Mapping the Protein Interaction Network for TFIIB-Related Factor Brf1 in the RNA Polymerase III Preinitiation Complex

Abstract

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 ...

A Region of Bdp1 Necessary for Transcription Initiation That Is Located within the RNA Polymerase III Active Site Cleft

“…Based on the binding analysis with the C128 N-terminal fragment, we conclude that Bdp1 region II is possibly localized in the Pol III active site cleft. Supporting this conclusion, Bdp1 region II also interacts with the Brf1 N-terminal half proposed to be positioned in the Pol III active site cleft as well (17).…”

“…On the basis of the results of DNA cross-linking and DNase I and hydroxyl radical protection analyses, both Brf1 and Bdp1 approximately localize on the promoter DNA from bases Ϫ40 to Ϫ2 upstream of the transcription start site (referred to as the ϩ1 base), while TBP binds the DNA from bases Ϫ20 to Ϫ31 (8-15). The two termini of Brf1 have different functions, with the C-terminal half (amino acids [aa] 165 to 596) nucleating the TBP-Brf1-Bdp1 complex upstream of the transcription start site and the N-terminal half positioning within the Pol III active site cleft to assist with promoter opening (16)(17)(18).In the yeast Saccharomyces cerevisiae, Bdp1 is a 594-amino-acid polypeptide with an apparent molecular mass of 90 kDa by gel electrophoresis (14,19). The C-terminal region contains a conserved SANT (SWI3, ADA2, N-CoR, and TFIIIB BЉ; aa 415 to 472) domain that generally functions as a DNA-binding module (Fig.…”

“…On the basis of the results of DNA cross-linking and DNase I and hydroxyl radical protection analyses, both Brf1 and Bdp1 approximately localize on the promoter DNA from bases Ϫ40 to Ϫ2 upstream of the transcription start site (referred to as the ϩ1 base), while TBP binds the DNA from bases Ϫ20 to Ϫ31 (8-15). The two termini of Brf1 have different functions, with the C-terminal half (amino acids [aa] 165 to 596) nucleating the TBP-Brf1-Bdp1 complex upstream of the transcription start site and the N-terminal half positioning within the Pol III active site cleft to assist with promoter opening (16)(17)(18).…”

BRF1 mutations alter RNA polymerase III–dependent transcription and cause neurodevelopmental anomalies

“…The N-terminal region of BRF1 has a role in DNA binding and Pol III recruitment (Kassavetis et al 1998), is homologous to the Pol II transcription factor TFIIB, and contains an N-terminal zinc ribbon domain and two cyclin domains (Khoo et al 2014). The BRF1 gene on human chromosome 14q32.33 encodes several functional isoforms, some of which lack the zinc ribbon domain (McCulloch et al 2000).…”

Eukaryotic transcription initiation machinery visualized at molecular level

“…5 In Saccharomyces cerevisiae, the N-terminal domain of Brf1 (TFIIB-related factor 1) participates in a network of protein-protein interactions resembling that of TFIIB in Pol II PIC, whereas its cyclin repeats domain contacts the Pol III-specific subunit C34. 37 The N-terminal region of yeast Bdp1 was positioned within the active center cleft of Pol III PIC, contacting Pol III subunits C128, C37, and Brf1. 38 TFIIIC is composed of two subcomplexes that are connected by the interaction between Tfc4/t131 and Tfc3/t138.…”