Structural Biology of RNA Polymerase III: Subcomplex C17/25 X-Ray Structure and 11 Subunit Enzyme Model

Jasiak, Anna J.; Armache, Karim‐Jean; Märtens, Birgit; Jansen, Ralf‐Peter; Cramer, Patrick

doi:10.1016/j.molcel.2006.05.013

Cited by 86 publications

(93 citation statements)

References 53 publications

(76 reference statements)

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“…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.…”

Section: C82 and C34supporting

confidence: 83%

“…To elucidate protein interactions for the Pol III active center cleft, we replaced surface residues of the C160 clamp core and clamp head domains with the nonnatural amino acid photo-cross-linker p-benzoyl-L-phenylalanine (BPA). The sites were chosen at presumed interfaces of the 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.…”

Section: C82 and C34mentioning

confidence: 99%

See 1 more Smart Citation

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

Herzog²,

Jennebach

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Transcription initiation by eukaryotic RNA polymerase (Pol) III relies on the TFIIE-related subcomplex C82/34/31. Here we combine crosslinking and hydroxyl radical probing to position the C82/34/31 subcomplex around the Pol III active center cleft. The extended winged helix (WH) domains 1 and 4 of C82 localize to the polymerase domains clamp head and clamp core, respectively, and the two WH domains of C34 span the polymerase cleft from the coiled-coil region of the clamp to the protrusion. The WH domains of C82 and C34 apparently cooperate with other mobile regions flanking the cleft during promoter DNA binding, opening, and loading. Together with published data, our results complete the subunit architecture of Pol III and indicate that all TFIIE-related components of eukaryotic and archaeal transcription systems adopt an evolutionarily conserved location in the upper part of the cleft that supports their functions in open promoter complex formation and stabilization. R NA polymerase III (Pol III) is the largest eukaryotic RNA polymerase, composed of 17 subunits with a total molecular weight of ∼0.7 MDa (1). Pol III synthesizes certain small untranslated RNAs (e.g., tRNAs, 5S rRNA, U6 snRNA, and 7SL RNA) involved in RNA processing and translation and in protein translocation (2, 3). Human Pol III mutations have been implicated in a neurodegenerative disorder, hypomyelinating leukodystrophy (4-6).The three eukaryotic RNA polymerases share a similar 12-subunit core as represented by the X-ray structure of yeast Pol II (1). In addition to the core, Pol III contains five specific subunits forming two subcomplexes, C37/53 and C82/34/31. The C37/53 subcomplex participates in promoter opening, transcription termination, and polymerase reinitiation (7-9). The C34 subunit of the C82/34/31 subcomplex plays a role in open complex formation and in recruiting Pol III to the preinitiation complex (PIC) through interaction with TFIIB-related factor 1 (Brf1) (10-13). The human RPC62/39/32 subcomplex, homologous to the yeast C82/34/31 complex, is dissociable and required for promoter-specific initiation (11).The two Pol III-specific subcomplexes contain structural domains homologous to domains in the Pol II transcription factors TFIIF and TFIIE, including the TFIIF-related dimerization module in the C37/53 subcomplex and several TFIIE-related winged helix (WH) domains in subunits C82 and C34 (14-20). TFIIE is composed of two subunits, Tfa1 and Tfa2, in yeast, or TFIIEα and TFIIEβ in humans. Whereas Tfa1 bears an extended WH (eWH) domain in its N-terminal region, Tfa2 has two adjacent WH domains (21,22). Two adjacent Tfa2-related WH domains are also present in the C34 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 C-terminal region of the A49 subunit (18). The crystal structure of the human C82 homolog RPC62 contains four Tfa1-like eWH domains (eWH1-4) that are structurally organized aroun...

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Section: C82 and C34supporting

confidence: 83%

Section: C82 and C34mentioning

confidence: 99%

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

Herzog²,

Jennebach

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…The structure of the Rpb7/Rpb4 complex is very similar to the archaeal EЈF dimer structure (22,17) and to the RNAPIII counterpart of these subunits, C17/C25 (37,38). Their similar structure strongly argues for their functional equivalence.…”

Section: Discussionmentioning

confidence: 75%

The RPB7 Orthologue E′ Is Required for Transcriptional Activity of a Reconstituted Archaeal Core Enzyme at Low Temperatures and Stimulates Open Complex Formation

Naji

Grünberg

Thomm

2007

Journal of Biological Chemistry

120

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RNA polymerases from Archaea and Eukaryotes consist of a core enzyme associated with a dimeric E F (Rpb7/Rpb4) subcomplex but the functional contribution of the two subunit subcomplexes to the transcription process is poorly understood. Here we report the reconstitution of the 11-subunit RNA polymerase and of the core enzyme from the hyperthermophilic Archaeon Pyrococcus furiosus. The core enzyme showed significant activity between 70 and 80°C but was almost inactive at 60°C. E stimulated the activity of the core enzyme at 60°C, dramatically suggesting an important role of this subunit at low growth temperatures. Subunit F did not contribute significantly to catalytic activity. Permanganate footprinting at low temperatures dissected the contributions of the core enzyme, subunit E , and of archaeal TFE to open complex formation. Opening in the ؊2 and ؊4 region could be achieved by the core enzyme, subunit E stimulated bubble formation in general and opening at the upstream end of the transcription bubble was preferably stimulated by TFE. Analyses of the kinetic stabilities of open complexes revealed an unexpected E -independent role of TFE in the stabilization of open complexes. Transcription in cells of Archaea andBacteria is catalyzed by a single RNA polymerase (RNAP), 2 whereas eukaryotic cells contain three different types of RNAPs (I, II, and III) that carry out specialized functions. Despite their morphological similarity to Bacteria, Archaea have a transcriptional machinery that is more akin to the eukaryotic machinery (1-3).Like eukaryotes, Archaea use extrinsic transcription factors for initiation. The process of transcription initiation in Archaea can be dissected in three steps. The general transcription factor TATA-binding protein (TBP) interacts with the archaeal TATA box, transcription factor B (TFB) stabilizes the binding of TBP to the promoter. The TBP-TFB promoter complex mediates recruitment of RNAP (4, 5). These extrinsic factors show the major structural features of eukaryotic TBP and TFIIB and interact with DNA and RNAP in a similar fashion as their eukaryotic counterparts (6, 7). This exceptional degree of similarity between the archaeal and eukaryotic transcriptional machineries extends also to the RNAP.Archaeal RNAP consist of 11 or 12 different subunits (8 -10) that display a high level of primary sequence similarity to the subunits present in eukaryotic RNAPII. With the exception of subunits RPB8 and RPB9, orthologs of other RNAPII subunits have been identified in all archaeal genomes studied so far. A recent in silico study revealed a high degree of conservation of subunits shared by pol II and Archaea in particular in the regions of RNAP comprising the catalytic center and proteinprotein binding studies showed a similar pattern of subunit interactions between the subunits of pol II and of Pyrococcus RNAP (11). Although very similar to eukaryotic RNAP in subunit composition and transcription initiation factor requirement the archaeal machinery is less complex than the eukaryotic machine...

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“…In this regard, it is appropriate to recall the isolation of 24 singlesite mutations in RET1 (encoding C128) that are moderately or strongly defective in termination and the demonstration that some of these mutations cause the corresponding pol IIIs to elongate transcripts more rapidly (61,62), apparently comparable with pol III lacking C53 and C37. Based on the pol II homology-derived structural model of pol III (37), all of the termination-deficient mutations of Shaaban et al (62) can be mapped to the lobe and fork domains of C128. Eight mutations in the lobe are clustered in the N-terminal half of helix ␣9 and the ␣8-␣9 loop (corresponding to Rpb2 residues 338 -354).…”

Section: Discussionmentioning

confidence: 99%

The C53/C37 Subcomplex of RNA Polymerase III Lies Near the Active Site and Participates in Promoter Opening

Kassavetis

Prakash²,

Shim

2010

Journal of Biological Chemistry

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The C53 and C37 subunits of RNA polymerase III (pol III) form a subassembly that is required for efficient termination; pol III lacking this subcomplex displays increased processivity of RNA chain elongation. We show that the C53/C37 subcomplex additionally plays a role in formation of the initiation-ready open promoter complex similar to that of the Brf1 N-terminal zinc ribbon domain. In the absence of C53 and C37, the transcription bubble fails to stably propagate to and beyond the transcriptional start site even when the DNA template is supercoiled. The C53/C37 subcomplex also stimulates the formation of an artificially assembled elongation complex from its component DNA and RNA strands. Protein-RNA and protein-DNA photochemical cross-linking analysis places a segment of C53 close to the RNA 3 end and transcribed DNA strand at the catalytic center of the pol III elongation complex. We discuss the implications of these findings for the mechanism of transcriptional termination by pol III and propose a structural as well as functional correspondence between the C53/C37 subcomplex and the RNA polymerase II initiation factor TFIIF. RNA polymerase III (pol III)3 transcribes genes encoding short RNA products that are essential for protein synthesis (e.g. tRNAs, 5 S rRNA), RNA processing (e.g. U6 small nuclear RNA, the RNA subunit of RNase P), protein transport (7SL RNA of the signal recognition particle), and diverse other functions, particularly in higher eukaryotes (1). Pol III is brought to the transcriptional start sites of its genes through interactions with its central 3-subunit initiation factor, TFIIIB (subunits Brf1, TBP, and Bdp1). TFIIIB, in turn, is assembled on DNA upstream of the transcriptional start site in a largely sequenceindependent process by its six-subunit assembly factor, TFIIIC, which binds to two promoter elements (boxA and boxB) located downstream of the transcriptional start site. An additional initiation factor, TFIIIA, serves solely as the adaptor for assembling TFIIIC on 5 S rRNA genes. Vertebrates contain an additional form of TFIIIB in which its paralogue Brf2 replaces Brf1 for transcription of a class of genes that use the SNAP c complex in place of TFIIIC as their TFIIIB-assembly factor (2, 3). Once it has been assembled onto DNA, Saccharomyces cerevisiae TFIIIB suffices for recruiting pol III for robust and accurately initiating transcription in vitro (4); at least in yeast, this also appears to be the case in vivo (5). The complexity of pol III contrasts with the apparent simplicity of its core transcription apparatus (the TFIIIB⅐DNA complex and diverse assembly factors). Five pol III subunits have no counterparts in the considerably less complex pol II (with its merely 12 subunits). Three of these pol III subunits (C82, C34, and C31) form a subassembly that interacts with the TFIIIB⅐DNA complex and is required for specifically initiating transcription (6). Two subunits, C53 and C37, form a subassembly that limits the processivity of pol III elongation and contributes to the abil...

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Structural Biology of RNA Polymerase III: Subcomplex C17/25 X-Ray Structure and 11 Subunit Enzyme Model

Cited by 86 publications

References 53 publications

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

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

The RPB7 Orthologue E′ Is Required for Transcriptional Activity of a Reconstituted Archaeal Core Enzyme at Low Temperatures and Stimulates Open Complex Formation

The C53/C37 Subcomplex of RNA Polymerase III Lies Near the Active Site and Participates in Promoter Opening

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