Transcription initiation by mammalian RNA polymerase II is effected by multiple common factors interacting through minimal promoter elements and regulated by gene-specific factors interacting with distal control elements. Minimal promoter elements that can function independently or together, depending on the specific promoter, include the upstream TATA box and a pyrimidine-rich initiator (Inr) overlapping the transcription start site. The binding of TFIID to the TATA element promotes the assembly of other factors into a preinitiation complex but factors which function at the Inr have not been defined. We show here that a novel factor (TFII-I) binds specifically to Inr elements, supports basal transcription from the adenovirus major late promoter and is immunologically related to the helix-loop-helix activator USF. We further show that TFII-I also binds to the upstream high-affinity USF site (E box), that USF also binds to the Inr, and that TFII-I and USF interact cooperatively at both Inr and E box sites. Thus, TFII-I represents a novel type of transcription initiation factor whose interactions at multiple promoter elements may aid novel communication mechanisms between upstream regulatory factors and the general transcriptional machinery.
The transcription factor TFII-I has been shown to bind independently to two distinct promoter elements, a pyrimidine-rich initiator (Inr) and a recognition site (E-box) for upstream stimulatory factor 1 (USF1), and to stimulate USF1 binding to both of these sites. Here we describe the isolation of a cDNA encoding TFII-I and demonstrate that the corresponding 120 kDa polypeptide, when expressed ectopically, is capable of binding to both Inr and E-box elements. The primary structure of TFII-I reveals novel features that include six directly repeated 90 residue motifs that each possess a potential helix-loop/span-helix homology. These unique structural features suggest that TFII-I may have the capacity for multiple protein-protein and, potentially, multiple protein-DNA interactions. Consistent with this hypothesis and with previous in vitro studies, we further demonstrate that ectopic TFII-I and USF1 can act synergistically, and in some cases independently, to activate transcription in vivo through both Inr and the E-box elements of the adenovirus major late promoter. We also describe domains of USF1 that are necessary for its independent and synergistic activation functions.
Earlier in vitro studies identified USF as a cellular factor which activates the adenovirus major late (Ad‐ML) promoter by binding to an E‐box motif located at position −60 with respect to the cap site. Purified USF contains 44 and 43 kDa polypeptides, and the latter was found (by cDNA cloning) to be a helix‐loop‐helix protein. In this report, we demonstrate a 25‐to 30‐fold stimulation of transcription via an upstream binding site by ectopic expression of the 43 kDa form of USF (USF43) in transient transfection assays. More recent data have also revealed alternate interactions of USF43 at pyrimidine‐rich (consensus YYAYTCYY) initiator (Inr) elements present in a variety of core promoters. In agreement with this observation, we show here that USF43 can recognize the initiator elements of the HIV‐1 promoter, as well as those in the Ad‐ML promoter, and that ectopic expression of USF43 can stimulate markedly the corresponding core promoters (TATA and initiator elements) when analyzed in transient co‐transfection assays. Mutations in either Inr 1 or Inr 2 reduced the USF43‐dependent transcription activity in vivo. In addition, in vitro transcription assays showed that mutations in either or both of the Inr 1 and Inr 2 sequences of the HIV‐1 and Ad‐ML promoters could affect transcription efficiency, but not the position of the transcriptional start site. These results indicate that USF43 can stimulate transcription through initiator elements in two viral promoters, although the exact mechanism and physiological significance of this effect remain unclear.
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