Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators.

Abstract

Transcriptional stimulation by the model activator GAL4-VP16 (a chimeric protein consisting of the DNA-binding domain of the yeast activator GAL4 and the acidic activation domain of the herpes simplex virus protein VP16) involves a series of poorly understood protein-protein interactions between the VP16 activation domain and components of the RNA polymerase II general transcription machinery. One of these interactions is the VP16-mediated binding and recruitment of transcription factor TFIIB. However, TATA box-binding protein (TBP)-associated factors (TAFs), or coactivators, are required for this interaction to culminate in productive transcription complex assembly, and one such TAF, Drosophila TAF40, reportedly forms a ternary complex with VP16 and TFIIB. Due to TFIIB's central role in gene activation, we sought to directly visualize the surfaces of this protein that mediate formation of the ternary complex. We developed an approach called protease footprinting in which the broad-specificity proteases chymotrypsin and alkaline protease were used to probe binding of 32P-end-labeled TFIIB to GAL4-VP16 or TAF40. Analysis of the cleavage products revealed two regions of TFIIB protected by VP16 from protease attack, one of which overlapped with a region protected by TAF40. The close proximity of the VP16 and TAF40 binding sites on the surface ofTFIIB suggests that this region could act as a regulatory interface mediating the effects of activators and coactivators on transcription complex assembly.The RNA polymerase II general transcription machinery is composed of six or more general factors called TFIIA through TFIIH (1). One of the key issues in the field is to understand how an upstream activator interacts with these factors to stimulate preinitiation complex assembly and transcription (2-7). The chimeric activator GAL4-VP16 (8-10) is a good model for studying this problem because of its potency in mammalian cell transfection assays (8) and in cell-free transcription systems (11-13). VP16 binds several potential targets in affinity chromatography studies (3-7, 14, 15), but the interaction with TFIIB is of particular interest because of its avidity (4, 5) and because VP16 recruits TFIIB to a promoter in assays of transcription complex assembly (4, 16). The interaction has also been validated by genetic studies: a point mutation in VP16, for example, simultaneously abolishes its interaction with TFIIB and its ability to stimulate transcription (4,17). Similarly, deletion mutations at the carboxyl ends of the two 77-aa repeats in TFIIB eliminate binding to VP16 (18)(19)(20), and point mutations in one of these regions, a putative basic amphipathic a-helix at the end of the first repeat, weaken TFIIB's interaction with VP16 and eliminate GAL4-VP16-responsive transcription in vitro (18 MATERIALS AND METHODSPlasmid Constructs. pETlld-C-HMK was constructed by inserting an oligonucleotide encoding a heart muscle kinase phosphorylation site (HMK) at the BamHI site of pETlid (Novagen). Nco I-BamHI fragments encod...

Interaction between the Retinoid X Receptor and Transcription Factor IIB Is Ligand-dependent in Vivo

“…An alteration in digestion pattern was also observed for TFIIB in the presence of bacterially expressed RXR but not GST control protein (not shown). These data are further support for the interaction of RXR with TFIIB and are consistent with either an induced conformational change in TFIIB or protection from proteolytic cleavage due to binding of RXR to TFIIB (82,83).…”

“…Using in vitro translated TFIIB and baculovirus/or bacterially expressed RXR proteins, we observed that RXR induced an altered proteolytic digestion pattern of TFIIB. These data suggest that one possible action of nuclear receptors may be to induce a conformational change in TFIIB which, as suggested for VP16, then exposes other binding interfaces of TFIIB to other transcription factors, possibly TAF II 40 (82,85), TBP (9, 31, 86, 87), TFIID (8, 34, 85, 88), TFIIF, and RNAPII (89). These results raise the question of whether gene activation by nuclear hormone receptors might result not simply from a passive assembly of transcriptional proteins due to acidic, proline-rich, or glutamine-rich regions, but rather as the active alteration of protein interfaces of TFIIB which in turn present protein interfaces with affinity to other transcription factors, thus influencing assembly of the PIC.…”

“…Since it had been shown that the acidic activator, VP-16, induced a conformational change in TFIIB (82,83) and bound to a similar region of TFIIB, we performed a partial proteolysis assay to test whether RXR may induce a conformational change within TFIIB. Using in vitro translated TFIIB and baculovirus/or bacterially expressed RXR proteins, we observed that RXR induced an altered proteolytic digestion pattern of TFIIB.…”

The VP16 Activation Domain Interacts with Multiple Transcriptional Components as Determined by Protein-Protein Cross-linking in Vivo

“…First, unlike the case for SAGA, TBP and TFIIB are not recruited by the Gal4 activator bound to its genomic sites in the absence of a TATA element (23,24). Second, the VP16 activation domain interacts with surfaces of TBP (64,65) and TFIIB (31,66) that are critical for promoter binding, and mutations that abolish the interactions in vitro do not significantly affect the level of transcriptional activity in vivo (67,68). This suggests that the VP16 activation domain may not interact with TBP and TFIIB in the context of a preinitiation complex, and in this regard our observed crosslinking occurs primarily (and perhaps exclusively) when the proteins are not bound to DNA.…”

Transcriptional regulation by Modulo integrates meiosis and spermatid differentiation in male germ line

“…This full-size Modulo variant contains the N-terminal acidic domain, the structure of which is characteristic for the acidic activators (21) that facilitate assembly of the core transcription machinery on the promoter and recruitment of chromatinremodeling factors (22). Known acidic activators interact with the TFIID complex and facilitate interaction of TFIID with TFIIA and TFIIB (23)(24)(25)(26)(27)(28). In testes, TFIID is represented by a specific variant encoded by the meiotic-arrest genes of the sa group (7).…”