TFIIA Has Activator-dependent and Core Promoter Functions in Vivo

Abstract

The physiological role of TFIIA was investigated by analyzing transcription in a yeast strain that contains a TATA-binding protein (TBP) mutant (N2-1) defective for interacting with TFIIA. In cells containing N2-1, transcription from a set of artificial his3 promoters dependent on different activators is generally reduced by a similar extent, indicating that TFIIA function is largely nonselective for activators. In addition, TATA element utilization, a core promoter function, is altered at his3 promoters dependent on weak activators. Genomic expression analysis reveals that 3% of the genes are preferentially affected by a factor of 4 or more. Chimeras of affected promoters indicate that the sensitivity to the TFIIA-TBP interaction can map either to the upstream or core promoter region. Unlike wild-type TBP or TFIIA, the N2-1 derivative does not activate transcription when artificially recruited to the promoter via a heterologous DNA binding domain, indicating that TFIIA is important for transcription even in the absence of an activation domain. Taken together, these results suggest that TFIIA plays an important role in both activatordependent and core promoter functions in vivo. Further, they suggest that TFIIA function may not be strictly related to the recruitment of TBP to promoters but may also involve a step after TBP recruitment. Initiation of RNA polymerase (pol)1 II transcription requires the assembly of a large complex of proteins that must interact at the promoter in a productive manner (1, 2). Formation of this complex is accelerated by activators that bind to the promoter and aid in recruitment of the components in the complex. The first step in promoter recognition is binding of TFIID to the TATA element. TFIID is a multiprotein complex containing TATA-binding protein (TBP) and TBP-associated factors (3). TFIIA stabilizes the TBP-TATA interaction (4 -7) by interacting directly with the TBP and DNA flanking the TATA element (8, 9). TFIIA also counteracts several negative regulators of transcription that specifically target TBP. It inhibits the abilities of Mot1 and NC2 to dissociate TBP from the TATA element (10 -13), and it blocks the inhibition of TBP binding to the TATA element by the N-terminal domain of TBP-associated factor 130 (14). Thus, there are several mechanisms by which TFIIA functions at core promoters in vitro.Although TFIIA is not required for in vitro transcription using highly purified components, activated transcription is often stimulated by TFIIA. This is in accord with the observations that TFIIA can interact directly with activation domains in vitro (15-17) and that TFIIA is required for activator-dependent stabilization of the TFIID⅐TATA complex (16, 18 -20). A simple model is that the activator-dependent TFIID⅐TFIIA complex is formed rapidly and stably on the TATA element, thereby serving as an efficient scaffold for the remainder of the initiation complex. Alternatively, TFIIA could act as a coactivator, conveying information between the activator and TBP. In this regard, in...

The Stability of the TFIIA-TBP-DNA Complex Is Dependent on the Sequence of the TATAAA Element

“…When the amount of RNA initiating from ϩ1 (which is driven by the noncanonical element, T C ) and ϩ13 (driven by TATA) start sites were compared, the N2-1 strain showed a preferential increase in transcription initiating from ϩ13, even when the overall level of transcription was low (33). This is in stark contrast to the utilization of the HIS3 promoter elements occurring in wild type cells, which shows similar levels of output (22).…”

“…Depend on TFIIA Activity-Recent work characterizing a TBP allele defective for interaction with TFIIA (the N2-1 derivative of TBP) has shown that the TFIIA-TBP interaction is important for HIS3 core element utilization (33). When the amount of RNA initiating from ϩ1 (which is driven by the noncanonical element, T C ) and ϩ13 (driven by TATA) start sites were compared, the N2-1 strain showed a preferential increase in transcription initiating from ϩ13, even when the overall level of transcription was low (33).…”

“…interaction is having a more dramatic effect on the noncanonical element (T C ) than on the canonical element (33). To further test the importance of the TFIIA-TBP interaction at the HIS3 promoter, we examined the effect of introducing a fusion construct of a subunit of TFIIA (Toa2) to TBP (34).…”

Positive and negative functions of the SAGA complex mediated through interaction of Spt8 with TBP and the N-terminal domain of TFIIA

“…Several approaches have been taken to determine which genes require TFIIA for expression in vivo. These include TFIIA subunit depletion, mutation of TFIIA subunits to impair interaction with TBP, or mutation of TBP to inhibit interaction with TFIIA (Kang et al 1995;Ozer et al 1998a;Liu et al 1999;Stargell et al 2000). These studies generally agree that TFIIA is important for transcription of a specific subset of genes, although there is not yet agreement on the exact subset of genes requiring TFIIA for normal expression.…”

Efficient Binding of NC2·TATA-binding Protein to DNA in the Absence of TATA

“…The general transcription factor TFIIA accelerates and stabilizes binding of TBP to TATA boxes (19 -21). It also functions as a co-activator for some transcriptional activators (22)(23)(24)(25)(26). TFIIA further functions as an antagonist of NC2 (also called Dr1/DRAP) (27,28).…”