TFIIIB subunit locations on U6 gene promoter DNA mapped by site‐specific protein–DNA photo‐cross‐linking

Abstract

Edited by Ivan SadowskiRNA polymerase III-transcribed U6 snRNA genes have gene-external promoters that contain TATA boxes. U6 TATA sequences are bound by TFIIIB that in Drosophila contains the three subunits TBP, Brf1, and Bdp1. The overall structure of TFIIIB is still not well understood. We have therefore studied the mode of TFIIIB binding to DNA by site-specific protein-DNA photo-cross-linking. The results indicate that a portion of Brf1 is sandwiched between Bdp1 and TBP upstream of the TATA box. Furthermore, Bdp1 traverses the DNA under the N-terminal stirrup of TBP to interact with the DNA (and very likely Brf1) downstream of the TATA sequence.Keywords: Bdp1; Brf1; Protein-DNA photo-cross-linking; RNA polymerase III transcription; TATA box-binding protein; TFIIIB Eukaryotic RNA polymerase III (Pol III) synthesizes a wide variety of small RNA molecules involved in diverse metabolic processes, including components of the protein synthesis apparatus (tRNA and 5S rRNA) and the spliceosome (U6 snRNA). The Pol III promoters can be divided into three distinct types: Type I (5S rRNA) and Type II (e.g. tRNA) promoters are gene-internal and usually are TATA-less [1][2][3]. Type III promoters (e.g., U6) on the other hand are geneexternal and inevitably contain a TATA sequence [1][2][3][4][5][6][7]. The only general transcription factor that is required at all three types of Pol III promoters is TFIIIB. TFIIIB is considered to be the Pol III initiation factor per se because once bound to the DNA, TFIIIB is able to recruit Pol III for multiple rounds of transcription initiation [8].TFIIIB is composed of three subunits, normally the TATA box-binding protein (TBP), Brf1, and Bdp1 [3]. However, heterogeneity can exist in TFIIIB at different Pol III promoters. For example, in Drosophila melanogaster, the TBP-related factor TRF1 was found to be utilized for Pol III transcription in place of TBP [9]. Recent work in our lab confirmed that TRF1 replaced TBP as a subunit of TFIIIB for tRNA gene transcription in fruit flies. Interestingly, however, we also found that transcription of U6 snRNA genes by Pol III instead utilized TBP rather than TRF1 [10]. Thus, different types of Pol III promoters in insects can differentially utilize either TBP (at Type III promoters) or TRF1 (at Type I and Type II promoters) as a component of TFIIIB.Brf1, another subunit of TFIIIB, is related to the Pol II general transcription factor TFIIB. The N-terminal half of Brf1 is homologous to the zinc ribbon domain and the two cyclin-fold repeats of TFIIB, but Brf1 has an extended C-terminus that averages over 360 amino acid residues in length among yeast, fruit flies, and humans [3].Interestingly, U6 transcription in mammals requires a unique and distinct form of Brf known as Brf2 [11,12]. Human Brf2 contains an N-terminal domain homologous to those found in TFIIB and Brf1, and it Abbreviations TFIIIB, transcription factor IIIB; TBP, TATA box binding protein; TRF1; TBP-related factor 1; TFIIB, transcription factor IIB; Brf1, TFIIB-related factor 1; Brf...

Molecular mechanisms of Bdp1 in TFIIIB assembly and RNA polymerase III transcription initiation

“…In the TFIIIB–DNA complex, Bdp1SANT lays at the interface between the DNA region immediately upstream of the TATA box, the TBP N-terminal stirrup, and the Brf2 TBP anchor domain—the high-affinity TBP-binding site at the very C terminus of Brf2 4 —in agreement with recent crosslinking studies 16, 21 (Fig. 1d).…”

Assembly of SNAPc, Bdp1, and TBP on the U6 snRNA Gene Promoter in Drosophila melanogaster

“…The upper and lower strand patterns are highly consistent with each other under the assumption that Bdp1 interacts primarily with a single face of the DNA over the region examined in these experiments. The cross-linking of Bdp1 that occurred on both DNA strands at the positions near the TATA box was not surprising based on results we previously obtained with TFIIIB alone (28) and results observed by others from yeast TFIIIB cryo-EM structures (17).…”

“…In previous work, we used site-specific protein-DNA photo-cross-linking assays to map the nucleotide positions of the U6 promoter that are in close proximity to the three different subunits of DmSNAPc when DmSNAPc alone is bound to the DNA (33). In a more recent study, we similarly mapped the nucleotide positions where each of the three subunits of TFIIIB map to the U6 promoter DNA when TFIIIB alone is bound to the DNA (28). To extend these data, we have now investigated the binding pattern of the DmSNAPc-Bdp1-TBP ternary protein complex on the U6 promoter DNA.…”

“…Previously, by using site-specific protein-DNA photo-cross-linking assays, we identified nucleotide positions where each of the individual DmSNAPc subunits cross-linked as part of the complex to U6 snRNA gene promoter DNA (14,33). Likewise, we reported interactions of the TFIIIB subunits (in the absence of DmSNAPc) with specific nucleotides in the U6 snRNA gene promoter (28). Those studies revealed both the linear positions (translational location along the DNA helix) and rotational positions (face of the DNA double helix) occupied by each of the DmSNAPc and TFIIIB subunits on the DNA.…”

Molecular mechanism of promoter opening by RNA polymerase III

“…During the preparation of this manuscript, the human TBP-Brf2 (residues 64-407)-Bdp1 (residues 286-407) crystal structure14 became available, giving us additional confidence in assigning the density N-terminal to the Bdp1-SANT domain. Finally, the prominent density running underneath the DNA was assigned to Bdp1 residues 275-320 based on available protein-DNA crosslinks16,58 that placed a region of Bdp1 on the opposite side of TBP and was mapped to Bdp1 residues 299-315. Reported photo-crosslinks of Bdp1 K281 to Brf1 and Bdp1 residues 291-295 to the C128 protrusion gave additional confidence26.…”