Three Functional Classes of Transcriptional Activation Domains

Abstract

We have studied the abilities of different transactivation domains to stimulate the initiation and elongation (postinitiation) steps of RNA polymerase II transcription in vivo. Nuclear run-on and RNase protection analyses revealed three classes of activation domains: Sp1 and CTF stimulated initiation (type I); human immunodeficiency virus type 1 Tat fused to a DNA binding domain stimulated predominantly elongation (type IIA); and VP16, p53, and E2F1 stimulated both initiation and elongation (type IIB). A quadruple point mutation of VP16 converted it from a type IIB to a type I activator. Type I and type IIA activators synergized with one another but not with type IIB activators. This observation implies that synergy can result from the concerted action of factors stimulating two different steps in transcription: initiation and elongation. The functional differences between activators may be explained by the different contacts they make with general transcription factors. In support of this idea, we found a correlation between the abilities of activators, including Tat, to stimulate elongation and their abilities to bind TFIIH.Stimulation of eukaryotic gene expression requires sequence-specific factors with DNA binding domains and activation domains that interact with the general transcription factors (GTFs) and recruit RNA polymerase II (pol II) to the promoter (3, 65). In vivo the transcriptionally active form of pol II is probably a holoenzyme complex which contains a number of the GTFs as well as other polypeptides (36). Different activation domains interact with different GTFs, including TFIIB (44), TFIID (17,19,63), and TFIIF (76). These interactions are thought to recruit, stabilize, and/or modify the activity of the pol II holoenzyme.We recently demonstrated that the activation domains of p53, VP16, and E2F1 bind directly to TFIIH (50a, 72). TFIIH is a multisubunit factor, different forms of which are required for both transcription and nucleotide excision repair of DNA (9). TFIIH is the only GTF which has enzymatic activities: it has two helicase subunits and a cyclin-dependent protein kinase subunit which phosphorylates the pol II large-subunit C-terminal domain (CTD) (12,52,58,60). Both of these enzymatic activities are implicated in steps in transcription which occur shortly after initiation of the RNA chain. First, a helicase is required for efficient formation of open complexes and for promoter clearance on linear templates in vitro (20). Second, when paused polymerases resume elongation on several Drosophila genes in vivo, the CTD becomes phosphorylated, suggesting a possible role for TFIIH kinase in regulating elongation (50, 70). Furthermore, inhibitors of the TFIIH kinase inhibit elongation under activated (74), but not basal (57), transcription conditions.In vivo, rate-limiting steps after initiation have been well documented for a number of genes. For example, polymerases stall 20 to 40 bases downstream of the start sites in the Drosophila hsp70 and human c-myc genes (38, 51, 64) and terminate...

Modular organization of the E2F1 activation domain and its interaction with general transcription factors TBP and TFIIH

“…VP16 stimulates elongation, as well as initiation, by RNA polymerase II (Yankulov et al, 1994) and, indeed, other experiments have indicated that the abilities of various activators, including E2F1, to bind TFIIH correlate with their e ects on elongation downstream of the promoter (Blau et al, 1996). The tat transactivator protein of HIV-1 stimulates transcription elongation and interacts with TFIIH (Blau et al, 1996). Tat has also recently been shown to directly stimulate TFIIH to phosphorylate the carboxy-terminal heptapeptide repeat domain (CTD) of the largest subunit of RNA polymerase II (Parada and Roeder, 1996).…”

“…E2F1 is one of a growing number of activator proteins, including VP16, that have not been shown to bind TFIIH (Xiao et al, 1994;Tong et al, 1995;Blau et al, 1996). VP16 stimulates elongation, as well as initiation, by RNA polymerase II (Yankulov et al, 1994) and, indeed, other experiments have indicated that the abilities of various activators, including E2F1, to bind TFIIH correlate with their e ects on elongation downstream of the promoter (Blau et al, 1996).…”

Transcriptional activators and activation mechanisms

“…By comparing the RNAP density along a gene, it is possible to gain information about which step, initiation or elongation, an activator may stimulate. Using this and other analyses, Blau et al (Blau et al, 1996) concluded that, while some activators (e.g., Sp1 and CTF) work primarily on the initiation step, others (e.g., Tat) work primarily on the elongation step. Another class of activators (e.g., VP16, p53 and E2F1) can work on both initiation and elongation.…”

“…Studies to compare the roles of different activators suggest that synergy may reflect combinatorial actions on distinct steps of transcription (Blau et al, 1996). By comparing the RNAP density along a gene, it is possible to gain information about which step, initiation or elongation, an activator may stimulate.…”

“…Another class of activators (e.g., VP16, p53 and E2F1) can work on both initiation and elongation. An analysis of these activators revealed that synergy was only achieved between those that work on different steps of transcription (Blau et al, 1996).…”