Heterologous protein production late in Saccharomyces cerevisiae fermentations is often desirable because it may help avoid the unintentional selection of more rapidly growing, non-protein-expressing cells or allow for the expression of toxic proteins. Here, we describe the use of the MET25 promoter for the production of human serum albumin (HSA) and HSA-fusion proteins in S. cerevisiae. In media lacking methionine, the MET25 promoter yielded high expression levels of HSA and HSA fused to human glucagon, human growth hormone, human interferon alpha, and human interleukin-2. More importantly, we have shown that this system can be used to delay heterologous protein production until late log phase of the growth of the culture and does not require the addition of an exogenous inducer.

Posttranslational modification of general transcription factors may be an important mechanism for global gene regulation. The general transcription factor IIA (TFIIA) binds to the TATA binding protein (TBP) and is essential for high-level transcription mediated by various activators. Modulation of the TFIIA-TBP interaction is a likely target of transcriptional regulation. We report here that Toa1, the large subunit of yeast TFIIA, is phosphorylated in vivo and that this phosphorylation stabilizes the TFIIA-TBP-DNA complex and is required for high-level transcription. Alanine substitution of serine residues 220, 225, and 232 completely eliminated in vivo phosphorylation of Toa1, although no single amino acid substitution of these serine residues eliminated phosphorylation in vivo. Phosphorylated TFIIA was 30-fold more efficient in forming a stable complex with TBP and TATA DNA. Dephosphorylation of yeast-derived TFIIA reduced DNA binding activity, and recombinant TFIIA could be stimulated by in vitro phosphorylation with casein kinase II. Yeast strains expressing the toa1 S220/225/232A showed reduced high-level transcriptional activity at the URA1, URA3, and HIS3 promoters but were viable. However, S220/225/232A was synthetically lethal when combined with an alanine substitution mutation at W285, which disrupts the TFIIA-TBP interface. Phosphorylation of TFIIA could therefore be an important mechanism of transcription modulation, since it stimulates TFIIA-TBP association, enhances high-level transcription, and contributes to yeast viability.Eukaryotic RNA polymerases require the formation of a multiprotein preinitiation complex near the promoter start site for efficient transcription initiation to occur (reviewed in references 8, 42, 49, and 63). The composition of the preinitiation complex may vary among promoters, but the best-studied model promoters indicate that the preinitiation complex consists of the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. The formation of the preinitiation complex and the subsequent recruitment of RNA polymerase II to the promoter start site can be rate limiting for transcription in vitro and in vivo and are subject to regulation by activators and repressors. Precisely how activators and repressors modulate the formation, stability, and organization of the preinitiation complex remains the subject of considerable investigation (reviewed in reference 47).One of the first steps in promoter recognition and preinitiation complex assembly is the association of TFIID with the TATA box (2, 36). TFIID is a multiprotein complex that consists of the TATA binding protein (TBP) and TBP-associated factors (3, 16). The general transcription factors TFIIA and TFIIB can bind directly to TBP and stabilize its association with the TATA box (14,20,38,57). The formation of the TFIID-TFIIA-TFIIB complex can be rate limiting for several model promoters in vitro and in vivo (6,33,55,59). Activators stabilize the association of TBP with the TATA element directly or by enhancin...

Transcription factor IIA (TFIIA) is a positive acting general factor that contacts the TATA-binding protein (TBP) and mediates an activator-induced conformational change in the transcription factor IID (TFIID) complex. Previously, we have found that phosphorylation of yeast TFIIA stimulates TFIIA⅐TBP⅐TATA complex formation and transcription activation in vivo. We now show that human TFIIA is phosphorylated in vivo on serine residues that are partially conserved between yeast and human TFIIA large subunits. Alanine substitution mutation of serine residues 316 and 321 in TFIIA ␣␤ reduced TFIIA phosphorylation significantly in vivo. Additional alanine substitutions at serines 280 and 281 reduced phosphorylation to undetectable levels. Mutation of all four serine residues reduced the ability of TFIIA to stimulate transcription in transient transfection assays with various activators and promoters, indicating that TFIIA phosphorylation is required globally for optimal function. In vitro, holo-TFIID and TBP-associated factor 250 (TAF II 250) phosphorylated TFIIA on the ␤ subunit. Mutation of the four serines required for in vivo phosphorylation eliminated TFIID and TAF II 250 phosphorylation in vitro. The NH 2 -terminal kinase domain of TAF II 250 was sufficient for TFIIA phosphorylation, and this activity was inhibited by full-length retinoblastoma protein but not by a retinoblastoma protein mutant defective for TAF II 250 interaction or tumor suppressor activity. TFIIA phosphorylation had little effect on the TFIIA⅐TBP⅐TATA complex in electrophoretic mobility shift assay. However, phosphorylation of TFIIA containing a ␥ subunit Y65A mutation strongly stimulated TFIIA⅐TBP⅐TATA complex formation. TFIIA-␥Y65A is defective for binding to the ␤-sheet domain of TBP identified in the crystal structure. These results suggest that TFIIA phosphorylation is important for strengthening the TFIIA⅐TBP contact or creating a second contact between TFIIA and TBP that was not visible in the crystal structure. TFIIA1 was identified originally as an activity required for optimal in vitro transcription with HeLa cell nuclear extracts and various viral and cellular templates (1) (reviewed in Refs. 2-5). TFIIA was isolated as three polypeptides from human and Drosophila embryo extracts, and the two largest subunits are proteolytic cleavage products of a single open reading frame (6 -13). TFIIA could be purified as a TATA-binding protein (TBP)-associated factor, although it could also be found as a chromatographically separate complex from TBP (7). In addition to binding directly to TBP, TFIIA stabilized the binding of TBP with TATA DNA in electrophoretic mobility shift assays (EMSA) (14, 15). In transcription reactions reconstituted with partially purified general factors and coactivators, TFIIA is essential for activator-mediated transcription and has a weak stimulatory effect on basal transcription lacking exogenous activators. In transcription reactions reconstituted with recombinant and affinity-purified general factors and coactivato...