Mapping the Principal Interaction Site of the Brf1 and Bdp1 Subunits of Saccharomyces cerevisiae TFIIIB

Abstract

The Brf1 subunit of the central RNA polymerase (pol) III transcription initiation factor TFIIIB is bipartite; its N-terminal TFIIBrelated half is principally responsible for recruiting pol III to the promoter and for promoter opening near the transcriptional start site, whereas its pol III-specific C-terminal half contributes most of the affinities that hold the three subunits of TFIIIB together. Here, the principal attachment site of Brf1 for the Bdp1 subunit of TFIIIB has been mapped by a combination of structure-informed, site-directed mutagenesis and photochemical protein-DNA cross-linking. A 66-amino acid segment of Brf1 is shown to serve as a two-sided adhesive surface, with the side chains projecting away from its extended interface with TATA-binding protein anchoring Bdp1 binding. An extensive collection of N-terminal, C-terminal, and internal deletion proteins has been used to demarcate the interacting Bdp1 domain to a 66-amino acid segment that includes the SANT domain of this subunit and is phylogenetically the most conserved region of Bdp1.The RNA polymerase (pol) 3 III transcription apparatus of yeast comprises, in addition to the polymerase itself, three transcription initiation factors, TFIIIA, -B, and -C.The three-subunit TFIIIB is the core initiation factor required and sufficient for recruiting pol III to the promoter and accurately initiating transcription. The large six-subunit TFIIIC places TFIIIB on DNA upstream of the transcriptional start site and protects the generally very small pol III transcription units from encroachment by repressive chromatin (1, 2). TFIIIA is the 5 S rRNA gene-specific factor that places TFIIIC on the transcription unit-internal promoter of these genes (reviewed in Refs. 3-5).The work that is presented here deals with the structure of TFIIIB, which is composed of three subunits: the TATA-binding protein (TBP), Brf1, and Bdp1. TBP and Brf1 bind tightly and co-purify, whereas Bdp1 dissociates during later steps of conventional column chromatography. (The two separated fractions of TFIIIB have been designated BЈ and BЉ, and B double prime has been adopted as the eponym of the Bdp1 subunit.) The 596-amino acid Brf1 is a fusion protein, with an N-terminal half that is related to the pol II transcription initiation factor TFIIB and a pol III-specific C-terminal half. A conserved segment of the C-terminal half (homology domain II; amino acids 439 -515) constitutes the Brf1 high affinity binding site for the evolutionarily highly conserved core of TBP (comprising all but the N-terminal 60 amino acids of the Saccharomyces cerevisiae protein); the N-terminal half of Brf1 engages TBP through a separate lower affinity interaction.The N-and C-terminal halves of Brf1 also provide separate Bdp1-docking sites, with the C-proximal homology domain II contributing the higher Bdp1 affinity. TBP makes only a minor direct contribution to bringing Bdp1 into TFIIIB. Thus, the pol III-specific half of Brf1 appears to contribute the interactions that principally hold TFIIIB together. Ind...

Comparative overview of RNA polymerase II and III transcription cycles, with focus on RNA polymerase III termination and reinitiation

“…Photocrosslinking studies used to analyze the interaction between TFs and the promoters of a 5S rRNA gene and a tRNA gene have shown that the Tfc4 subunit of TFIIIC associates with the region around the transcription start site (TSS), which is consistent with proximal flexibility, 20 while the 90 kDa subunit of TFIIIC, TFC6, associates with the terminator element. 47 Nagarajavel et al 10 analyzed genome-wide 'bootprints' of TFIIIC and TFIIIB in vivo, with single nucleotide resolution, using ChIP-seq.…”

“…20 Once a TFIIIB-DNA complex is formed, it is sufficient to recruit RNAP III and direct transcription, suggesting that TFIIIB is the transcription initiation factor proper for RNAP III while the others function as prerecruitment assembly factors, as was demonstrated in vitro and in vivo. 21,22 Interactions between Brf1 and the integral RNAP III subunits C34 and C17 are important for RNAP III recruitment.…”

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

“…Brf1c exists mostly as a scaffold that holds together the three TFIIIB subunits (12, 26). In particular, structural analysis of the Brf1-TBP-DNA complex indicated that homology block II is positioned along the convex and lateral surfaces of TBP and that the block also interacts with Bdp1 (5,6,10,22,[26][27][28]. The homology blocks are separated by two nonconserved connecting regions that we refer to as C linkers 1 and 2 (Fig.…”

“…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)(5)(6)(7)(8)(9)(10). The TFIIIB-DNA assembly is required for subsequent Pol III recruitment and transcript initiation.…”

Without a License, or Accidents Waiting to Happen

“…The principal attachment sites between TBP, Brf1, and Bdp1 were determined by chopping and slicing; the N-terminal half of Brf1 and its TFIIB paralog were shown to bind to overlapping TBP sites (129); the structure of the stronger TBP-binding segment of Brf1, which is part of its C-terminal half, in complex with TBP and DNA was also solved (130). The structure, in turn, led to an in vitro mutation screen showing that the TBP-interacting Brf1 segment serves as a kind of double-sided adhesive mediating attachment of TBP to Bdp1 (131). Cellular functions are executed by spatially organized multiprotein complexes, each of whose subunits may comprise multiple domains, securing the network of interactions that determine assembly as well as function.…”