Bidirectional binding of the TATA box binding protein to the TATA box

Abstract

By selective attachment of a DNA cleavage agent to specific residues in the yeast TATA box binding protein (yTBP), we demonstrate that, in solution, yTBP binds to the TATA boxes of both the adenovirus major late promoter and the yeast CYC1 promoter with only a modest preference in orientation and binds well to several overlapping binding sites. The general factors TFIIA and TFIIB each increase the rotational and translational selectivity of yTBP but are not sufficient, at least individually, to confer a unique polarity to the preinitiation complex. We conclude that TBP alone cannot define the productive orientation of general factor assembly on a promoter.The TATA box binding protein (TBP) is required by all three RNA polymerases for the promoter-specific initiation of transcription (1). All eukaryotic TBP-DNA complexes observed in crystal structures show the conserved C-terminal domain of TBP (TBP c ) bound to the TATA box in a single orientation that is consistent with the assembly of a preinitiation complex with a unique polarity (2-8). The binding of TBP to the TATA box is thought to orient the complex correctly on the promoter and nucleate preinitiation complex formation (3, 9, 10). Several groups have proposed reasons why the highly symmetric TBP c molecule binds to a nearly symmetric TATA box in only one of two possible orientations in the crystal. These reasons include amino acid and electrostatic differences between the C-and N-terminal repeats of TBP c coupled with the differential deformability of each half of the TATA box (2,3,11,12). Despite these factors that could favor the orientation of TBP c observed in the crystal, the pseudo-2-fold symmetry of TBP c and of many TATA boxes is intriguing. Approximately 80% of the amino acids that contact DNA are identical in the two halves of TBP c , and molecular modeling studies reveal no unfavorable interactions when TBP is bound in the opposite orientation (13). Also, bidirectional transcription from a TATA box and forward transcription from a reverse TATA box can be observed, suggesting that perhaps TBP and͞or the preinitiation complex can function bidirectionally (14 -17). These issues led us to ask to what extent TBP binds to the TATA box in a unique orientation in solution.The affinity cleavage method (18, 19) permits determination of the orientation of a protein bound to its DNA target site and the effects of other factors on this orientation (20). We used this technique to examine the orientation of TBP bound to a TATA box in solution. Using affinity cleavage, we discovered that TBP binds to the TATA boxes of the adenovirus major late promoter (AdMLP) and CYC1 promoters with only a modest preference (⌬G obs ϭ 0.2 Ϫ 0.3 kcal⅐mol Ϫ1 ) in orientation. The general transcription factors TFIIB and TFIIA skew the ratio of TBP binding in favor of the generally accepted polarity but do not fix it to a unique orientation. The high degree of polarity achieved in regulated transcription (16,17,21) suggests that other promoterspecific factors define the po...

Function and Structural Organization of Mot1 Bound to a Natural Target Promoter

“…If TBP binds with a preferred orientation appropriate for PIC assembly, FeBABE tethered to the C-terminal stirrup is predicted to preferentially cleave the DNA at the upstream edge of the TATA box and farther upstream with one helical turn periodicity (7,8). Indeed, FeBABE cleavage was seen for the top strand of the AdMLP at the upstream end of the TATA box and one helical turn farther up the DNA (Fig.…”

“…Because TBP stirrups bind at the ends of the TATA box sequence, the location of DNA cleavage reveals the orientation of the bound protein (8). If TBP binds with a preferred orientation appropriate for PIC assembly, FeBABE tethered to the C-terminal stirrup is predicted to preferentially cleave the DNA at the upstream edge of the TATA box and farther upstream with one helical turn periodicity (7,8).…”

“…TBP is a saddle-shaped protein that interacts with DNA as a monomer (6,7). Because TBP can straddle DNA in either of two directions, its binding polarity determines the orientation of the assembled PIC and hence the direction in which transcription initiates (8). TBP recruitment to promoters is regulated by chromatin, activators, and co-activators that contact TBP directly, and general factors that modulate TBP binding and activity (1, 2, 4, 9 -11).…”

Molecular and Cellular Biology

“…This behavior of TFIIB not only provides strong support for the HBV composite P(A)S-TATA box in sustaining promoter characteristics but also was helpful for mapping the orientation of this TATA box (27). It was reported that the TATA box can in fact act bidirectionally, and the involvement of auxiliary TFIIA and TFIIB is essential to determine the correct orientation (7). Based on these data we defined the TATA box orientation to be at the viral positive strand, where all the other viral promoters have been mapped.…”

Opposite Orientations of a Transcription Factor Heterodimer Bind DNA Cooperatively with Interaction Partners but Have Different Effects on Interferon-β Gene Transcription

“…Some functions long thought to be unidirectional, such as promoterdirected gene transcription, have been recently found to be bidirectional (22,23). The structural basis for this bidirectional transcription remains to be established, but could be rooted in the bidirectional binding of transcription regulatory proteins as well as the TATA box-binding protein at their recognition sites (24).…”