Transcriptional activator protein

Alternatively, one interesting drug delivery technique exploits the active transport of certain naturally-occurring and relatively small biomacromolecules across the cellular membrane. For instance, the nuclear transcription activator protein (Tat) from HIV type 1 (HlV-1) is a 101-amino acid protein that must interact with a 59-base RNA stem-loop structure, called the traus-activation region (Tar) at the 5 end of all nascent HlV-1 mRNA molecules, in order for the vims to replicate. HIV-Tat is actively transported across the cell membrane, and localizes to the nucleus [28]. It has been found that the arginine-rich Tar-binding region of the Tat protein, residues 49-57 (Tat+9 57), is primarily responsible for this translocation activity [29]. 

Figure 5.1. Yeast two-hybrid system. Interaction of proteins X and Y upstream of a reporter gene leads to transcriptional activation. Protein X is part of a fusion protein that binds to a site on DNA upstream of the reporter gene by means of a DNA binding domain. Protein Y is part of a fusion protein that contains a transcriptional activation domain. Interaction of proteins X and Y places the activation domain in the vicinity of the reporter gene and stimulates its transcription.

A bacterial two-hybrid system has been developed that, similar to the yeast system, functions via activation of transcription (Dove and Hochschild, 1998 Joung et al., 2000). RNA polymerase (RNAP) in E. coli consists of an enzymatic core composed of the a, (3, and (3 subunits in the stoichiometry a2(3(3, and one of several c factors that enable the enzyme to recognize specific promoters (Heilman and Chamberlin, 1988). Many bacterial transcriptional activator proteins bind the promoters they regulate and interact directly with subunits of RNAP. The most commonly observed contact is between activator proteins and the a subunit of RNAP (Ebright and Busby, 1995). The function of the a subunit is to initiate the assembly of RNAP by forming a dimer (Igarashi et al., 1991). 

FIG. 2. Model of the interlocked feedback loop mechanism in Drosophila. The perjtim loop (left) and dClk loop (right) are shown. Transcriptional activator genes, black italics transcriptional activator proteins, black capitals transcriptional repressor genes, grey italics transcriptional repressor proteins, grey capitals. Filled arrows, transcriptional activation open... 

As in procaryotes, there are transcription activating proteins, the transcriptional activators, as well as proteins which inhibit transcription, the transcriptional repressors. Of the two classes, the activators are the most extensively studied and characterized. The mechanism of eucaryotic repressors has only been clarified in a few cases. 

Figure 16.1. Schematic representation of the yeast two-hybrid system for evaluation of protein-protein interactions. Haploid yeast of a and a cells can mate to form (a/a) diploid cells. (A) If two test proteins, PT1 and PT2—expressed in (a/a) diploid cells as fusion proteins of DNA binding domains (DAB) and activation domains (AD) of yeast gene-transcript activator proteins—bind to each other, the binding interaction allows the diploid cells to grow in histidine selection media. Histidine selection media is permissive for diploid cells that express the HISS reporter gene only if PT1 and PT2 interact. (B) If PT1 and PT2 do not interact, no HISS gene product is expressed and the hybrid cell cannot grow in histidine media.

Each of the monomeric proteins c-jun and c-fos, as well as other members of the leucine zipper family, has an N-terminal DNA-binding domain rich in positively charged basic amino acid side chains, an activation domain that can interact with other proteins in the initiation complex, and the leucine-rich dimerization domain.363 The parallel coiled-coil structure (Fig. 2-21) allows for formation of either homodimers or heterodimers. However, cFos alone does not bind to DNA significantly and the cjun/cFos heterodimer binds much more tightly than does cjun alone.364 The yeast transcriptional activator protein GCN4 binds to the same 5 -TGACTCA sequence as does the mammalian AP-1 and also has a leucine zipper structure.360 364 365... 

The DNA binding ability of peptide nanostructures has also been reported by the artificial dimerization of peptide sequences corresponding to the contact region of the transcriptional activator protein GCN4. Cuenoud and Schepartz... 

Hope, I.A. Struhl, K. (1987). GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA. The EMBO Journal 6, 2781-4. 

Table 2.1 Examples of DNA sequence specific transcription factors that regulate stress inducible promoters now being used in therapeutic applications. Shown are the stress inducers, transcriptional activating proteins and consensus response elements recognized by the transacting proteins...

As outlined in Fig. 7.1, the Y2H system consists of two protein chimeras, and a reporter gene downstream from the binding site for the transcriptional activator. If the two proteins of interest (X and Y) interact, they effectively dimerize the DNA-binding protein chimera (DBD-X) and the transcription activation protein chimera (AD-Y). Dimerization of the DBD and the transcription AD helps to recruit the transcription machinery to a promoter adjacent to the binding site for the transcriptional activator, thereby activating transcription of the reporter gene. 

Christensen, J., Cotmore, S. F., and Tattersall, P. (1995). Minute virus of mice transcriptional activator protein NSl binds directly to the transactivation region of the viral P38 promoter in a strictly ATP-dependent manner./. Virol. 69, 5422-5430. 

Irrespective of these advances, the limited understanding of the genetic systems that control bacterial responses to pollutant species and the specificity of the interaction between the transcription activator protein and its chemical effector intrinsically restrict the construction and use of these... 

The Tat protein was one of the first nonclassical transport vectors to be recognized. A nuclear transcription activating protein of the HIV-1 retrovirus,90 Tat was initially... 

Moye-Rowley W.S., Harshman, K.D., and Parker, G.S. (1989) Yeast YAPl encodes a novel form of the jun family of transcriptional activator proteins. Genes and Development, 3, 283—292. 

Transannular heterobivalent inhibitor 399 Transcription activator protein 1428 Transcription gene silencing (TGS) 571 Transdermal therapeutic system 1382 Transfectants... 

Chen, Y. C., Liang, Y. C., Lin-Shiau, S. Y, Ho, C.-T., and Lin, J. K. 1999. Inhibition of TPA-induced protein kinase C and transcription activator protein-1 binding activities by theaflavin-3,3 -digallate from black tea in NIH3T3 cells. J. Agric. Food Chem. 47 1415-21. 

Chen Y., Ebright Y. W., Ebright R. H. (1994) Identification of the target of a transcription activator protein by protein-protein photocrosslinking. Science 265 90. 

Zhou Y., Pendergrast P. S., Bell A., Williams R., Busby S., EbrightR. H. (1994) The functional subunit of a dimeric transcription activator protein depends on promoter architecture. EMBO J. 13 4549. 

For a laboratory new to the three-hybrid assay, we recommend beginning with the yeast two-hybrid system, which is based on reconstitution of a eukaryotic transcriptional activator protein. Not only is this assay straightforward to practice but also all the necessary strains and plasmids are commercially available. As discussed below, however, there are potential advantages to working in E. coli or using a nontranscription-based assay. Several E. coli-based transcription assays and general protein complementation assays (PCA) have now been developed as two-hybrid assays. Notably, while the E. coli transcription assays have proven amenable to the introduction of small molecule CIDs, the PCAs have not. 

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