Redundant Cooperative Interactions for Assembly of a Human U6 Transcription Initiation Complex

Abstract

The core human U6 promoter consists of a proximal sequence element (PSE) located upstream of a TATA box. The PSE is recognized by the snRNA-activating protein complex (SNAP c ), which consists of five types of subunits, SNAP190, SNAP50, SNAP45, SNAP43, and SNAP19. The TATA box is recognized by TATA box binding protein (TBP). In addition, basal U6 transcription requires the SANT domain protein Bdp1 and the transcription factor IIB-related factor Brf2. SNAP c and mini-SNAP c , which consists of just SNAP43, SNAP50, and the N-terminal third of SNAP190, bind cooperatively with TBP to the core U6 promoter. By generating complexes smaller than mini-SNAP c , we have identified a 50-amino-acid region within SNAP190 that is (i) required for cooperative binding with TBP in the context of mini-SNAP c and (ii) sufficient for cooperative binding with TBP when fused to a heterologous DNA binding domain. We show that derivatives of mini-SNAP c lacking this region are active for transcription and that with such complexes, TBP can still be recruited to the U6 promoter through cooperative interactions with Brf2. Our results identify complexes smaller than mini-SNAP c that are transcriptionally active and show that there are at least two redundant mechanisms to stably recruit TBP to the U6 transcription initiation complex.An important event during activation of transcription is the establishment of a stable transcription initiation complex. For RNA polymerase II, transcription initiation complexes appear to be partially disassembled at each round of transcription, losing transcription factor IIB (TFIIB), RNA polymerase II, and TFIIF as well as, in some cases, TFIIE and TFIIH (23,24,33,34). Other factors including TFIIA, TATA box binding protein (TBP), some of the TFIID TBP-associated factors (TAFs), and, in a crude extract containing mediator, TFIIH, TFIIE, and mediator itself, are left behind and form a scaffold for reinitiation of transcription (33). In the case of RNA polymerase III transcription, initiation complexes preformed on 5S-and tRNA-type promoters can direct several rounds of transcription in the presence of concentrations of sarcosyl that inhibit formation of new initiation complexes, suggesting that the complexes are stable for several rounds of transcription (8,9).The human U6 snRNA promoter is a type 3 RNA polymerase III promoter characterized by gene-external promoter elements, which can be divided into enhancer and core regions. The enhancer region, referred to as the distal sequence element, is located about 200 bp upstream of the transcription start site and contains binding sites for the POU domain protein Oct-1 and the zinc finger protein STAF (see reference 7 for a review). Both of these factors can activate U6 transcription and can be localized to the U6 promoter region in vivo by chromatin immunoprecipitation experiments (14, 20-22, 26, 28, 35).The U6 core promoter region consists of a proximal sequence element (PSE) and a TATA box located about 50 and 25 bp, respectively, upstream of the transcription s...

Cytoskeletal Filamin A Differentially Modulates RNA Polymerase III Gene Transcription in Transformed Cell Lines

“…Fig. 5 shows that knockdown of FLNA did not change the occupancy of BRF1 at the U6 RNA gene promoter; this is likely because BRF2 replaces BRF1 in transcription factor III B in U6 RNA gene expression (24). However, loss of FLNA still enhanced the recruitment of TFIIIC2 at the U6 RNA gene promoter.…”

Cooperation between Small Nuclear RNA-activating Protein Complex (SNAPC) and TATA-box-binding Protein Antagonizes Protein Kinase CK2 Inhibition of DNA Binding by SNAPC

“…SNAP C contains at least five proteins called SNAP190 (PTF␣), SNAP50 (PTF␤), SNAP45 (PTF␦), SNAP43 (PTF␥), and SNAP19 (27)(28)(29)(30)(31)(32)(33). The largest subunit SNAP190 plays a centrally important role in human snRNA gene transcription first by serving as the scaffold for SNAP C assembly though interactions with most other members of SNAP C (34,35). Once the complex is assembled, SNAP190 further recognizes the PSE through its Myb DNA binding domain (30) and is also the direct target for Oct-1 (23,36).…”

“…The TATA box is recognized by the TBP component of the Brf2⅐TFIIIB complex (35, 38 -41). SNAP C , through its SNAP190 subunit, stimulates TBP binding to the U6 TATA box as an early critical step in RNA polymerase III transcription (35,38).…”

Co-expression of multiple subunits enables recombinant SNAPC assembly and function for transcription by human RNA polymerases II and III

“…The central region of SNAP190 may negatively regulate SNAP C DNA binding, but interestingly, auto-inhibition of DNA binding can be overcome by interactions between the central region of SNAP190 and Oct-1 during activated transcription by RNA polymerases II and III [15]. The C-terminal region of SNAP190 interacts with SNAP45 [25], which may stabilize DNA binding of SNAP C in the context of the natural complex [15]. However, DNA binding by SNAP C does not require SNAP45 or SNAP19 as DNA binding activity can be reconstituted using a partial recombinant SNAP C (hereafter referred to as mini-SNAP C ) that contains SNAP43, SNAP50, and the N-terminal region of SNAP190 encompassing amino acids 1-505 [15].…”

“…SNAP C is composed of at least five subunits, SNAP19 [17], SNAP43 [18,19], SNAP45 [19,20], SNAP50 [21,22], and SNAP190 [23]. SNAP190, the largest of the subunits, provides the fundamental scaffold for assembly of the complex and directly interacts with all other SNAP C subunits [24] and with TBP [25,26]. SNAP190 also contains an unusual DNA binding domain in its N-terminal region consisting of four and a half myb domain repeats that is required for SNAP C DNA binding to the PSE [23] and for cooperative promoter recruitment of TBP to the TATA box during U6 transcription [26].…”