BRFU, a TFIIB-like factor, is directly recruited to the TATA-box of polymerase III small nuclear RNA gene promoters through its interaction with TATA-binding protein.

Shona Murphy,Pavel C̆abart

doi:10.1074/jbc.m108515200

Abstract

The human snRNA genes transcribed by RNA polymerase II (pol II) and III (pol III) have different core promoter elements. Both gene types contain similar proximal sequence elements (PSEs) but differ in the absence (pol II) or presence (pol III) of a TATA-box, which, together with the PSE, determines the assembly of a pol III-specific pre-initiation complex. BRFU is a factor exclusively required for transcription of the pol III-type snRNA genes. We report that recruitment of BRFU to the TATA-box of these promoters is TATA-binding protein (TBP)-dependent. BRFU in turn stabilizes TBP on TATA-containing template and extends the TBP footprint both upstream and downstream of the TATA element. The core domain of TBP is sufficient for BRFU.TBP.DNA complex formation and for interaction with BRFU off the template. We have mapped amino acid residues within TBP and domains of BRFU that mediate this interaction. BRFU has no specificity for sequences flanking the TATA-box and also forms a stable complex on the TATA-box of the pol II-specific adenovirus major late promoter (AdMLP). Furthermore, pol III-type transcription can initiate from an snRNA gene promoter containing an AdMLP TATA-box and flanking sequences. Therefore, the polymerase recruitment is not simply determined by the sequence of the TATA-box and immediate flanking sequences.

Highlights

Introduction of the adenovirus major late promoter (AdMLP)TATA-box and Flanking Sequences into the snRNA Promoter Retains pol III TranscriptionSpecificity—The observation that AdMLP TATA-box and flanking sequences have no selective exclusion effect on recruitment of BRFU versus TFIIB prompted us to perform the following functional assay
TATA-binding protein (TBP) is required for transcription of both types of snRNA gene and is likely to be recruited to the TATAless polymerase II (pol II)-specific promoters by interaction with PTF binding to the proximal sequence elements (PSEs) [3]
TBP Recruits BRFU to the TATA-box, and the TBP Core Domain Is Sufficient for BRFU1⁄7TBP1⁄7DNA Complex Formation on pol III snRNA Promoters—In vitro studies on mRNA promoters indicate that the PIC assembles in an ordered stepwise fashion where TFIID binding to the TATA-box nucleates the assembly of a complex containing TFIIA and TFIIB, which in turn recruits other basal factors and pol II (reviewed by Orphanides et al [24])

Summary

Here we show that BRFU interacts with TBP to form a

This paper is available on line at http://www.jbc.org complex on TATA-containing templates and have mapped amino acid residues within TBP and domains of BRFU that mediate this interaction. We found that BRFU, unlike TFIIB, appears to have no specificity for sequences outside the binding site for TBP. The data presented here provide an insight into an important step in nucleation of a pol III- specific snRNA transcription initiation complex

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION