserved sequence C-terminal to C34 winged-helix domain 2. 91

Our cross-linking results for Brf1c identified additional Bdp1 and TBP interactions 92

in the C-linker 1 region. Mutational analysis indicated that a Bdp1-binding block 93

in C-linker 1 is required for optimal cell growth and in vitro transcription activity. 94

Overall, our work provides a precise mapping of the network of protein-protein 95

interactions for Brf1 and further elucidates the domain architecture of the Pol III 96

PIC. 97

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

Materials and Methods 98

Yeast strains and plasmids 99

Yeast strains used for this study were derived from BY4705 with chromosomal 100

disruptions of individual genes by the KanMX4 cassette, yielding Brf1 shuffle 101

strain YSK1 [MAT

ade2::his3G his3

200 leu2

met15

lys2

trp1

63 ura3

102

(brf1::KanMX4) Brf1-pRS316 (URA3+)] and C34 shuffle strain YLy3 [MAT

103

ade2::his3G his3

200 leu2

met15

lys2

trp1

63 ura3

(Rpc34::KanMX4) 104

Rpc34-pRS316 (URA3+)] (34, 35). Brf1 and Rpc34 (C34) were separately cloned 105

into yeast 2 micron vector pRS425 with LEU2 selection marker (36). Both genes 106

were driven by yeast ADH1 promoter. Brf1 was either V5- or 13-Myc-epitope 107

tagged at the C-terminus via the QuikChange II Site-Directed Mutagenesis Kit 108

(Stratagene), yielding plasmids pSK1 (Adh1-Brf1cV5-pRS425) and pSK2 (Adh1-109

Brf1c13Myc-pRS425), respectively. C34 was C-terminally V5-tagged, yielding 110

pYL5 (Rpc34cV5-pRS425). Each of the constructed plasmids was used to 111

generate individual mutant plasmids containing single “TAG” (amber) nonsense 112

codon substitution at intended amino acid positions. To generate yeast strains for 113

incorporating non-natural amino acids p-benzoyl-L-phenylalanine (BPA) into Brf1 114

and C34, we applied plasmid shuffling to transform individual amber plasmids 115

into yeast YSK1 together with the plasmid pLH157 encoding a suppressor 116

tRNACUA (corresponding to TAG amber codon) and a BPA-tRNA synthetase (16, 117

37). 118

For Brf1 mutagenesis study, the gene encoding Brf1 along with its 119

endogenous promoter was cloned into the vector pRS315 with a single HA 120

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

epitope tag at the C-terminus, yielding pSK3 (Brf1-HA, ars cen, LEU2) (38). All 121

Brf1 mutant plasmids were generated based on pSK3, and the plasmids were 122

transformed into the Brf1 shuffle strain to generate mutant strains by the 5-FOA 123

drop-out method. For cells growth assay, both the WT and mutant strains were 124

grown in YPD till OD600 1.0, and the cell cultures were subsequently diluted with 125

the dilution range of 10-2, 10-4 and 10-6. The diluted cells were spotted on the 126

synthetic complete glucose plate lacking leucine, and the growth phenotypes at 127

temperatures 16 °C, 25 °C, 30 °C, and 37 °C were monitored. The incubation 128

time for cell growth at 30 °C and 37 °C was 3 days. For subsequent biochemical 129

studies, yeast whole cell extract (WCE) is prepared. Detailed procedures for 130

preparation of WCE from individual BPA-incorporated or mutant yeast strains 131

have been described previously (14, 39). 132

133

PIC isolation and BPA photo-crosslinking 134

The Pol III pre-initiation complex (PIC) was isolated using the immobilized 135

template assay (IMT) with yeast WCE and DNA template containing either the U6 136

snRNA or SUP4 tRNA promoter immobilized on Streptavidin magnetic beads 137

(DynaI) as previously described (14, 39). For the BPA photo-crosslinking 138

experiment, 800 µg of WCE was incubated with 4 µg of DNA template 139

immobilized on 200 µg of DynaI beads (Invitrogen) at 30oC for 30 min. Each 140

reaction was washed three times with transcription buffer containing 20 mM 141

KHepes (pH7.9), 80 mM KCl, 5 mM MgCl2, 1 mM EDTA, 2%(vol/vol) glycerol, 142

and 0.01% Tween 20. After washing, the reaction was divided into two fractions, 143

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

one that would receive UV irradiation (+UV) and the other that would serve as a 144

control (-UV). UV irradiation was conducted with a total energy of 6500 µJ/cm2 in 145

a Spectrolinker XL-1500 UV oven (Spectronics). The samples were then 146

resuspended in NuPAGE sample buffer (Invitrogen) for SDS-PAGE and Western 147

analysis. The Western blot was visualized with the LICOR Odyssey infrared 148

imaging system using fluorescent dye-labeled secondary antibodies. 149

150

In vitro transcription 151

In vitro transcription was conducted with the IMT assay as described above. After 152

washing, the isolated PICs were resuspended in 17 µL of transcription buffer 153

containing 200 ng α-amanitin, 4 units of RNase inhibitor (Promega), and 1 mM 154

DTT. A mixture of NTPs (3 µL) was subsequently added, and the resulting 155

reaction mixture contains 500 µM each of ATP, UTP, CTP, 50 µM GTP and 0.16 156

µM [α-32P] GTP (3000 Ci/mmol). After allowing the reaction to proceed at 30oC 157

for 30 min, transcription was quenched by adding 180 µL of 0.1 M sodium 158

acetate, 10 mM EDTA, 0.5% SDS and 200 µg/mL glycogen. The transcripts were 159

extracted by phenol/chloroform and ethanol precipitated, separated on 6% (wt/vol) 160

denaturing urea polyacrylamide gel and visualized by autoradiogram. 161

Restoration of transcription activity was conducted by adding recombinant Brf1 162

(160 ng) into the Brf1 mutants WCE. 163

164

Immunoprecipitation 165

Brf1 wild-type (WT) and mutant WCEs containing Bdp1 C-terminal Flag-tag and 166

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

Brf1 C-terminal HA-tag were used for immunoprecipitation (IP). WCE (1 mg) was 167

mixed with 50 µL of anti-Flag agarose beads (Sigma) in the extract dialysis buffer 168

containing 20 mM KHEPES pH 7.9, 100 mM KCl, 5 mM MgCl2, 1 mM EDTA, 169

and 20% glycerol and incubated overnight at 4oC. Following 3 washes with 500 170

µL of extract dialysis buffer, the bound proteins were eluted by boiling the beads 171

at 95oC for 5 min in 20 µL of 4X NuPAGE buffer (Invitrogen). The eluted proteins 172

were resolved by SDS-PAGE and analyzed by Western blot analysis probing with 173

the following antibodies, anti-Flag (probed for Bdp1), anti-HA (probed for Brf1), 174

anti-TBP, and anti-τ131 (Tfc4; TFIIIC subunit). 175

176

C34 purification and FeBABE conjugation 177

Expression and purification of C34 was as described previously (39). To avoid 178

off-target FeBABE conjugation, three endogenous cysteines were altered to non-179

cysteine residues as follows: Cys124Ala, Cys244Ala and Cys260Ser. All single-180

cysteine C34 variants were derived from the non-cysteine C34. FeBABE 181

conjugation was performed as described previously (14). 182

183

Hydroxyl radical cleavage with C34-FeBABE conjugate 184

Hydroxyl radical probing in the Pol III PIC was conducted based on the 185

previously established protocol using a C82 mutant WCE allowing dissociation of 186

the C82/34/31 subcomplex from the polymerase core (39). In a IMT reaction, 400 187

µg yeast WCE containing C-terminally Flag3-tagged Brf1 and the C82 deletion-188

mutant (50-52) was incubated with 0.72 µg of recombinant C31, 2 µg of 189

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

recombinant C82, and 0.94 µg of C34-FeBABE conjugate in a 200 µL reaction 190

containing 2 µg of SUP4 tRNA promoter DNA template. The PICs on beads were 191

washed three times with transcription buffer. After washing, samples were 192

resuspended in 7.5 µL of transcription buffer. The following reagents were added 193

sequentially: 2.5 µL of 50% (vol/vol) glycerol, 1.25 µL of 50 mM sodium ascorbate, 194

and 1.25 µL of H2O2 mix [0.24% (vol/vol) H2O2, 10mM EDTA]. The hydroxyl 195

radical cleavage reaction was conducted at 30oC for 8 min and quenched by 196

adding 4.5 µL NuPAGE LDS sample buffer (Invitrogen) and 1 µL of 1M DTT. The 197

protein cleavage sites in Brf1 were determined based on the method described 198

previously (14). In vitro transcription analysis was also conducted in parallel. The 199

C34-FeBABE conjugates restored transcription activity similar to that of the wild-200

type (data not shown). 201

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

Results 202

Brf1 N-terminal domain interacts with Pol III in a similar mode as the TFIIB-203

Pol II complex 204

To map the protein-protein interaction network of Brf1, we applied the 205

nonsense suppression method to incorporate BPA site-specifically to the entire 206

Brf1 (37, 40). We generated individual yeast strains each containing a single TAG 207

amber codon in the Brf1 coding sequence for BPA replacement at the designated 208

amino acid positions. A total of 197 strains were created as listed in Table S1. We 209

isolated yeast WCEs from these Brf1-BPA strains and conducted the immobilized 210

template (IMT) assay coupled with UV-irradiation to allow site-specific photo-211

cross-linking in the isolated PICs. The cross-linking samples were subsequently 212

applied to SDS-PAGE and Western blotting analyses, and protein cross-links 213

were determined based on the appearance of additional low-mobility gel bands 214

generated by UV-irradiation. As demonstrated in the Western analysis (Figure 215

1B), BPA substitution in residues Gly44 and Gln62 in the N-linker region of Brf1 216

generated protein cross-links of the size of ~240 kDa (Fig. 1B; lanes 4 and 6). By 217

subtracting the apparent molecular weight of Brf1, the polypeptide cross-linked to 218

Brf1 was estimated to have molecular weight in the range of 160 to 180 kDa. We 219

confirmed this crosslinked polypeptide to be the largest subunit C160 of Pol III by 220

repeating the photo-cross-linking experiment using WCEs containing C-terminally 221

HA-tagged C160 and probing with anti-HA antibody (Fig. 1B; lanes 10 and 12). 222

Cross-linking to the second largest subunit C128 of Pol III was also observed for 223

BPA substitution in residues Arg85 and Arg149 of the first cyclin fold and residue 224

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

Asn18 of the ribbon fold (Fig. 1C and data not shown). 225

As summarized in Figure 1A, Brf1-C160 and -C128 cross-links are 226

distributed respectively in the N-linker and ribbon/cyclin repeat subdomains. The 227

cross-linking pattern suggests a TFIIB-like binding mode as in the Pol II-TFIIB 228

structural model (20). In the Pol II-TFIIB model, the linker region of TFIIB, 229

including B-reader and B-linker motifs, are positioned in the polymerase active 230

center contacting the lid, rudder, and clamp coiled-coil motifs of Rpb1 231

(homologous to C160). In addition, the first cyclin fold of TFIIB is in close contact 232

with the wall and protrusion domains of Rpb2 (homologous to C128), and the 233

ribbon fold of TFIIB contacts both Rpb1 and Rpb2 in the RNA exit tunnel (16, 20). 234

To further investigate this TFIIB-like binding mode, we conducted another BPA 235

cross-linking analysis in the wall domain of C128. As demonstrated in Figure 1D, 236

a BPA substitution at His801 of the wall domain generated a cross-link with Brf1, 237

supporting the localization of Brf1 on C128. Although further structural and 238

biochemical analyses are required to determine the structural region of Brf1 in 239

contact with the wall domain of C128, our combined photo-cross-linking results 240

with BPA substituted C128 and Brf1 suggest that the Brf1 N-terminal domain 241

likely has a TFIIB-Pol II binding mode in the PIC. 242

In addition to cross-linking with the two largest subunits of Pol III, we also 243

observed Brf1-C17 cross-linking for residues Lys5 and His8 in the zinc-binding 244

knuckle of the ribbon domain (Fig. 1A; data not shown). Since C17 dimerizes 245

with C25 to form the stalk subcomplex that localizes adjacent to the RNA exit 246

tunnel (41), the Brf1-C17 cross-link suggests a potential functional link between 247

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

Brf1 and the stalk in transcription initiation. Furthermore, we observed a Brf1-TBP 248

cross-link at Lys211 at the H2’ helix of the second cyclin fold (Fig. 1A & 1E). This 249

cross-link supports the structural model for the binding of cyclin fold repeats with 250

the TBP-DNA complex, where the loop between H2’ and H3’ helices of the 251

second cyclin fold interacts with the C-terminal stirrup and the C-terminus of TBP 252

(42). 253

254

Brf1 cyclin fold repeat subdomain connects with C34 for Pol III recruitment 255

Our BPA cross-linking analysis for Brf1n revealed subdomain-specific 256

interactions with C160, C128, and TBP, suggesting that Brf1n organizes TFIIB-257

like domain architecture in the PIC. Based on previous studies with yeast two-258

hybrid and pull-down analyses, Brf1 also contains a Pol III-system specific 259

interaction with the C34 subunit of the Pol III complex. However, the interaction 260

site for C34 was not precisely mapped as the studies were involved either with 261

the full-length Brf1 protein or with the cyclin fold repeats (aa. 90-262) (22, 43). 262

Consistent with the low-resolution protein mapping data, we observed a weak 263

cross-link between Brf1 and the C34 subunit of Pol III at residue Tyr99 of the H2 264

helix in the first cyclin fold (Figure 2A). 265

Our previous cross-linking analysis on Pol III identified inter-subunit 266

interactions that localize C34 N-terminal winged-helix domains WH1 and WH2 267

above the Pol III active center cleft and the C-terminal region beneath the 268

polymerase clamp domain (Figure 2B). However, it remains unclear how C34 269

provides additional Pol III-Brf1 interaction for Pol III recruitment as indicated in 270

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

previous studies (22, 29, 44). To address this, we incorporated BPA in C34 to 271

map Brf1 binding sites. BPA substitution at Glu169, located at the connecting 272

region between WH2 and the predicted WH3, resulted in a weak cross-link with 273

Brf1 (Figure 2C). Surprisingly, Glu169 is located near the amino acid stretch 274

Asp171-Glu173 that is functionally important for Pol III recruitment (44). 275

To further characterize the C34-Brf1 interaction, we applied site-directed 276

hydroxyl radical analysis to probe the structural region of Brf1 near the C34 277

WH2/3 connecting region. We generated C34 single cysteine mutants to 278

conjugate the hydroxyl radical reagent FeBABE at the amino acid positions 279

Leu170 and Ile172. The FeBABE-conjugated C34 variants were applied to the 280

IMT assay for hydroxyl radical protein cleavage analysis in the PIC. In Figure 2D, 281

a Brf1 cleavage fragment was commonly generated by the C34-FeBABE 282

conjugates (Figure 2D; lanes 2, 3, and 4). By comparing with the molecular 283

weight ladder generated from in vitro translated Brf1 peptide fragments, the 284

cleavage site was determined to be in the H4’ helix of Brf1n second cyclin fold. In 285

summary, the combined cross-linking and hydroxyl radical analyses suggest an 286

interaction between the WH2/3 connecting region of C34 and the cyclin fold 287

repeats of Brf1n. As the biochemical probing results were weak, we suspect that 288

C34 might not strongly interact with Brf1 in the PIC. However, as previous studies 289

suggested that BPA is a less efficient cross-linking reagent due to its geometry 290

requirement for hydrogen abstraction by benzophenone (45), the weak C34-Brf1 291

crosslinking could also be attributed to the poor cross-linking efficiency of BPA. 292

293

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

Brf1 C-terminal domain contains extended Bdp1 and TBP binding region 294

The homology block II of Brf1c serves as the dominant binding site for both 295

TBP and Bdp1, and this block adopts a “vine-on-a-tree” conformation to interact 296

with TBP from the convex surface to the lateral surface of the first structural 297

repeat (6, 27, 28). Consistent with the protein interaction model, our BPA cross-298

linking analysis conducted in homology block II revealed cross-links with Bdp1 299

and TBP. As indicated in the summary of Brf1c cross-linking (Figure 3A) and 300

illustrated in Figure 3B, BPA-substitution at residue His473 generates two cross-301

links confirmed to be TBP and Bdp1, indicating simultaneous interactions with 302

both proteins. Similar simultaneous cross-linking was also observed for BPA 303

substitution at the neighboring residue Ala472 (data not shown). In the homology 304

block II-TBP-DNA ternary complex structure, His473 and Ala472 belong to the 305

helix H23 that interacts with the convex surface of the TBP first structural repeat. 306

Our cross-linking results therefore further suggest the localization for Bdp1 on the 307

TBP convex surface. 308

Additional Bdp1 and TBP cross-links were also observed for BPA 309

substitutions in the connecting region between homology blocks I and II, which 310

we refer to as C-linker 1. As shown in Figure 3C and summarized in Figure 3A, 311

BPA incorporated at residues Lys319 and Lys335 generated Bdp1 cross-linking. 312

In contrast to the Brf1-Bdp1 cross-links that are clustered closer to homology 313

block I, Brf1-TBP cross-linking occurs at residues widely distributed in C-linker 1 314

(Figure 3D and summarized in Figure 3A). 315

316

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

The Bdp1-binding block is important for transcription initiation 317

On the basis of extensive TBP and Bdp1c interactions revealed by BPA 318

cross-linking, we introduced a series of truncations and point mutations in Brf1c. 319

Internal truncations and point mutations were initially introduced in homology 320

block I resulting in cell lethality. In contrast, most of the mutations in C-linker 1 321

resulted in yeast strains without observable temperature-dependent growth 322

defects. However, mutations in the sequence block Gln311-Arg350, which 323

provided multiple cross-linking with Bdp1 (Figure 3A), conferred a temperature-324

sensitive growth phenotype. As demonstrated in Figure 4A, the yeast strains with 325

either Leu332Glu point mutation or del (Glu331-Tyr340) internal truncation 326

showed slow cell growth at the non-permissive temperature 37°C. We isolated 327

WCEs from these two mutant strains and conducted a co-immunoprecipitation 328

assay to analyze Brf1-Bdp1 binding. As shown in Figure 4B and 4C, both Brf1c 329

mutants severely compromised the binding with Bdp1, supporting our cross-link 330

data. We further analyzed this newly identified Bdp1-binding block by in vitro 331

transcription and PIC formation assays on the SUP4 DNA template. Both 332

mutations severely compromised transcription activity (Figure 4D, lanes 2 and 3). 333

The mutations also caused reduced Bdp1 and Brf1 protein levels in the isolated 334

PICs from the IMT assay (Figure 4E, lanes 2 and 3), indicating that both 335

mutations affect stable association of Bdp1 and Brf1 in the PIC. Our results thus 336

suggest that this Bdp1-binding block provides important structural support for 337

stabilizing Brf1 and Bdp1 in the PIC. 338

339

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

Discussion 340

In the Pol III transcription machinery, Brf1 together with TBP and Bdp1 341

constitutes transcription factor TFIIIB for Pol III recruitment and open promoter 342

complex formation. Using site-specific biochemical probing analyses in this study, 343

we precisely mapped the network of protein interactions for Brf1 in the PIC. Our 344

cross-linking results suggest that the Brf1 N-terminal domain organizes a TFIIB-345

like domain architecture in the PIC. In contrast, the C-terminal half of Brf1 serves 346

mainly as the interface to hold TBP and Bdp1 for TFIIIB complex. An open 347

promoter model for the Pol III PIC is thus derived based on the x-ray structures of 348

Pol II-TFIIB, TFIIB cyclin folds-TBP-DNA, and Brf1 homology block II-TBP-DNA 349

complexes (Figure 5) (20, 28, 46). In the model, the ribbon and the cyclin fold 350

repeat subdomains are respectively localized in the RNA exit tunnel and on the 351

wall domain of polymerase. TBP contacts a 8-bp-long DNA sequence that starts 352

from 30 bases upstream of the transcription start site, and the Brf1 cyclin folds 353

clamp the second stirrup of TBP and interact with DNA sequences flanking the 354

TBP-binding region. Brf1 N-linker region was not modeled due to the lack of 355

structural information. However, this region likely interacts with the open 356

promoter region as well as structural motifs of the active center based on our 357

Brf1-C160 cross-linking and its functional role, together with the ribbon 358

subdomain, in DNA opening (11, 13, 47, 48). 359

The domain architecture of Pol III derived from our previous study 360

localizes the TFIIE-like C82 and C34 subunits on the polymerase clamp (Figure 361

5). The WH2 domain of C34 is in close contact with the clamp coiled-coil and 362

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

further interacts with the upstream edge of the transcription bubble, which is a 363

10~12 base strand-separated promoter region spanning upstream beginning 364

from the transcription start site (39). With the localization of C34 WH2 domain, 365

the functionally important connecting region immediately C-terminal to WH2 is 366

likely positioned adjacent to the Brf1 cyclin fold repeat subdomain. Our site-367

specific cross-linking and hydroxyl radical data support this interaction. Further, 368

this C34 connecting region likely contributes to additional upstream C34-DNA 369

interaction based on the Pol III-DNA topography analysis indicating co-370

localization of C34 and Brf1 in the promoter region spanning ~20 bases upstream 371

of the transcription start site (49, 50). In the Pol II PIC, the TFIIB cyclin folds were 372

found to interact with Tfg1 and Tfg2 subunits of the transcription factor TFIIF (15), 373

which is positioned on the lobe and protrusion domains of polymerase (40). 374

Compared to TFIIE, which also interacts with the polymerase clamp, the 375

localization of TFIIF is on the opposite side of the polymerase cleft. Therefore, 376

the cyclin repeats domain is involved in establishing specific interactions with 377

polypeptides on the polymerase active center cleft for respective transcription 378

systems. 379

Our cross-linking data indicate Brf1c mainly serves as a bipartite interface 380

for TBP and Bdp1. Specifically, our analysis extends Bdp1- and TBP-binding 381

sites to the C-linker 1 region, and we identified a functionally important Bdp1-382

binding sequence block. Although this Bdp1-binding block contains low sequence 383

homology, secondary structure analysis indicates consensus -helical secondary 384

structures in this region. Furthermore, this Bdp1-binding block contains the amino 385

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

acid sequence Gly328-Glu329-Gln330-Glu331-Leu332 (GEXEL) that was 386

previously reported to be a conserved short motif in Brf1c (25). The structural 387

region of Bdp1 that interacts with this sequence block remains to be determined. 388

In addition to TBP and Bdp1 interactions, we observed a weak C34 cross-link for 389

BPA substitution at Gln549 adjacent to homology block III (data not shown). This 390

C34 cross-link supports a previous genetic interaction analysis that mapped Brf1-391

C34 interaction to homology blocks II and III (29). 392

The domain architecture of the PIC derived from this study explains 393

respective functional roles in DNA opening for ribbon and N-linker and in 394

organizing TFIIIB-pol III-DNA complex for the cyclin fold repeats subdomain and 395

the C-terminal domain. In the eukaryotic Pol I system, the TFIIB-related factors 396

TAF1B in human and Rrn7 in yeast also contain TFIIB-like ribbon and cyclin 397

repeat subdomains in their N-terminal domains and unique C-terminal domains 398

specific for respective polymerases (23, 24). Genetic analysis for TAF1B 399

indicated that the zinc ribbon and the connecting region (N-terminal linker) mainly 400

function in post-recruitment step(s), reminiscent of Brf1 (23). Although the 401

analysis for domain localization is not available, a conserved binding mechanism 402

may exist for these Pol I factors as suggested by our study for Brf1. 403

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

Acknowledgements 404

We thank Dr. George Kassavetis (UC San Diego) for advices on biochemical 405

probing experiments. We thank Yue-Chang Chou for protein purification. We 406

thank AndreAna Peña for English editing. This work was supported by the grant 407

NSC 100-2311-B-001-013-MY3 from National Science Council, R.O.C. and the 408

Career Development Award to H.-T.C. from Academia Sinica. 409

410

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

References 411

1. Dieci, G., G. Fiorino, M. Castelnuovo, M. Teichmann, and A. Pagano. 2007. The 412

expanding RNA polymerase III transcriptome. Trends Genet 23:614-622. 413

2. Geiduschek, E. P., and G. A. Kassavetis. 2001. The RNA polymerase III 414

transcription apparatus. J Mol Biol 310:1-26. 415

3. Schramm, L., and N. Hernandez. 2002. Recruitment of RNA polymerase III to its 416

target promoters. Genes Dev 16:2593-2620. 417

4. Ishiguro, A., G. A. Kassavetis, and E. P. Geiduschek. 2002. Essential roles of Bdp1, 418

a subunit of RNA polymerase III initiation factor TFIIIB, in transcription and tRNA 419

processing. Mol Cell Biol 22:3264-3275. 420

5. Kassavetis, G. A., C. Bardeleben, A. Kumar, E. Ramirez, and E. P. Geiduschek. 421

1997. Domains of the Brf component of RNA polymerase III transcription factor 422

IIIB (TFIIIB): functions in assembly of TFIIIB-DNA complexes and recruitment of 423

RNA polymerase to the promoter. Mol Cell Biol 17:5299-5306. 424

6. Kassavetis, G. A., R. Driscoll, and E. P. Geiduschek. 2006. Mapping the principal 425

interaction site of the Brf1 and Bdp1 subunits of Saccharomyces cerevisiae TFIIIB. 426

J Biol Chem 281:14321-14329. 427

7. Kumar, A., A. Grove, G. A. Kassavetis, and E. P. Geiduschek. 1998. Transcription 428

factor IIIB: the architecture of its DNA complex, and its roles in initiation of 429

transcription by RNA polymerase III. Cold Spring Harb Symp Quant Biol 63:121-430

129. 431

8. Kumar, A., G. A. Kassavetis, E. P. Geiduschek, M. Hambalko, and C. J. Brent. 432

1997. Functional dissection of the B" component of RNA polymerase III 433

transcription factor IIIB: a scaffolding protein with multiple roles in assembly and 434

initiation of transcription. Mol Cell Biol 17:1868-1880. 435

9. Librizzi, M. D., M. Brenowitz, and I. M. Willis. 1998. The TATA element and its 436

context affect the cooperative interaction of TATA-binding protein with the TFIIB-437

related factor, TFIIIB70. The Journal of biological chemistry 273:4563-4568. 438

10. Saida, F. 2008. Structural characterization of the interaction between TFIIIB 439

components Bdp1 and Brf1. Biochemistry 47:13197-13206. 440

11. Kassavetis, G. A., A. Kumar, G. A. Letts, and E. P. Geiduschek. 1998. A post-441

recruitment function for the RNA polymerase III transcription-initiation factor IIIB. 442

Proc Natl Acad Sci U S A 95:9196-9201. 443

12. Kassavetis, G. A., A. Kumar, E. Ramirez, and E. P. Geiduschek. 1998. Functional 444

and structural organization of Brf, the TFIIB-related component of the RNA 445

polymerase III transcription initiation complex. Mol Cell Biol 18:5587-5599. 446

13. Hahn, S., and S. Roberts. 2000. The zinc ribbon domains of the general 447

transcription factors TFIIB and Brf: conserved functional surfaces but different 448

roles in transcription initiation. Genes Dev 14:719-730. 449

14. Wu, C. C., Y. C. Lin, and H. T. Chen. 2011. The TFIIF-like Rpc37/53 dimer lies at 450

the center of a protein network to connect TFIIIC, Bdp1, and the RNA polymerase 451

III active center. Molecular and cellular biology 31:2715-2728. 452

15. Chen, H. T., and S. Hahn. 2004. Mapping the location of TFIIB within the RNA 453

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

polymerase II transcription preinitiation complex: a model for the structure of 454

the PIC. Cell 119:169-180. 455

16. Chen, H. T., and S. Hahn. 2003. Binding of TFIIB to RNA polymerase II: Mapping 456

the binding site for the TFIIB zinc ribbon domain within the preinitiation complex. 457

Mol Cell 12:437-447. 458

17. Bushnell, D. A., K. D. Westover, R. E. Davis, and R. D. Kornberg. 2004. Structural 459

basis of transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 Angstroms. 460

Science 303:983-988. 461

18. Liu, X., D. A. Bushnell, D. Wang, G. Calero, and R. D. Kornberg. 2010. Structure 462

of an RNA polymerase II-TFIIB complex and the transcription initiation 463

mechanism. Science 327:206-209. 464

19. Kostrewa, D., M. E. Zeller, K. J. Armache, M. Seizl, K. Leike, M. Thomm, and P. 465

Cramer. 2009. RNA polymerase II-TFIIB structure and mechanism of transcription 466

initiation. Nature 462:323-330. 467

20. Sainsbury, S., J. Niesser, and P. Cramer. 2012. Structure and function of the 468

initially transcribing RNA polymerase II-TFIIB complex. Nature. 469

21. Colbert, T., and S. Hahn. 1992. A yeast TFIIB-related factor involved in RNA 470

polymerase III transcription. Genes Dev 6:1940-1949. 471

22. Khoo, B., B. Brophy, and S. P. Jackson. 1994. Conserved functional domains of 472

the RNA polymerase III general transcription factor BRF. Genes Dev 8:2879-2890. 473

23. Naidu, S., J. K. Friedrich, J. Russell, and J. C. Zomerdijk. 2011. TAF1B is a TFIIB-474

like component of the basal transcription machinery for RNA polymerase I. 475

Science 333:1640-1642. 476

24. Knutson, B. A., and S. Hahn. 2011. Yeast Rrn7 and human TAF1B are TFIIB-477

related RNA polymerase I general transcription factors. Science 333:1637-1640. 478

25. Martinez, M. J., and K. U. Sprague. 2003. Cloning of a putative Bombyx mori 479

TFIIB-related factor (BRF). Arch Insect Biochem Physiol 54:55-67. 480

26. Kassavetis, G. A., C. A. Joazeiro, M. Pisano, E. P. Geiduschek, T. Colbert, S. Hahn, 481

and J. A. Blanco. 1992. The role of the TATA-binding protein in the assembly and 482

function of the multisubunit yeast RNA polymerase III transcription factor, TFIIIB. 483

Cell 71:1055-1064. 484

27. Colbert, T., S. Lee, G. Schimmack, and S. Hahn. 1998. Architecture of protein and 485

DNA contacts within the TFIIIB-DNA complex. Mol Cell Biol 18:1682-1691. 486

28. Juo, Z. S., G. A. Kassavetis, J. Wang, E. P. Geiduschek, and P. B. Sigler. 2003. 487

Crystal structure of a transcription factor IIIB core interface ternary complex. 488

Nature 422:534-539. 489

29. Andrau, J. C., A. Sentenac, and M. Werner. 1999. Mutagenesis of yeast TFIIIB70 490

reveals C-terminal residues critical for interaction with TBP and C34. J Mol Biol 491

288:511-520. 492

30. Ferri, M. L., G. Peyroche, M. Siaut, O. Lefebvre, C. Carles, C. Conesa, and A. 493

Sentenac. 2000. A novel subunit of yeast RNA polymerase III interacts with the 494

TFIIB-related domain of TFIIIB70. Mol Cell Biol 20:488-495. 495

31. Moir, R. D., K. V. Puglia, and I. M. Willis. 2000. Interactions between the 496

tetratricopeptide repeat-containing transcription factor TFIIIC131 and its ligand, 497

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

TFIIIB70. Evidence for a conformational change in the complex. J Biol Chem 498

275:26591-26598. 499

32. Moir, R. D., K. V. Puglia, and I. M. Willis. 2002. Autoinhibition of TFIIIB70 binding 500

by the tetratricopeptide repeat-containing subunit of TFIIIC. J Biol Chem 277:694-501

701. 502

33. Moir, R. D., I. Sethy-Coraci, K. Puglia, M. D. Librizzi, and I. M. Willis. 1997. A 503

tetratricopeptide repeat mutation in yeast transcription factor IIIC131 (TFIIIC131) 504

facilitates recruitment of TFIIB-related factor TFIIIB70. Mol Cell Biol 17:7119-7125. 505

34. Brachmann, C. B., A. Davies, G. J. Cost, E. Caputo, J. Li, P. Hieter, and J. D. Boeke. 506

1998. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a 507

useful set of strains and plasmids for PCR-mediated gene disruption and other 508

applications. Yeast 14:115-132. 509

35. Wach, A., A. Brachat, R. Pohlmann, and P. Philippsen. 1994. New heterologous 510

modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. 511

Yeast 10:1793-1808. 512

36. Christianson, T. W., R. S. Sikorski, M. Dante, J. H. Shero, and P. Hieter. 1992. 513

Multifunctional yeast high-copy-number shuttle vectors. Gene 110:119-122. 514

37. Chin, J. W., T. A. Cropp, J. C. Anderson, M. Mukherji, Z. Zhang, and P. G. Schultz. 515

2003. An expanded eukaryotic genetic code. Science 301:964-967. 516

38. Sikorski, R. S., and P. Hieter. 1989. A system of shuttle vectors and yeast host 517

strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. 518

Genetics 122:19-27. 519

39. Wu, C. C., F. Herzog, S. Jennebach, Y. C. Lin, C. Y. Pai, R. Aebersold, P. Cramer, 520

and H. T. Chen. 2012. RNA polymerase III subunit architecture and implications 521

for open promoter complex formation. Proc Natl Acad Sci U S A 109:19232-19237. 522

40. Chen, H. T., L. Warfield, and S. Hahn. 2007. The positions of TFIIF and TFIIE in the 523

RNA polymerase II transcription preinitiation complex. Nat Struct Mol Biol 524

14:696-703. 525

41. Jasiak, A. J., K. J. Armache, B. Martens, R. P. Jansen, and P. Cramer. 2006. 526

Structural biology of RNA polymerase III: subcomplex C17/25 X-ray structure and 527

11 subunit enzyme model. Mol Cell 23:71-81. 528

42. Nikolov, D. B., H. Chen, E. D. Halay, A. A. Usheva, K. Hisatake, D. K. Lee, R. G. 529

Roeder, and S. K. Burley. 1995. Crystal structure of a TFIIB-TBP-TATA-element 530

ternary complex. Nature 377:119-128. 531

43. Werner, M., N. Chaussivert, I. M. Willis, and A. Sentenac. 1993. Interaction 532

between a complex of RNA polymerase III subunits and the 70-kDa component of 533

transcription factor IIIB. J Biol Chem 268:20721-20724. 534

44. Brun, I., A. Sentenac, and M. Werner. 1997. Dual role of the C34 subunit of RNA 535

polymerase III in transcription initiation. EMBO J 16:5730-5741. 536

45. Tate, J. J., J. Persinger, and B. Bartholomew. 1998. Survey of four different 537

photoreactive moieties for DNA photoaffinity labeling of yeast RNA polymerase 538

III transcription complexes. Nucleic acids research 26:1421-1426. 539

46. Tsai, F. T., and P. B. Sigler. 2000. Structural basis of preinitiation complex 540

assembly on human pol II promoters. Embo J 19:25-36. 541

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

47. Kassavetis, G. A., G. A. Letts, and E. P. Geiduschek. 1999. A minimal RNA 542

polymerase III transcription system. EMBO J 18:5042-5051. 543

48. Kassavetis, G. A., G. A. Letts, and E. P. Geiduschek. 2001. The RNA polymerase III 544

transcription initiation factor TFIIIB participates in two steps of promoter opening. 545

EMBO J 20:2823-2834. 546

49. Bartholomew, B., D. Durkovich, G. A. Kassavetis, and E. P. Geiduschek. 1993. 547

Orientation and topography of RNA polymerase III in transcription complexes. 548

Mol Cell Biol 13:942-952. 549

50. Bartholomew, B., G. A. Kassavetis, and E. P. Geiduschek. 1991. Two components 550

of Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) are stereospecifically 551

located upstream of a tRNA gene and interact with the second-largest subunit of 552

TFIIIC. Molecular and cellular biology 11:5181-5189. 553

554

555

556

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

Figure Legends 557

Figure 1. Brf1n BPA photo-crosslinking. (A) Schematic of Brf1 domain 558

architecture and summary of Brf1n BPA photo-crosslinking. Residue numbers for 559

the boundaries of individual subdomains are marked. NL, N-linker; CL-1&2, C-560

linker 1&2. BPA-substituted residues are color coded according to respective 561

cross-linked polypeptides indicated below the horizontal connecting lines. Lower 562

panel: models of the ribbon fold (left) and the Brf1c homology block II-TBP-DNA 563

complex (right). The magenta sphere in the ribbon model indicates the zinc ion. 564

TBP is displayed with the molecular surface model in light green. Others are 565

shown as backbone trace with Brf1c homology block II in brown, Brf1n cyclin 566

folds in orange, template DNA (TS) in dark blue, and non-template DNA (NTS) in 567

cyan. BPA-substituted residues with confirmed cross-linking targets are 568

highlighted with spheres. The hydroxyl radical cleavage site (Ala246±5aa) in 569

Brf1n by C34-FeBABE is indicated. (B) Western analysis of Brf1-C160 photo-570

cross-linking. BPA-substituted residues are indicated above the lanes. Brf1-C160 571

cross-linking was identified using anti-V5 antibodies (Brf1) (lanes 1-6) and 572

confirmed with anti-HA antibodies (C160) (lanes 7-12), respectively. Triangles are 573

placed next to the cross-linking gel bands. All cross-linking bands in subsequent 574

figures are marked by triangles. WCE, yeast whole cells extract; UV + or −, with 575

or without UV irradiation; WT, wild-type Brf1 with no BPA replacement; *, non-576

specific background band. (C) Brf1-C128 photo-crosslinking. Brf1-C128 cross-577

linking band was visualized with anti-V5 antibody (Brf1) (lanes 1-4) and 578

confirmed with anti-HA antibody (C128) (lanes 5-8). (D) C128-Brf1 photo-579

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

crosslinking. C128-Brf1 cross-linking band was visualized with anti-Myc antibody 580

(C128) (lanes 1-4) and confirmed with anti-Flag antibody (Brf1) (lanes 5-8). The 581

BPA position in C128 additionally cross-links to C82. (E) Brf1-TBP photo-582

crosslinking at BPA substituted residue Lys211 in the second cyclin fold of Brf1. 583

The cross-linked Brf1-TBP was probed with anti-V5 antibody (Brf1) (lane 1-4) and 584

confirmed by anti-TBP antibody (lane 5-8). 585

586

Figure 2. Brf1n cyclin folds interact with C34. (A) Brf1-C34 photo-cross-linking 587

from BPA-substitution at residue Tyr99 of Brf1. The cross-linking was visualized 588

with an antibody against V5 (Brf1) (left panel) and was verified with C34 589

antiserum (right panel). Cross-linking bands are marked with triangles. The 590

bands marked with asterisks (*) are background bands, which appear to be UV-591

specific. (B) Schematic of C34 domain architecture. As highlighted in the 592

sequence of the connecting region between WH2 and 3 domains, Asp171 and 593

Glu173 mutations affect transcription initiation. (C) Western analysis of C34-Brf1 594

cross-linking. BPA-substitution is at the residue Glu169 of C34. Crosslink was 595

visualized by probing with anti-V5 antibody (C34) (left panel) and the identity of 596

the C34-Brf1 cross-linking band was verified by probing with Brf1 antiserum (right 597

panel). Asterisk (*) marks a non-specific background band. (D) Determination of 598

C34-FeBABE hydroxyl radical cleavage site in Brf1. The hydroxyl radical 599

cleavage peptide fragment is revealed in the Western blot analysis with anti-Flag 600

antibody, and the cleavage site is determined to be in the cyclin fold repeat 601

subdomain of Brf1 as indicated. The non-cysteine (nonCys) mutant of C34 does 602

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

not contain any cysteine residue for FeBABE conjugation and served as the 603

negative control. Non-specific bands are marked with asterisks. 604

605

Figure 3. BPA photo-cross-linking in Brf1c. (A) Summary of Brf1c BPA photo-606

crosslinking. (B) Western analysis of cross-linking for BPA-substitution at His473 607

of Brf1. The cross-linking results were probed with anti-V5 antibody (Brf1) (left 608

panel), anti-Flag antibody (TBP) (middle panel), and anti-Bdp1 antibody (right 609

panel). The cross-linking bands are marked with triangles. A slight upper mobility 610

shift for the Brf1-TBP cross-link in the middle panel was caused by the use of 611

Flag epitope tagging in TBP. (C) Western analysis of Brf1-Bdp1 cross-linking at 612

Lys319 and Lys335 in the C-linker 1 (CL-1) region. The Western blot was probed 613

with anti-Myc antibody for Myc-epitope tagged Brf1 and anti-Bdp1 antibody to 614

confirm the Bdp1 polypeptide in the cross-linked fusion (lanes 8 and 10). (D) 615

Western analysis of Brf1-TBP cross-linking for BPA-substitution at residues 616

Ser420, Gln424 and Asn418 of Brf1c. 617

618

Figure 4. Mutational analysis of Brf1c homology block I and C-linker 1. (A) Cell 619

growth phenotype was analyzed by the serial dilution spot assay. Both 620

Leu332Glu and del (Glu331-Tyr340) mutants showed slower growth at 37oC. (B) 621

Western blot analysis of co-immunoprecipitation for Brf1 Leu332Glu and del 622

(Glu331-Tyr340) mutants. Co-IP was conducted with anti-Flag agarose to 623

precipitate Flag-tagged Bdp1 and co-immune precipitated polypeptides were 624

probed with respective antibodies indicated on the left. (C) IP-anti-Flag results 625

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

are quantified and plotted with WT signals set to 1. Errors bars indicate s.e.m. 626

from four independent experiments. (D) Transcription activity of Brf1 mutants. As 627

indicated, WCEs from wild-type (WT) or mutant yeast strains were used in the in 628

vitro transcription assay. The autoradiograms show the SUP4 pre-tRNA transcript 629

(upper panel) and SnR6 transcript (lower panel). rBrf1, recombinant wild-type 630

Brf1. (E) Immobilized template analysis. Proteins in the isolated Pol III PICs from 631

the IMT assay were probed with antibodies as indicated on the left. The relative 632

protein levels for Brf1 and Bdp1 are listed below each gel band. 633

634

Figure 5. Model of the Pol III open promoter complex. (A) The structural model 635

contains Pol III, Brf1, TBP, and open promoter DNA based on the Pol II-TFIIB-636

TBP open promoter complex (19, 20) and the Brf1c homology block II-TBP-DNA 637

structure (28). Subdomains of Brf1 are displayed with the backbone trace model 638

and are color-coded: Brf1n cyclin repeats in orange and Brf1c homology block II 639

in brown. The molecular surface model of TBP is colored pale green. The Pol III 640

core structure is shown as the white molecular surface, and the magenta sphere 641

in the active center denotes the magnesium ion. Pol III-specific subunits are 642

displayed as follows: C34 WH1 and WH2, magenta backbone trace; C82, tan 643

molecular surface; C37/53 subcomplex, light blue molecular surface. DNA is 644

represented by the phosphate backbone trace with the template strand in blue 645

and non-template strand in cyan. Positions of DNA base-pairs -38/-39 and -21 on 646

the non-template strand (relative to the transcription start site as +1) are also 647

indicated. The localization for the WH3 domain of C34 is indicated as the dashed 648

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

oval line in black. The atomic coordinate file for the Pol III PIC model is available 649

upon request. (B) Same as in (A) with rotation as indicated. The molecular 650

surfaces for Pol III core, C37/53 subcomplex, and C82 are semi-transparent. As 651

highlighted with the spheres in the Brf1 cyclin repeats model, Glu98 and Tyr99 652

provide Brf1-C34 BPA-cross-linking and Ala246 (±5aa) is the hydroxyl radical 653

cleavage site by the FeBABE-conjugated C34. The dashed circle represents the 654

potential localization for the connecting region between the WH2 and WH3 655

domains of C34. 656

657

658

on March 4, 2018 by guesthttp://mcb.asm.org/Downloaded from

BRF1 120 MCB.00910 13.full

Navigation menu

Versions of this User Manual:

Views

Navigation