Reliable glycosylation reactions that allow for the stereo- and regioselective installation of glycosidic linkages are paramount to the chemical synthesis of glycan chains. The stereoselectivity of glycosylations is exceedingly difficult to control due to the reaction's high degree of sensitivity and its shifting, simultaneous mechanistic pathways that are controlled by variables of unknown degree of influence, dominance, or interdependency. An automated platform was devised to quickly, reproducibly, and systematically screen glycosylations and thereby address this fundamental problem. Thirteen variables were investigated in as isolated a manner as possible, to identify and quantify inherent preferences of electrophilic glycosylating agents (glycosyl donors) and nucleophiles (glycosyl acceptors). Ways to enhance, suppress, or even override these preferences using judicious environmental conditions were discovered. Glycosylations involving two specific partners can be tuned to produce either 11:1 selectivity of one stereoisomer or 9:1 of the other by merely changing the reaction conditions.

Biochemical analyses have suggested potential targets for transcriptional activation domains, which include several components of the RNA polymerase II machinery, as well as the chromatin template. Here we examine the mechanism of transcriptional activation in yeast cells by connecting a heterologous DNA-binding domain (LexA) to the TATA-binding protein (TBP). LexA-TBP efficiently activates transcription from a promoter containing a LexA operator upstream of a TATA element. Activation is promoter-specific and is sensitive to mutations on the DNA-binding surface of TBP; hence it is not due to a fortuitous activation domain on TBP. Thus a promoter-bound protein lacking an activation domain can stimulate transcription if it is directly connected to TBP. This suggests that recruitment of TBP to the promoter can be a rate-limiting step for transcription in vivo, and that interactions between activation domains and factors that function after TBP recruitment can be bypassed for activation.

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The transcription factor nuclear factor-B (NF-B) is activated by a diverse number of stimuli including tumor necrosis factor-␣, interleukin-1, UV irradiation, viruses, as well as receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR). NF-B activation by the tumor necrosis factor receptor (TNFR) involves the formation of a multiprotein complex termed a signalosome. Although previous studies have shown that the activated EGFR can induce NF-B, the mechanism of this activation remains unknown. In this study, we identify components of the signalosome formed by the activated EGFR required to activate NF-B and show that, although the activated EGFR uses mechanisms similar to the TNFR, it recruits a distinct signalosome. We show the EGFR forms a complex with a TNFR-interacting protein (RIP), which plays a key role in TNFR-induced NF-B activation, but not with TRADD, an adaptor protein which serves to recruit RIP to the TNFR. Furthermore, we show that the EGFR associates with NF-B-inducing kinase (NIK) and provide evidence suggesting multiprotein complex formation between the EGFR, RIP, and NIK. Using a dominant negative NIK mutant, we show that NIK activation is required for EGFR-mediated NF-B induction. We also show that a S32/36 IB␣ mutant blocks EGFR-induced NF-B activation. Our studies also suggest that a high level of EGFR expression, a frequent occurrence in human tumors, is optimal for epidermal growth factorinduced NF-B activation. Finally, although protein kinase B/Akt has been implicated in tumor necrosis factor and PDGF-induced NF-B activation, our studies do not support a role for this protein in EGFR-induced NF-B activation.The engagement of the epidermal growth factor receptor by its cognate ligand results in the generation of a number of intracellular signals (1). The initial changes induced by ligand binding are receptor dimerization, activation of the kinase activity of the receptor, and autophosphorylation of the receptor on tyrosine residues (2, 3). Autophosphorylation of the receptor results in the creation of docking sites for a number of secondary signaling proteins bearing specific protein interaction domains such as the Src homology 2 domain, which interact specifically with phosphorylated tyrosine residues (4). As a consequence of this interaction, these secondary signaling proteins may themselves become activated and trigger a number of downstream signals. These signaling cascades result in the activation of a number of transcription factors such as AP-1 and STATS (5).The NF-B 1 family of transcription factors plays an important role in inflammatory responses (6). A diverse number of stimuli including cytokines such as TNF␣ and IL-1, UV irradiation, and lipopolysaccharide are known to activate NF-B. In unstimulated cells NF-B is sequestered in the cytoplasm by the IB family of proteins (7). Binding of IB to NF-B masks nuclear localization signals on NF-B and prevents its translocation to the nucleus (8). Stimulation of cells with a diverse array of stimuli results in phos...