binding could result in a general conformational change that allows the bound protein to activate transcription, alter natively these two functions could be served by separate and independent domains. Domain swap experiments suggest that the latter is typically the case. The critical tests to address this question was first performed in the yeast Saccharomyces cerevisiae.

The yeast GAL1 gene is a member of a group of genes involved in galactose metabolism. Transcription of GAL1 is controlled by the DNA-binding transactivator protein Gal4. Gal4 binds to a 17bp enhancer element (termed upstream activator sequence, or UAS in yeast) located upstream of the Gal1 promoter via its N-terminal DNA binding domain (DBD; Gal4 amino acids 1-73). Gal4 activates GAL1 transcription through a C-terminal 34 aa activation domain (AD). To systematically test the contributions of the Gal4 AD and DBD sequences to GAL1 gene transcription activation, and to ask whether the Gal4 DBD uniquely contributed to such transcription activation, a series of domain swap experiments were performed (Figure 1). The amino terminal 73-amino-acid DBD of Gal4 was removed and replaced with the DBD of LexA, an E. coli DNA-binding protein. This domain swap resulted in a chimeric molecule (lexA DBD-Gal4 AD) that did not bind to the GAL1 UAS, and did not activate transcription of the GAL1 gene as might be expected (see Figure 38–18). If, however, the lexA operator—the DNA sequence normally bound by the LexA DBD—was inserted upstream of the promoter of theGAL gene to replace the normal GAL1 enhancer, the hybrid LexA Gal4 AD fusion protein bound to this chimeric gene (at the substituted lexA operator) and activated transcription of GAL1. This general experiment has been repeated many times with a large array of different DBDs and ADs. Collectively, such data demonstrates that the DBDs and ADs of many transcription factors can function independently.

Fig1. Domain-swap experiments demonstrate the independent nature of DNA-binding and transcription activation domains. The yeast GAL1 gene contains an upstream activating sequence/enhancer (UAS_GAL /Enhancer) that is bound by the multi domain DNA binding regulatory transcriptional activator protein Gal4. Gal4, like the lambda cI protein is modular, and contains an N-terminal DNA binding domain (DBD) and a C-terminal activation domain (AD). When the intact Gal4 transcription factor binds the GAL1 UAS_GAL enhancer, activation of GAL1 gene transcription ensues [(A); Active]. Control experiments demonstrate that all three GAL1 gene specific components [ie. cis- and trans-active components: UAS_GAL DNA enhancer, Gal4 DBD and Gal4 AD) are required for active transcription of the natural GAL1 gene, as expected [(B), (C), (D), (E), (F)-all Inactive]. A chimeric protein, in which the DBD of Gal4 is replaced with the DBD of the E coli-specific operator DNA binding protein LexA fails to stimulate GAL1 transcription because the LexA DBD cannot bind to the UAS_GAL /Enhancer [(G); Inactive]. By contrast, the LexA DBD-Gal4 AD fusion protein does activate GAL1 transcription when the LexAoperator (the natural target for the LexA DBD) is inserted upstream of the GAL1 promoter region, replacing the normal UAS_GAL /Enhancer [(H); Active].

The hierarchy involved in assembling gene transcription activating complexes includes proteins that bind DNA and transactivate; others that form protein–protein complexes which bridge DNA-binding proteins to transactivating proteins; and others that form protein–protein complexes with components of coregulators or the basal transcription apparatus. A given protein may thus have several modular surfaces or domains that serve different functions (Figure 2). As described in Chapter 36, the primary purpose of these molecules is to facilitate the assembly and/or activity of the basal transcription apparatus on thecis-linked promoter. Not shown here, but DNA-binding repressor proteins are organized similarly with separable DBDs and silencing domains, SDs.

Fig2. Proteins that regulate transcription have several domains. This hypothetical transcription factor has a DBD that is distinct from a ligand-binding domain (LBD) and several activation domains (ADs) (1-4). Other proteins may lack the DBD or LBD and all may have variable numbers of domains that contact other proteins, including coregulators and those of the basal transcription complex.

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مواضيع ذات صلة

Regulation of Messenger RNA Stability Provides Another Control Mechanism

Eukaryotic Genes Can Be Amplified or Rearranged During Development or in Response to Drugs

Several Structural Motifs Compose the DNA-Binding Domains of Regulatory Transcription Factor Proteins

Reporter Genes Are Used to Define Enhancers & Other Regulatory Elements That Modulate Gene Expression

Certain DNA Elements Enhance or Repress Transcription of Eukaryotic Genes

Epigenetic Mechanisms Contribute Importantly to the Control of Gene Transcription

The Chromatin Template Contributes Importantly to Eukaryotic Gene Transcription Control

The Genetic Switch of Bacteriophage Lambda (λ) Provides Another Paradigm for Understanding the Role of Conditional Regulatory Protein-DNA Interactions in Transcriptional Control in Eukaryotic Cells

Analysis of Lactose Metabolism in E. coli Led to the Discovery of the Basic Principles of Gene Transcription

Biologic Systems Exhibit Three Types of Temporal Responses to a Regulatory Signal

Regulated Expression of Genes is Required for Development, Differentiation, & Adaptation

Posttranslational Processing Affects the Activity of many Proteins