RNA Polymerase III Subunit Architecture and Implications for Open Promoter Complex Formation

RNA polymerase III subunit architecture and implications for open promoter complex formation

Chih-Chien Wua,b, Franz Herzogc, Stefan Jennebachd, Yu-Chun Lina, Chih-Yu Paia, Ruedi Aebersoldc,e, Patrick Cramerd, and Hung-Ta Chena,1

aInstitute of Molecular Biology, Academia Sinica, Taipei, Taiwan 115, Republic of China; bDepartment of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan 112, Republic of China; cDepartment of Biology, Institute of Molecular Systems Biology, Eidgenössiche Technische Hochschule Zurich, CH-8093 Zurich, Switzerland; dGene Center and Department of Biochemistry, Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, 81377 Munich, Germany; and eFaculty of Science, University of Zurich, CH-8006 Zurich, Switzerland

Edited by E. Peter Geiduschek, University of California at San Diego, La Jolla, CA, and approved October 3, 2012 (received for review July 11, 2012) Transcription initiation by eukaryotic RNA polymerase (Pol) III relies subcomplexes and their contacts with the 12-subunit polymerase on the TFIIE-related subcomplex C82/34/31. Here we combine cross- core. The C37/53 dimerization module was positioned into the linking and hydroxyl radical probing to position the C82/34/31 electron density adjacent to the lobe domain of the C128 subunit subcomplex around the Pol III active center cleft. The extended on one side of the polymerase cleft, similar to the localization of winged helix (WH) domains 1 and 4 of C82 localize to the polymerase the TFIIF dimerization module and the A49/34.5 dimerization domains clamp head and clamp core, respectively, and the two WH module on Pol II and Pol I, respectively (23–25). This structural domains of C34 span the polymerase cleft from the coiled-coil region arrangement was supported by site-specific photo-cross-linking of the clamp to the protrusion. The WH domains of C82 and C34 and hydroxyl radical probing analyses that provided direct posi- apparently cooperate with other mobile regions flanking the cleft tional mapping for protein interactions (8). during promoter DNA binding, opening, and loading. Together with The C82/34/31 subcomplex was proposed to occupy a large published data, our results complete the subunit architecture of Pol electron density region between the clamp and the stalk of the III and indicate that all TFIIE-related components of eukaryotic and polymerase core, on the side of the cleft opposite the lobe (20, 24, archaeal transcription systems adopt an evolutionarily conserved 25). By fitting the crystal structure of hRPC62 into the yeast Pol location in the upper part of the cleft that supports their functions in III cryo-EM envelope, Lefèvre et al. proposed a model in which open promoter complex formation and stabilization. eWH2, eWH3, and the coiled-coil region are positioned on the clamp and eWH1 and eWH4 are exposed to solvent for single- NA polymerase III (Pol III) is the largest eukaryotic RNA stranded DNA binding (20). An alternative orientation was Rpolymerase, composed of 17 subunits with a total molecular proposed by Fernández-Tornero et al. that placed eWH4 closer weight of ∼0.7 MDa (1). Pol III synthesizes certain small untranslated to the stalk of the polymerase core (26). Thus, it currently RNAs (e.g., tRNAs, 5S rRNA, U6 snRNA, and 7SL RNA) involved remains unclear how C82 and the C82/34/31 subcomplex are po- in RNA processing and translation and in protein translocation (2, 3). sitioned on Pol III. To resolve this issue, protein–protein inter- Human Pol III mutations have been implicated in a neurodegener- actions between the C82/34/31 subcomplex and the Pol III core ative disorder, hypomyelinating leukodystrophy (4–6). must be mapped. fi The three eukaryotic RNA polymerases share a similar 12-sub- Here we site-speci cally inserted photo-cross-linking amino fi unit core as represented by the X-ray structure of yeast Pol II (1). In acids into C160 and C82 and identi ed protein regions involved in addition to the core, Pol III contains five specific subunits forming C82-polymerase core interactions by site-directed hydroxyl radical two subcomplexes, C37/53 and C82/34/31. The C37/53 subcomplex probing. Our data reveal that C82 is anchored on the Pol III clamp participates in promoter opening, transcription termination, and via its domains eWH1 and eWH4. Mass spectrometric (MS) – polymerase reinitiation (7–9). The C34 subunit of the C82/34/31 analysis of peptides obtained by tryptic digestion of lysine lysine cross-linked Pol III is consistent with these results, and additionally subcomplex plays a role in open complex formation and in recruiting – Pol III to the preinitiation complex (PIC) through interaction with locates the coiled-coil stalk of C82 at the polymerase clamp stalk TFIIB-related factor 1 (Brf1) (10–13). The human RPC62/39/32 junction near the ABC27 (Rpb5) and ABC23 (Rpb6) subunits of subcomplex, homologous to the yeast C82/34/31 complex, is disso- the Pol III core. Cross-linking-MS analysis also positions the two adjacent WH domains of C34 above the active site cleft. The ciable and required for promoter-specificinitiation(11). The two Pol III-specific subcomplexes contain structural domains resulting detailed interaction map completes the subunit architecture of Pol III and provides insights into the function of the C82/ homologous to domains in the Pol II transcription factors TFIIF 34/31 subcomplex during transcription initiation. and TFIIE, including the TFIIF-related dimerization module in the C37/53 subcomplex and several TFIIE-related winged helix (WH) Results domains in subunits C82 and C34 (14–20). TFIIE is composed of α β C82 and C34 Reside on the Polymerase Clamp. To elucidate protein two subunits, Tfa1 and Tfa2, in yeast, or TFIIE and TFIIE in interactions for the Pol III active center cleft, we replaced surface humans. Whereas Tfa1 bears an extended WH (eWH) domain in residues of the C160 clamp core and clamp head domains with the its N-terminal region, Tfa2 has two adjacent WH domains (21, 22). nonnatural amino acid photo-cross-linker p-benzoyl-L-phenylala- Two adjacent Tfa2-related WH domains are also present in the C34 nine (BPA). The sites were chosen at presumed interfaces of the subunit and its human homolog RPC39. Pol I contains the A49/34.5 subcomplex that features a TFIIF-like dimerization module and

a tandem WH domain that contains two Tfa2-like WH folds in the Author contributions: C.-C.W., S.J., R.A., P.C., and H.-T.C. designed research; C.-C.W., F.H., C-terminal region of the A49 subunit (18). The crystal structure of S.J., Y.-C.L., and C.-Y.P. performed research; C.-C.W., F.H., and S.J. analyzed data; and C.-C.W., the human C82 homolog RPC62 contains four Tfa1-like eWH P.C., and H.-T.C. wrote the paper. domains (eWH1–4) that are structurally organized around a C- The authors declare no conﬂict of interest. terminal coiled-coil stalk (20). A Tfa1-related eWH domain is also This article is a PNAS Direct Submission. present in the archaeal transcription factor E (TFE) (22). 1To whom correspondence should be addressed. E-mail: [email protected]. Recent cryo-EM studies on the overall structural organization This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of the yeast Pol III suggested locations of the Pol III-speciﬁc 1073/pnas.1211665109/-/DCSupplemental.

19232–19237 | PNAS | November 20, 2012 | vol. 109 | no. 47 www.pnas.org/cgi/doi/10.1073/pnas.1211665109 Downloaded by guest on October 1, 2021 1C; summarized in Table S1). Additionally, we found that four consecutive residues (Asp304 to Ile307) in the coiled-coil motif of the clamp core yielded a cross-link to C82 and another cross-link to C34 (Fig. 1 B and C). Cross-linking to C34 is also located from Ala206 to Pro209 in the clamp head, and His207 and Asn208 shows simultaneous cross-linking to C82 (Fig. 1C). Taken together, our cross-linking results indicate that the C82/34/31 subcomplex is mainly anchored on the clamp domain of C160 via its C82 subunit, and that C34 contributes to contacts along the rim of the clamp.

C82 Is Involved in a Protein Interaction Network. Our C160 cross- linking results indicate that C82 and C34 reside on the clamp domain but did not sufﬁce to position the domains of these subunits on the Pol III core. We therefore conducted an extensive BPA cross-linking analysis for C82 that is summarized in Fig. S1A and Table S2. The data reveal C82–C31 and C82–C34 intrasubcomplex cross-links. Representative C82–C31 cross-links are shown in Fig. S1B. C31-cross-linked residues mostly lie within the C82 eWH1 and eWH2 domains, and one resides in its coiled-coil domain (Fig. S1A). By contrast, C34-cross-linked residues are scattered in eWH3 and eWH4, except for one in eWH2 (Fig. S1 A and C). This C82–C34 binding interface is consistent with the previously proposed C82–C34 interaction obtained by protein pull-down experiments (20). C82 shows distinct interaction sur- faces for C31 and C34, which suggests a mainly linear connectivity for the three subunits, consistent with a previous mass spectrometric analysis (28). Concerning the interaction of C82 with the Pol III core, residues in eWH1, eWH4, the ﬁrst insertion of eWH3, and the C- terminal coiled-coil stalk yield C82–C160 cross-links (Fig. 2A and BIOCHEMISTRY

Fig. 1. C160 BPA photo-cross-linking indicates C82 and C34 reside on the C160 clamp domain. (A) Western blot of C82–C160 cross-links. Amino acid positions in C160 clamp replaced by BPA are indicated above the lanes. C160 and cross-linking bands were visualized by probing with anti-Myc antibody (C160-Myc, Left), and the cross-linking bands are conﬁrmed to be C160–C82 fusion by probing Flag-tagged C82 (Right). C160 WCE, C160 whole-cell extract; UV + or −, with or without UV irradiation; WT, wild-type C160 with no BPA replacement. (B) C34-C160 cross-links. C160 and C34–C160 cross-linking bands were visualized by anti-Myc antibody (Left) and anti-HA antibody to reveal N-terminally HA-tagged C34 (Right). As indicated, these C160-BPA derivatives also generate simultaneous C82-C160 cross-links. (C) Positions in C82– and C34–C160 cross-links in polymerase clamp. The yeast Pol III core- C37/53 surface model is colored white. Magenta sphere: magnesium ion in the polymerase active site. As indicated, C160 residues yielding C82 and C34 cross-links are shown in red and brown, respectively. The residues cross- linking to both C82 and C34 are colored mauve.

Pol III core with the C82/34/31 subcomplex based on previous Pol III models (24, 25, 27). We conducted photo-cross-linking by using yeast whole-cell extracts containing C160-BPA derivatives in the immobilized template assay to probe potential protein targets within the Pol III PIC. C160-cross-linked polypeptides were found Fig. 2. C82 BPA photo-cross-linking. (A) Western blot of C82–C160 cross- to have estimated molecular weights of 80 and 30 kDa, corre- linking. BPA-substituted residues in C82 are indicated above. C82 and cross- linking fusion bands are revealed by anti-V5 antibody (Left). The Flag-epitope sponding approximately to the sizes of C82 and C34 or C31. We tagged C160 in the cross-linking bands is revealed by anti-Flag antibody epitope-tagged the possible candidates and confirmed the cross- (Right). Asterisks indicate unidentified cross-linked polypeptides. (B) Amino linked polypeptides by identifying the epitope-containing cross- acid positions cross-linked to C160 in the yeast C82 model. The yeast C82 linked fusion in Western blot analysis, as described (8). Fig. 1A homology model (displayed as the ribbon model of Cα trace) is generated by shows Western blot analysis revealing C160–C82 cross-links the Modeler program (42) using the human RPC62 structure (PDB 2XUB) (20) fi as the template. The dashed line indicates the missing residues (aa547–567 in identi ed by antibodies against epitope-tagged C160 and C82 C82) in the eWH4. C82 structural domains are colored in red (eWH1), green (anti-Myc for C160 and anti-Flag for C82). (eWH2), blue (eWH3), orange (eWH4), and salmon (coiled-coil). The color This analysis reveals C82 cross-linking to C160 residues spanning scheme is used for all following figures. Residues cross-linking to C160 and from the upstream clamp core to the downstream clamp head (Fig. C25 are highlighted with cyan and olive spheres, respectively.

Wu et al. PNAS | November 20, 2012 | vol. 109 | no. 47 | 19233 Downloaded by guest on October 1, 2021 Fig. S1A). By mapping the positions cross-linked to C160 on the in Fig. S2A). The reconstituted subcomplex was subsequently ti- yeast C82 homology model based on the human RPC62 struc- trated into a C82 mutant yeast whole-cell extract (Fig. S2B) for Pol ture, we reveal a protein interaction surface for C160 binding III PIC formation with the immobilized template assay. All that comprised eWH1, eWH4, and the coiled-coil stalk of C82 FeBABE-tethered C82 variants are shown to restore transcription (Fig. 2B). In addition to C160 and intrasubcomplex cross-links, activity (Fig. S2C). Fig. 3A shows hydroxyl radical cleavage of C160 Leu620-BPA near the tip of C82 coiled-coil showed simulta- from individual C82 variants with FeBABE tethered at Cys posi- neous cross-links to C25 of the polymerase stalk and C53 of the tions in the eWH1 and eWH4 domains. By mapping the cleavage C37/53 subcomplex (Fig. S1 A and D). This result is consistent sites to the Pol III core structural model, we found that eWH1 and with our previous data indicating that the C53 N terminus eWH4 are respectively localized to the clamp head and clamp core extends toward the polymerase stalk to allow for cross-linking to regions (Fig. 3B; summarized in Table S3). C82 and C25 (8). Cross-Linking-MS Analysis of Pol III. Although BPA cross-linking and C82 eWH1 and eWH4 Reside on the Polymerase Clamp. To more hydroxyl radical probing localized C82 eWH1/4 on the C160 precisely localize C82 domains on the C160 clamp domain, we clamp, additional structural restraints are required to further de- applied directed hydroxyl radical probing analysis with C82- fine the interface between C82 and the Pol III core for structural FeBABE conjugates. We purified a series of recombinant C82 modeling. To this end, we used lysine–lysine cross-linking followed single-cysteine variants to tether the ·OH-generating FeBABE by MS identification of the cross-linked peptides as recently de- reagent at defined Cys positions. These C82-FeBABE conjugates scribed for Pol I (23). A purified endogenous Pol III sample was were combined with purified recombinant C34 and C31 to re- subjected to cross-linking with disuccinimidyl suberate (DSS), constitute the C82/34/31 subcomplex (purified proteins are shown a reagent that reacts with primary amines present in lysine side

Fig. 3. Localization of C82 on the C160 clamp. (A) Directed hydroxyl radical cleavage of C160. Western blots of C160 cleavage from tethered FeBABE in C82 eWH1 and eWH4 are respectively shown in the left and right panels. C-terminally HA-tagged C160 fragments are revealed by anti-HA antibody. FeBABE positions in C82 are indicated above the lanes. Cleavage fragments in the lower-molecular-weight range are displayed with signals ampliﬁed (Bottom Right). Red asterisks mark identiﬁed cleavage fragments. Approximate locations of cleavage sites in C160 are indicated. (B) C82-Pol III core model. C82 (ribbon model) is manually docked as a rigid body on the Pol III core model (white surface model) based on directed hydroxyl radical cleavage and cross-linking-MS analyses. Hydroxyl radical cleavage regions in C160 clamp are highlighted in black covering 11 residues centered at the deduced cut site. (C) Cross-linked lysine pairs between C82 coiled-coil and subunits of the Pol III core. Green lines connect cross-linked lysine pairs from C82 (Lys594) to ABC27 (Lys171) and ABC23 (Lys72). The BPA substitution at C82 Leu620, highlighted as an olive sphere, cross-links to C25.

19234 | www.pnas.org/cgi/doi/10.1073/pnas.1211665109 Wu et al. Downloaded by guest on October 1, 2021 chain and protein N terminus. DSS cross-linking of Pol III was monitored by tryptic digestion and MS analysis. A complete list of cross-linked lysines and their corresponding peptide pairs is provided in Table S4. The cross-linking identiﬁed linkage pairs that validate a previous model of the Pol III core that was based on the Pol II structure (27, 29). In addition, distance restraints position the Pol III-speciﬁc subcomplex C37/53 on the Pol III core, supporting the previously proposed model for the C37/53 dimerization module on the lobe domain of C128 (Fig. S3 A and B) (8, 24, 25, 29). Cross-links also reveal the location of the C-terminal region of C37 in the active center cleft (Fig. S3B) and are generally consistent with contacts between the N-terminal region of C53 and the coiled-coil domain of C82 and the C17/25 stalk subcomplex (Fig. S1D), supporting previous BPA cross-linking and FeBABE analyses (8).

Model for C82 Binding to the Pol III Core. A schematic summary for C82 lysine–lysine cross-links is depicted in Fig. S4. We observed intramolecular cross-links to support the yeast C82 homology model based on the human RPC62 structure (29). In combination with the C82-FeBABE hydroxyl radical analysis (see above), we used intermolecular cross-links between C82 coiled-coil and the Pol III core subunits ABC27 (Rpb6) and ABC23 (Rpb5) to manually dock the C82 model on the Pol III core (Fig. 3 B and C). In the resulting model, the C82 domains eWH1 and eWH4 are located on the clamp head and clamp core in C160, respectively, whereas the C82 coiled-coil extends toward subunits ABC27 and ABC23. C82 coiled-coil is also positioned adjacent to the Pol III stalk, consistent with BPA cross-linking between the tip of C82 coiled-coil and C25 of the Pol III stalk (Fig. 3C). Further, the Fig. 4. Association of the C82/34/31 subcomplex with the Pol III core. (A) model satisfies all BPA photo-cross-linking results for C160 and Western blot analyses of coimmunoprecipitation and Pol III PIC formation B C with yeast whole-cell extract containing a C160 mutation. Coimmunopreci- C82 (Fig. S4 and ). pitation was conducted with anti-Flag agarose to immobilize Flag-epitope- To validate our model of C82 on the Pol III core, we generated tagged C82 (Flag C82 IP; Upper Left). Pol III PIC formation assay was con- yeast strains containing mutations in the predicted C160 clamp– ducted with the immobilized SUP4 tDNA (Upper Middle). The level of a TFIIIC C82 interface and tested their in vivo and in vitro phenotypes. We subunit Tfc4 in the PIC is also shown. As indicated, whole-cell extracts con- obtained a deletion mutant C160 Δ (35–41) that shows a slow- taining wild-type (WT) or internal deletion Δ (35–41) C160 were used. Single- BIOCHEMISTRY growth phenotype at elevated temperature but no significant round in vitro transcription of the isolated Pol III PIC was assayed (Lower). (B) change in the cellular C160 protein level (Fig. 4A, Input). In an Immunoblot and in vitro transcription signals were quantified from three immunoprecipitation assay by immobilizing C82, the C34 and C31 independent experiments with WT signals set to 1. Error bars indicate SD. (C) subunits of the subcomplex are coimmunoprecipitated, whereas Western blot analyses of coimmunoprecipitation and Pol III PIC formation with yeast whole-cell extracts containing C82 mutations. Similar to the the mutant C160 and another Pol III subunit C53 are partially A analyses in A, whole-cell extracts containing WT or the indicated mutations in dissociated (Fig. 4 , Flag C82 IP). By analyzing PIC formation on C82 eWH4 were used in coimmunoprecipitation, PIC formation, and in vitro immobilized SUP4 tDNA, C160 Δ (35–41) mutant showed ∼50% transcription analyses. Different from the analysis in A, immune pull-down reduction in Pol III recruitment (Fig. 4 A, PIC and B). In agreement was conducted by immobilizing Flag-epitope-tagged C128, and both single- with the defect in Pol III recruitment, we observed a concomitant round (SR) and multiple-round (MR) transcription were assayed (Lower). An decrease in transcription initiation for the C160 mutant based on asterisk indicates the read-through transcripts. (D) The results for PIC for- the single-round transcription analysis (Fig. 4 ALowerand B). mation and SR transcription from C are quantified and plotted with WT Further mutagenesis study on C82 allowed us to isolate two signals set to 1. Errors bars indicate SD from three independent experiments. internal deletion mutations in C82 eWH4 that confer temperature-sensitive growth defect. Coimmunoprecipitation analysis by for WH2, the adjacent WH1 is localized on the rim of the C160 immobilizing the Pol III core subunit C128 indicates that these clamp head based on five intrasubunit cross-links with WH2 and mutations lead to dissociation of C82 and C34 from the Pol III a single intersubunit cross-link with C82 eWH4. The positions for core (Fig. 4C). This association defect severely compromises Pol WH1 and WH2 are consistent with the localization of C34 on the III recruitment and causes complete loss of in vitro transcription C D polymerase clamp based on the BPA cross-linking results for C160 activity (Fig. 4 and ), in agreement with a role for the human C C82/34/31 homolog in transcription initiation (11). Taken to- (Figs. 5 and 1 ). Further, several cross-link pairs are observed be- gether, our mutational and functional analyses support the tween the C34 WH domains and the C37 C-terminal region, sup- proposed model for C82 binding to the Pol III core. porting the previously described location for the C37 C-terminal region near the Pol III active site (8) (Fig. S3C). C34 Bridges the Active Center Cleft. In addition to C82 positioning, Intersubunit cross-linked peptide pairs within the C82/34/31 we used lysine–lysine cross-linked peptide pairs to model the posi- subcomplex further suggest the positions of the C34 C-terminal tions for C34 WH domains. Homology models for C34 WH1 and region and C31 on C82. The C34 C-terminal region contacts C82 WH2 were generated based on the WH structures in mouse and eWH3, and C31 is positioned on the surface formed by C82 eWH1, human RPC39, respectively (Protein Data Bank accession numbers eWH2, and the coiled-coil stalk (Fig. S5 A and B). The C31 posi- 2DK8 and 2DK5). Based on a total of eight intersubunit cross-links, tion is also supported by cross-links with the C160 clamp head, the we docked the C34 WH2 model above the Pol III active center cleft C17/25 stalk, and ABC27 (Fig. S5A). Furthermore, the distinct to contact C128 protrusion, C160 clamp coiled-coil, and the eWH4 location of C34 and C31 and no observable cross-links between domain of the previously modeled C82 (Fig. 5). With the positioning C34 and C31 are in perfect agreement with the linear connectivity

Wu et al. PNAS | November 20, 2012 | vol. 109 | no. 47 | 19235 Downloaded by guest on October 1, 2021 initial strand separation and/or subsequent bubble stabilization. In accordance with this proposed mechanism, two amino acid residues in the WH2 domain of C34 that were previously reported to be involved in DNA binding and open complex formation (12, 20) may interact with the upstream edge of the transcription bubble (Fig. 6B). Our data and published results thus converge on a model that C34 is positioned over the active center cleft to stabilize the DNA bubble and aid in open complex formation. This model relies on C34 mobility, because promoter DNA must first be loaded into the cleft during the transition from the closed to the open complex, and this requires transient displacement of C34 from the location observed here. Weak evidence to support C34 mobility comes from the slightly different locations for C34 domains in Pol III and Pol III-DNA models derived by EM (20, 24, 25) but merits further exploration. C82 and C34 may cooperate with the TFIIB-related factor Brf1 Fig. 5. Model of C34 WH domains in Pol III based on lysine–lysine cross-links. to stimulate open complex formation. In our Pol III model, C82 Model of C34 and C82 on the Pol III core is shown on the left. C34 WH1 and WH2 eWH4 and C34 WH domains are in the vicinity of the polymerase ribbon models are colored purple. In the Pol III cleft view on the right, green lines clamp coiled-coil (Fig. 6A and Fig. S6C). In Pol II, the clamp connect intrasubunit and intersubunit cross-linked lysines (purple spheres with coiled-coil interacts with a region in TFIIB, the B-linker, which labels indicating subunit names and residue numbers). Brown patches on C160 was also implicated in DNA opening (37, 39). During Pol III clamp indicate the BPA-substituted residues in C160 that cross-linked to C34. initiation, Brf1 likely contacts C82 and C34, because C34 was shown to bind Brf1 during Pol III recruitment (10, 40). Situated at the upstream edge of the DNA bubble (Fig. 6B), C82, C34, and for the C82/34/31 subcomplex indicated by our C82 BPA-cross- – linking results and a previous MS study (28). Taken together, the Brf1 may provide essential protein DNA interactions for initial σ multiple experimental approaches have enabled us to arrive at the spontaneous strand separation, similar to the function of 2/3 in complete domain architecture for the 17-subunit Pol III enzyme. the bacterial system (30, 37, 39, 41). In particular, the C34 WH domains and C82 eWH4 could stabilize the emerging non- Discussion template DNA strand, whereas the template DNA strand slips fl Here we present the complete subunit domain architecture of Pol into the cleft and binds to its oor (31, 32, 37). On the downstream III, the largest eukaryotic RNA polymerase, derived from an in- side of the DNA bubble, C82 eWH1 could contact downstream A tegrative approach that combines nonnatural amino acid photo- DNA (Fig. 6 ). Further, the C37 C-terminal domain and another cross-linking, directed hydroxyl-radical probing, MS analysis of transcription factor, Bdp1, may also bind the DNA bubble (8, 33), C lysine–lysine chemically cross-linked peptides, and molecular because they localize near the Pol III active center (Fig. S3 ). modeling based on X-ray crystallographic structures. This work Our results suggest an evolutionary conservation of the mech- identified subunit–subunit interfaces within Pol III, and in par- anisms used by TFIIE-related components in the three eukaryotic ticular revealed the location of subcomplex C82/34/31 around the and the archaeal transcription initiation machineries. In Pol II, Pol III clamp and cleft. The C82 domains eWH1 and eWH4 are localized on the clamp head and clamp core, respectively, whereas the C82 C-terminal coiled-coil domain binds between the polymerase clamp and stalk and reaches near the subunits ABC27 and ABC23. This model differs from previously proposed relative orientations of the C82 human homolog structure on Pol III based on electron microscopic data alone (20, 24, 25). The C34 domain WH2 resides between the C160 coiled-coil motif on one side of the polymerase cleft and the C128 protrusion domain on the other side, whereas the C34 domain WH1 localizes to the clamp head. C31 is positioned adjacent to the clamp head and contacts C82 eWH1, eWH2, and its coiled-coil domain, ABC27, and the C17/25 stalk. The subunit domain model of Pol III derived here is generally consistent with the molecular envelope of the Pol III EM structure (24, 25) (Fig. S6 A and B). To derive implications for the function of C82 and C34 in Pol III initiation, we extended the Pol III model to a model of the Pol III open promoter complex (Fig. 6). The open promoter complex Fig. 6. Pol III architecture and open promoter complex. (A) Model for the model additionally contains the TATA box-binding protein TBP Pol III open promoter complex. The model of Pol III-Brf1-TBP-DNA open and the TFIIB-related factor Brf1 based on previous analysis of promoter complex was built based on the Pol II-TFIIB-TBP open complex (37) the related Pol II open complex (37). This model is consistent with and the Brf1-TBP-DNA structure (43). Template and nontemplate DNA previously reported protein–DNA cross-linking results, in which strands are in blue and cyan, respectively. The Pol III core-C37/53 model is C34 was cross-linked to the farthest upstream position at −21 with displayed as the white surface model. Ribbon models for Brf1 and TBP are respect to the transcription start +1, and C82 was cross-linked colored yellow-green and brown, respectively. The C82 surface model is − − + colored according to the domain color scheme as above, and both C34 WH both upstream at positions 8/ 7 and downstream at 11 (Fig. – 6B) (13, 38). The C34 location correlates well with its double- surface models are colored purple. (B) Protein DNA organization in the Pol III active center. The Pol III open promoter complex in A is displayed with stranded DNA binding ability and its functional roles in DNA fl different orientation to view the Pol III active center. The Pol III core is melting (12, 20). With their positions anking the DNA bubble of semitransparent and C34 is changed to the ribbon model. Lys135 and Lys138 the Pol III open complex model (Fig. 6B), the C34 WH domains in C34 WH2 are highlighted as black spheres. These two amino acids are potentially contribute their DNA binding residues to function in required for DNA binding and open complex formation (12, 20).

19236 | www.pnas.org/cgi/doi/10.1073/pnas.1211665109 Wu et al. Downloaded by guest on October 1, 2021 TFIIE has been mapped to the coiled-coil motif and the clamp transcription, hydroxyl radical cleavage, and cross-linking-MS analysis. head of Rpb1 (34, 35). In Pol I, the TFIIE-related A49 tandem Immobilized DNA templates containing either SUP4 tRNA or U6 snRNA winged-helix domain occupies a position above the active center genes were prepared as previously described (8). The Pol III core-C37/53 cleft similar to C34 (23). The archaeal TFIIE-like factor TFE was model was built as previously described (8). also localized near the clamp coiled-coil (36). Thus, all TFIIE- ACKNOWLEDGMENTS. We thank Cheng-Feng Lo and Jin-Cheng Lee for related components share the location within the upper active assistance with cloning and development of puriﬁcation strategies for C82, center cleft and a function in promoting formation and stabiliza- C34, and C31. This work was supported by National Science Council Grant tion of the transcription bubble. 100-2311-B-001-013-MY3 and a Career Development Award (to H.-T.C.) from Academia Sinica. P.C. was supported by Deutsche Forschungsgemeinschaft Materials and Methods Grant SFB646, TR5, Nanosystems Initiative Munich, the BioImaging Network, a European Research Council Advanced Grant, and a Jung-Stiftung grant. Detailed descriptions are available in SI Materials and Methods for plasmids F.H. is supported by a European Molecular Biology Organization long-term and yeast strains, BPA photo-cross-linking, preparation of proteins, in vitro fellowship and by European Commission Grant FP7-PEOPLE-IEF.

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