An activity (designated BTF1Y) in extracts of Saccharomyces cerevisiae can substitute for the human TATA box-binding factor BTF1 in a reconstituted transcription system containing the adenovirus 2 major late promoter, RNA polymerase B (II), and the basic transcription factors BTF2, BTF3, and STF. We have purified BTF1Y to homogeneity, using as assays reconstitution of in vitro transcription and DNase I footprinting on the TATA element. Both activities copurified with a 27-kDa polypeptide as determined by SDS/PAGE. Gel Accurate and specific initiation of transcription in vitro from minimal promoter elements of protein-coding genes requires at least four transcription factors in addition to RNA polymerase B (II) (1-5). The first step in transcription initiation corresponds to the binding of the factor BTF1 (the TATA box-binding factor, also known as TFIID and DB) to the TATA box element (6-8). This binding step is crucial in the initiation pathway, since it results in the formation of a stable "committed" complex onto which the other factors and RNA polymerase B assemble to form a multiprotein complex at the promoter proximal site (3, 4, 9-11).Although numerous attempts have been made to purify mammalian BTF1, notably from human HeLa cell extracts, purification was only partial (e.g., ref. MATERIALS AND METHODSPurification of BTF1Y Protein. S100 extract (34 mg of protein per ml) was prepared from frozen S. cerevisiae cells as described (19). After overnight dialysis against buffer DB [10 mM Tris'HCl, pH 7.9/5 mM MgCl2/0.5 mM dithiothreitol/17.4% (vol/vol) glycerol/0.2 mM phenylmethanesulfonyl fluoride (PMSF)] containing 0.05 M KCI, 125 ml of 2-folddiluted S100 was loaded onto a 450-tnl heparin-Ultrogel (IBF) column equilibrated in buffer DB containing 0.1 M KCI. After washing, proteins were eluted stepwise with buffer DB containing 0.15 M KCI, 0.37 M KCI (HEPO.37 fraction), and 0.6 M KCl. BTF1Y activity was found in the HEPO.37 fraction. After dialysis in buffer B (50 mM Tris-HCl, pH 7.9/8.7% glycerol/0.1 mM EDTA/0.5 mM dithiothreitol/0.1 mM PMSF) containing 0.08 M KCI, 2 liters of HEPO.37 fraction was loaded onto a 500-mI DEAE-Sephacel column. The flow-through fraction (DEFT80), which contained the BTF1Y activity, was made 50 mM potassium phosphate (pH 6.3) and loaded onto a sulfopropyl-5PW (Toyo Soda, Tokyo) HPLC column equilibrated in the same buffer. Proteins were eluted with a linear 0.08-0.60 M KCl gradient. Active BTF1Y fractions, which eluted at 0.4-0.5 M KCl (SPO.4 fraction), were dialyzed against buffer B (without PMSF) containing 0.05 M KCl, and 25 ml was loaded onto a heparin-5PW (Toyo Soda) HPLC column. Proteins were eluted with a linear 0.05-0.60 M KCI gradient, with BTF1Y activity recovered at -0.5 M KCl (HEPO.5 fraction). Ten milliliters of the HEPO.5 fraction was dialyzed against buffer C (50 mM Tris-HCl, pH 7.9/17.4% glycerol/0.1 mM EDTA/0.5 mM dithiothreitol/5 mM MgCI2) containing 0..1 M KCl and loaded onto a 400 /ul/heparin-Ultrogel column, equilibrated in the same buffer. Proteins w...

The state of the art regarding the six known sweet-tasting proteins (thaumatin, monellin, mabinlin, pentadin, brazzein and curculin) and the taste-modifying protein miraculin is reviewed. Their biochemical properties, molecular genetics and biotechnological production are assessed. All of these proteins have been isolated from plants that grow in tropical rainforests. They share no sequence homology or structural similarities. Nonetheless, one of them, thaumatin, shares extensive homology with certain non-sweet proteins found in other plants. The potential industrial applications of the sweet-tasting proteins are also discussed, placing special emphasis on the barriers that a recombinant product of these characteristics will have to overcome before it reaches the market.

Termination of transcription at tR1, the rho-dependent terminator between genes cro and cII of bacteriophage lambda, is mediated by interactions between rho protein and an RNA sequence element called rut. We show, using a filter retention assay technique, that rho protein binds with about 10-fold lower affinity to variants of cro RNA lacking both parts of rut or to normal cro RNA having one or the other part of rut bound to a complementary DNA oligonucleotide than it binds to unmodified cro RNA. These same variant and modified forms are nearly devoid of the strong rho ATPase cofactor activity of cro RNA. Estimates of binding energies of the rho-cro RNA interaction under different conditions reveal that termination function correlates with about 12.6 kcal of binding energy, of which two-thirds is due to nonelectrostatic interactions. The rut segment is shown to contribute about 1 kcal, nearly all to nonelectrostatic interactions. KCl is found to be more effective than potassium glutamate as a competitive counterion, and a decrease in 1.4 kcal of binding energy due to counterion competition correlates with a loss of termination and ATPase activities. In sum, the results indicate that the rut sequence contributes substantially to the overall binding affinity, that ionic interactions are also important, and that mere binding of rho to RNA is not sufficient for rho ATPase activation.

A protease-deficient strain of Aspergillus niger has been used as a host for the production of human tissue plasminogen activator (t-PA). In defined medium, up to 0.07 mg t-PA (g biomass)(-1) was produced in batch and fed-batch cultures and production was increased two- to threefold in two-phase batch cultures in which additional glucose was provided as a single pulse at the end of the first batch growth phase. Production was increased [up to 1.9 mg t-PA (g biomass)(-1)] by the addition of soy peptone to the defined medium. The rate of t-PA production in batch cultures supplemented with soy peptone (0.2 to 0.6 mg t-PA L(-1) h(-1)) was comparable to rates observed previously in high-producing mammalian or insect cell cultures. In glucose-limited chemostat culture supplemented with soy peptone, t-PA was produced at a rate of 0.7 mg t-PA L(-1) h(-1). Expression of t-PA in A. niger resulted in increased expression of genes (bipA, pdiA, and cypB) involved in the unfolded protein response (UPR). However, when cypB was overexpressed in a t-PA-producing strain, t-PA production was not increased. The t-PA produced in A. niger was cleaved into two chains of similar molecular weight to two-chain human melanoma t-PA. The two chains appeared to be stable for at least 16 h in culture supernatant of the host strain. However, in general, <1% of the t-PA produced in A. niger was active, and active t-PA disappeared from the culture supernatant during the stationary phase of batch cultures, suggesting that the two-chain t-PA may have been incorrectly processed or that initial proteolytic cleavage occurred within the proteolytic domain of the protein. Total t-PA (detected by enzyme-linked immunoassay) also eventually disappeared from culture supernatants, confirming significant extracellular proteolytic activity, even though the host strain was protease-deficient.

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