Infection

Streptavidin coated magnetic bead

FIGURE 26.15 Biopanning for RNA-Binding Proteins

To identify RNA-binding proteins (RNA-BP), a bait RNA (blue) is used that is linked to a biotinylated oligonucleotide. The bait RNA is incubated with a full-length phage display library. In this example, T7 gene 10B DNA is fused to a gene library that includes RNA binding protein genes (shown here in green). Those phage that express the full length RNA binding protein on the outside will bind to the RNA bait. This in turn is bound via the biotinylated oligonucleotide to magnetic beads coated with streptavidin. The captured phage is eluted from the magnetic beads by free biotin and is used to infect E. coli. Isolating the T7 DNA and sequencing the insert identifies the gene for the RNA binding protein.

Proteins can be screened to see which other proteins they bind using two hybrid analysis.

The test proteins (bait and prey) are fused separately to the two halves of a transcription factor. If the bait and prey bind each other they will reassemble the transcription factor and activate the genes it controls.

by association. It is assumed that the binding of a novel protein to one that is well characterized may provide some hint as to function.

Two-hybrid analysis depends on the modular structure of transcriptional activator proteins. Many of these proteins consist of two domains, a DNA binding domain and an activation domain. The DNA binding domain (DBD) recognizes a specific sequence in the DNA upstream of a promoter and the activation domain (AD) stimulates transcription by binding to RNA polymerase (Fig. 26.17). Provided that the two domains interact, they will activate transcription. It is not usually necessary for the two domains to be covalently joined to form a single protein.

In the two-hybrid system, both the DBD domain and the AD domain are fused to two other proteins (X and Y in Fig. 26.17). These two hybrid proteins are referred to as the "bait" (DBD-X) and the "prey" (AD-Y). If the bait captures the prey, i.e. if proteins X and Y interact, a complex will form and the gene will be activated. A convenient reporter gene is used to monitor for a successful interaction.

bait The fusion between the DNA binding domain of a transcriptional activator protein and another protein as used in two-hybrid screening prey The fusion between the activator domain of a transcriptional activator protein and another protein as used in two-hybrid screening

Tethering Technology Allows Small Molecule Screening

The converse problem is to isolate small molecules that bind to a given protein. In particular, a protein may have been chosen as a potential drug or antibiotic target. Consequently small molecules that bind to and inhibit the protein of interest are wanted. A library of small chemical molecules will be synthesized and screened for those that bind to the protein. This may be achieved by a variety of tedious procedures. However, a new approach known as tethering technology, developed by Sunesis corporation, allows greatly improved screening. This approach involves modifying the small molecule library by adding a chemical group that will act as a tether. The target protein is immobi lized and also modified with a tethering group. The two tethering groups are chosen so that they will form a cross link if they come into close contact. In the diagram shown (Fig. 26.16), a disulfide linkage is formed between the two tethering groups. The small molecules carry a short side chain that ends in a masked sulfhydryl group and the protein has a cysteine residue engineered into it whose sulfhydryl group is exposed on the surface close to the binding pocket. When a small molecule fits the binding site on the protein, a cross link can form between the tethering groups and the small molecule is trapped. The small molecule is later released for identification.

Introduce cysteine residue sh sh

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