Novel Inducible Eukaryotic Gene Expression Systems Based On Prokaryotic Elements

In recent years significant efforts have been directed to developing additional transcription systems which, along the same basic principle exploited by Bujard and colleagues (26), utilize well-known, ligand-induced, transcriptional activators taken from the bacterial world and convert them into eukaryotic transcription factors via simple manipulations such as the addition of ADs. The advantage of these systems is that they rely on DNA binding proteins that: a) do not interfere with eukaryotic transcriptional networks, b) recognize DNA bind ing sites that are not bound by mammalian transcription factors, and c) are capable of binding with high affinity distinct classes of small molecular weight ligands, some of them already used in the clinic. Although these new systems have not yet reached the same level of preclinical characterization of the Tet system, they show highly interesting properties for future translation to clinical applications.

A. A Switch Based on the Quorum-sensing Transcription Factor TraR

Bacteria are capable of ''sensing'' fluctuations in their population density, and respond by changing the pattern of gene expression. This response, called Quorum Sensing, is based on a simple mechanism consisting of a signal molecule, an acylated homoserine lactone (AHL) that accumulates in the immediate external environment, and a cognate transcription factor that it activates (70). Over 50 species of gram-negative bacteria produce AHLs that differ in the acyl side chain: an attractive feature of this two-component regulatory system is the existence of several variants in nature. The bacterial quorum sensing system represents a natural combinatorial library that could be exploited to generate reagents for the development of artificial eukaryotic gene regulation systems. To this end, it is necessary to reengineer the prokaryotic basic module, i.e., the signal and its cognate ''receptor,'' so it may function in eukaryotic cells.

TraR belongs to the LuxR family of transcriptional activators (70,71,72). Binding the small, diffusible Agrobacterium quorum-sensing signal 3-oxo-C8-HSL (Fig. 6) results in TraR activation and subsequent interaction with promoters containing one or more copies of an 18-bp inverted repeat called tra box (73-75). The 3-dimensional structure of TraR has recently been solved. The protein consists of two separate domains; an N-terminal domain with ligand-binding and dimer-izing properties, and a C-terminal domain that binds DNA (76,77). This modular structure makes the LuxR family of transcriptional activators a promising candidate for genetic manipulation.

A hybrid transcription factor was generated by fusing the activation domains VP16-F3 orp65 N-terminally to the cDNA fragment corresponding to the TraR wt protein (34,78). Both activation domains were separated from TraR by a short amino acid linker (79). In addition, a nuclear localization signal was added to the linker region of p65 in order to ensure nuclear localization of the chimeric protein. As in the case of authentic TraR, the chimeric transcription factors bind to DNA containing a tra box only in the presence of a cognate ligand, such as 3-oxo-C8-HSL (79). Importantly, 3-oxo-C8-HSL must be present during the protein synthesis reaction, in accordance with previous findings that the ligand is necessary for proper folding of TraR (80,81). In fact, the 3-dimensional structure shows that the ligand-binding site is deeply embedded in the protein core. This requirement brings the advantage that the system is totally silent in the absence of the ligand, but is

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