DNA-binding and transcriptional properties of human transcription factor TFIID after mild proteolysis.

Abstract

The existence of separable functions within the human class H general transcription factor TFIID was probed for differential sensitivity to mild proteolytic treatment. Independent of whether TFIID was bound to DNA or free in solution, partial digestion with either one of a variety of nonspecific endoproteases generated a proteaseresistant protein product that retained specific DNA recognition, as revealed by DNase I footprinting. However, in contrast to native TFIID, which interacts with the adenovirus major late (ML) promoter over a very broad DNA region, partially proteolyzed TFIID interacted with only a small region of the ML promoter immediately surrounding the TATA sequence. This novel footprint was very similar to that observed with the TATA factor purified from yeast cells. Partially proteolyzed human TFIID could form stable complexes that were resistant to challenge by exogenous templates. It could also nucleate the assembly of transcription complexes on the ML promoter with an efficiency comparable to that of native TFIID, yielding similar levels of transcription initiation. These results suggest a model in which the human TFHID protein is composed of at least two different regions or polypeptides: a protease-resistant "core," which by itself is sufficient for promoter recognition and basal transcriptional levels, and a protease-sensitive "tail," which interacts with downstream promoter regions and may be involved in regulatory processes.The promoter of protein-encoding (class II) genes is composed of multiple DNA elements. The minimum (or basal) promoter often contains a sequence referred to as the TATA box, whereas regulation is conferred by one or several upstream regulatory elements (for a review, see references 13 and 27). Recognition of the promoter by the general transcription factors leads to the formation of stable complexes that signal a gene for transcription by RNA polymerase II. Template challenge analyses have revealed that a particular transcription factor, designated TFIID, is the essential component of these stable complexes and that the TATA box sequence alone is sufficient for TFIID binding (4,26). Human TFIID is required for transcription of a number of cellular and viral genes in vitro (15). Binding of TFIID to the promoter DNA has been shown to be facilitated by an activity designated TFIIA (4, 6, 17), although an absolute TFIIA requirement for stable complex formation as well as for efficient transcription has not always been found (19,26 Recently, a protein was isolated from yeast which can substitute for human TFIID in a heterologous in vitro system reconstituted with the other general transcription factors and RNA polymerase II from HeLa cells (2, 3). This yeast TFIID is a single polypeptide of 27,000 daltons (10), which can form stable preinitiation complexes with various TATA boxes in the absence of any other transcription factor (3). DNase I footprinting has revealed a small (16 bp) region of interaction for yeast TFIID with the ML TATA box (2, 10), and the same nucleotides ...

A Domain of the even-skipped Protein Represses Transcription by Preventing TFIID Binding to a Promoter: Repression by Cooperative Blocking

“…If TFIID is added before CDGB, CDGB can still bind the GREs but cannot displace TFIID, and therefore there is no repression. Consistent with previously published data (47,52,53,59), these results also indicate that once TFIID is bound to the DNA, the disassociation rate for TFIID must be slow, or else CDGB would affect TFIID binding even when CDGB is added second. A final observation is that there was weak protection of sequences in the basal promoter by CDGB.…”

General Initiation Factors for Rna Polymerase Ii

“…Partially purified preparations of the native TATA factor from Drosophila protect a region extending from approximately -40 to +35 (with respect to the transcription initiation site) of the histone H3, histone H4, and actin 5C promoters (36). A partially purified preparation of the native TATA factor from human cells protects a similar region of the AdML and human histone H4 promoters (128)(129)(130), but protects only those sequences in the immediate vicinity of the TAT A box of the human HSP70 promoter (128). This difference in protection appears to correlate with the efficiency of promoter utilization in reconstituted transcription systems (128), and could reflect an inability of the native TAT A factor to interact specifically with the Initiator region of the HSP70 promoter.…”

The transcriptional activity of a muscle-specific promoter depends critically on the structure of the TATA element and its binding protein

“…However, the N-terminal domain of TBP is known to undergo a conformational change upon binding to the TATA motif (Hoffmann et al, 1990;Lieberman et al, 1991) and Van Dyke & Sawadogo (1990) have shown that the N-terminal domain of human TBP becomes insensitive to mild proteolysis upon DNA binding. A change in the conformation of the N-terminal domain of TBP upon binding to the IgH TATA sequence is an attractive hypothesis, since this domain of the protein has been proposed to be involved in the mediation of transcriptional activation on TATAcontaining promoters (Lescure et al, 1994;Zhou et al, 1991).…”