ternary elongation complex, where it likely functions in accurate termination. Our work explains how the Rpc37/53 dimer is anchored on the Pol III core and acts as a hub to integrate a protein network for initiation and termination.RNA polymerase III (Pol III) catalyzes the RNA synthesis of diverse untranslated transcripts, including precursor tRNAs, 5S rRNA, and certain small nuclear RNAs, such as U6 and 7SL RNAs. The other two nuclear RNA polymerases, Pol I and Pol II, transcribe pre-rRNA and mRNA, respectively. In humans, Pol III also participates in the transcription of a number of microRNAs (19) and plays a role in tumorigenesis (41,58). Among the nuclear RNA polymerases, Pol III is the largest enzyme, comprising 17 subunits with a total molecular mass of approximately 0.7 MDa; its core structure of 12 subunits is conserved among all nuclear Pols (18). The remaining five subunits are specific to Pol III and form two subcomplexes with distinct functions in transcription, Rpc82/34/31 and Rpc37/53. The former subcomplex interacts with the initiation transcription factor TFIIIB for the recruitment of polymerase and also participates in promoter opening and transcription initiation (6,50,55,56), whereas the latter participates in promoter opening and transcription termination and reinitiation (34,38). Previous yeast two-hybrid screenings provided an overview of protein-protein interactions among Pol III subunits (26), and electron microscopy analysis of Pol III suggested the location of the subcomplexes on the Pol III core structure (24,25,32,52). However, much more information on proteinprotein interactions between these subcomplexes and the initiation factors is necessary to understand the mechanisms of initiation, elongation, and termination and to determine how the mechanisms used by the Pol III machinery differ from those used by Pol I and II.A conserved dimerization module has recently been detected in the Pol I, II, and III machineries as part of Rpa49/ 34.5 of Pol I, the general transcription factor TFIIF of Pol II, and Rpc37/53 of Pol III (8,13,18,36). The presence of the TFIIF-like complexes is unique to the eukaryotic transcription systems. However, other than the conserved dimerization module and a winged-helix (WH) fold that is present as a single copy in each of the Tfg1 and Tfg2 subunits of TFIIF and as tandem repeats in Rpa49, the overall structural characteristics of the three TFIIF-like complexes differ; thus, their functions in transcription may also differ. For example, although all three of these TFIIF-like complexes are involved in transcription and elongation, they exert opposite effects on elongation, with Rpa49/34.5 and TFIIF increasing and Rpc37/53 reducing the elongation rate (36,38,59).Structural and biochemical analyses have suggested how individual TFIIF-like complexes establish protein interactions for their respective transcriptional activities. The TFIIF dimerization module, which resides in subunits Tfg1 and Tfg2, has been mapped to the Pol II lobe domain above the active site c...

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...

b TFIIB-related factor Brf1 is essential for RNA polymerase (Pol) III recruitment and open-promoter formation in transcription initiation. We site specifically incorporated a nonnatural amino acid cross-linker into Brf1 to map its protein interaction targets in the preinitiation complex (PIC). Our cross-linking analysis in the N-terminal domain of Brf1 indicated a pattern of multiple protein interactions reminiscent of TFIIB in the Pol active-site cleft. In addition to the TFIIB-like protein interactions, the Brf1 cyclin repeat subdomain is in contact with the Pol III-specific C34 subunit. With site-directed hydroxyl radical probing, we further revealed the binding between Brf1 cyclin repeats and the highly conserved region connecting C34 winged-helix domains 2 and 3. In contrast to the N-terminal domain of Brf1, the C-terminal domain contains extensive binding sites for TBP and Bdp1 to hold together the TFIIIB complex on the promoter. Overall, the domain architecture of the PIC derived from our cross-linking data explains how individual structural subdomains of Brf1 integrate the protein network from the Pol III active center to the promoter for transcription initiation. Eukaryotic RNA polymerase (Pol) III transcribes precursor tRNAs, 5S rRNA, small nuclear RNAs such as U6 and 7SK RNAs, and a number of small nucleolar RNAs and microRNAs (1). In the yeast Saccharomyces cerevisiae, the Pol III transcription apparatus consists of 17-subunit Pol III and three other transcription factors: single-polypeptide TFIIIA, three-subunit TFIIIB, and six-subunit TFIIIC (2, 3). TFIIIA and TFIIIC function as the promoter recognition factors, and TFIIIB is recruited to the promoter through TFIIIC. TFIIIB is composed of TFIIB-related factor Brf1, TATA box binding protein TBP, and SANT domain-containing subunit Bdp1. Previous biochemical studies indicated that Brf1 and TBP cooperatively assemble onto DNA upstream of the transcription start site and Bdp1 binds to the Brf1-TBP-DNA complex mainly through its SANT domain (4-10). The TFIIIB-DNA assembly is required for subsequent Pol III recruitment and transcript initiation. Both Brf1 and Bdp1 have been found to interact with Pol III and function in promoter opening (4,(11)(12)(13)(14).The N-terminal domain of yeast Brf1 (Brf1n; amino acids [aa] 1 to 286) contains a zinc ribbon fold (aa 3 to 34) and a cyclin fold repeat subdomain (aa 83 to 282) (Fig. 1A), both of which are homologous to those in the general transcription factor TFIIB of the Pol II system. On the basis of biochemical and structural analyses, TFIIB ribbon and cyclin fold repeats are, respectively, positioned in the RNA exit tunnel and in the wall domain of Pol II (15)(16)(17)(18)(19)(20). In addition, the connecting region between the TFIIB ribbon and the cyclin repeat domain has been structurally resolved to contain B reader and B linker motifs that interact with the Pol active center. On the basis of sequence comparison, the connecting region in Brf1n, which we refer to as the N linker, contains low sequence homology ...

Background A country’s health expenditure significantly improves its population health status. This study aims to examine the determinants of health expenditure in dictatorships. Methods We designed a mixed methods research approach. First, we used panel data from 1995 to 2014 covering 99 countries (n = 1488). Fixed effects regression models were fitted to determine how different types of authoritarianism relate to health expenditure. Second, we chose Ivory Coast to apply the synthetic control methods for a case study. We constructed a synthetic Ivory Coast, combining other dominant party regimes to resemble the values of health expenditure predictors for Ivory Coast prior to a regime change from a dominant party system to personalist dictatorships in 2000. Results We found that dominant party autocracies, compared with non-dominant party regimes, increased health expenditure (% of GDP) (1.36 percentage point increase, CI = 0.59–2.12). The marginal effect, however, decreased when an autocrat in this type of regime held elections (0.86 percentage point decrease, CI = 0.20–1.52). Furthermore, we found the difference in health expenditure between the actual Ivory Coast and its synthetic version starts to grow following the regime change in 2000 (in 2000, actual: 6.00%, synthetic: 6.04%; in 2001, actual: 4.85%, synthetic: 5.99%), suggesting a pronounced negative effect of the government transition on Ivory Coast health expenditure. Conclusion The findings suggest that different forms of dictatorship are associated with varying levels of health expenditure. Where dictatorships rely on popular support, as is the case with dominant party dictatorships, health expenditure is generally greater.