No abstract

Transcription factor IIA has been shown to interact with the TATA-binding protein and to act early during preinitiation complex formation. The human factor is composed of three subunits (oL, 13, 3,). A human cDNA clone encoding the largest subunit of TFIIA (oL) was isolated. The recombinant oL polypeptide, together with the 13 and 3' subunits, was capable of reconstituting TFIIA activity. Studies using antibodies raised against recombinant c~ polypeptide demonstrate that TFIIA can be an integral component of the preinitiation complex. We demonstrate that TFIIA not only interacts with TBP but also can associate with the TFIID complex. Functional assays establish that TFIIA has no apparent role in basal transcription but plays an important role in activation of transcription. Interestingly, amino acid sequence analyses of the 13-subunit demonstrate these residues to be entirely contained within the carboxyl terminus of the cDNA clone encoding the a-subunit.

There are four different genes encoding the cAMP-specific phosphodiesterase (PDE4) isozymes (A, B, C, and D). cAMP has been the only agent known to induce PDE4 gene expression. In the present study, we demonstrate, for the first time, that lipopolysaccharide (LPS) significantly and selectively stimulated PDE4B mRNA production in human monocytes. The LPS stimulation occurred very rapidly (in 30-45 min) and in a dose-dependent manner (0.01-100 ng/ml). We also demonstrate that LPS induction of PDE4B mRNA expression was inhibited strongly by interleukin (IL)-10. The inhibition with IL-10 was dose-dependent (0.1-10 ng/ml). IL-4 also inhibited the LPS induction, but to a lesser extent than IL-10. PDE4B mRNA expression was also stimulated by dibutyryl-cAMP. Interestingly, unlike LPS induction, the dibutyryl-cAMP induction of PDE4B mRNA expression was not inhibited by IL-10. By performing nuclear run-on and mRNA stability assays, we demonstrate further that IL-10 inhibited LPS-stimulated PDE4B mRNA synthesis by abolishing the gene transcription rather than by enhancing mRNA degradation. The present study suggests that PDE4B, as the only LPS-inducible PDE4 subtype, may be an appropriate target for discovering anti-inflammatory drugs.

Human transcription factor IIA (TFIIA) is composed of three subunits (a, f3, and y). TFIIA interacts with the TATA-box binding protein and can overcome repression of transcription. TFIIA was found to be necessary for VP16-mediated transcriptional activation through a coactivator function. We have separated the coactivator and antirepression activities of TFIIA. A TFIIA lacking the a subunit was isolated from HeLa cells. This "mini-TFIIA" interacts with the TATA-box binding protein and can overcome repression of transcription, but it is defective in transcriptional coactivator function.Initiation of transcription at promoters transcribed by RNA polymerase II (RNAPII) is complex. Different families of factors operate on these promoters (for a review, see ref. 1). One family of factors recognizes common promoter elements such as the TATA or the initiator motifs. These proteins are known as the basal, or general, transcription factors (GTFs) and include transcription factor IID (TFIID), TFIIB, TFIIE, TFIIF, and TFIIH (2). TFIID is the only GTF known to exhibit sequence-specific DNA binding activity, with specificity for the TATA element (3, 4). In vivo, TFIID exists as a large, multisubunit complex (5, 6), yet its DNA binding activity is intrinsic to only one polypeptide of 38 kDa, namely the TATA-box binding protein, TBP (7-9). The other subunits of TFIID are known as the TBP-associated factors, TAFs (10-12).A second class of factors that operate at promoters transcribed by RNAPII is composed of regulatory factors (13,14). One class of regulatory factors recognizes specific sequences located upstream or downstream of the TATA motifs (13-16). These factors can activate or repress transcription of specific genes upon binding to their sites. The molecular mechanisms by which regulatory factors regulate the activity of specific genes are poorly understood. Ultimately, regulatory factors encroach upon the basal transcription machinery to elicit their effect. In fact, some regulators have been shown to directly interact with components of the basal transcription machinery (1).Another growing family of transcription factors is known as coactivators (2, 17). Coactivators do not directly bind to promoter sequences (18), and they appear to function as "molecular bridges" between the basal transcription machinery and the promoter-bound activator. The prototype coactivators are the collection of TAFs in the TFIID complex (17). However, not all coactivators are subunits of the TFIID complex. Some coactivators are general, whereas others are activator-and tissue-specific. One such example is the B-cellspecific coactivator . Another collection of coactivators acts more generally, and they are required for optimal activation of transcription regardless of the activator type (23, 24). These general coactivators (cofactors) include some of the TAFs (25) (51,52). Moreover, the TBP-TFIIA interaction is important for activation of transcription in vivo (52). However, whether the different functions ascribed to TFIIA in activati...