298 7.6  High-Throughput Techniques

photobleaching of fluorescent dyes can also be used subsequently to determine the stoichi­

ometry of subunits within a specific prey protein, that is, how many repeating subunits there

are in that single molecule (see Chapter 8).

The most popular current technique for determining putative protein–​protein is the yeast

two-​hybrid assay (also known as two-​hybrid screening, the yeast two-​hybrid system, and

Y2H), which can also be adapted to probe for protein–​DNA and DNA–​DNA interactions.

This uses a specific yeast gene to act as a reporter for the interaction between a pair of specific

biomolecules, most usually proteins. The expression of a gene, that is, whether it is switched

“off” or “on” such that the “on” state results in its DNA sequence being transcribed into an

mRNA molecule that in turn is translated into a peptide or protein (see Chapter 2), is nor­

mally under the control of transcription factors, which bind to the promoter region of the

gene to either suppress transcription or activate it.

In the case of Y2H, an activating transcription factor is used. Activating transcription

factors typically consist of two subunits. One subunit is called the “DNA-​binding domain

(BD)” that binds to a region of the DNA, which is upstream from the promoter itself, called

the “upstream activating sequence.” Another is called the “activation domain (AD),” which

activates a transcription initiation complex (TIC) (also known as the preinitiation complex),

which is a complex of proteins bound to the promoter region, which results in gene expres­

sion being switched “on.” In YTH, two fusion proteins are first constructed using two separate

plasmids, one in which BD is fused to a specific bait protein and another in which AD is fused

with a candidate prey protein, such that the BD and AD subunits will only be correctly bound

to produce activation of the TIC if the bait and prey proteins themselves are bound together

(Figure 7.6b). In other words, the gene in question is only switched “on” if bait and prey bind

together.

In practice, two separate plasmids are transformed into yeast cells using different select­

able markers (see Section 7.4). Different candidate prey proteins may be tried from a library

of possible proteins, generating a different yeast strain for each. The reporter gene is typ­

ically selected to encode for an essential amino acid. Therefore, cells that are grown onto

agar plates that do not contain that specific essential amino acid will not survive. Colonies

that do survive are thus indicative of the bait and prey combination used being interaction

partners.

Y2H has implicitly high throughput since it utilizes cell colonies, each ultimately containing

thousands of cells. An improvement to the speed of throughput may come from using fluor­

escent protein tags on the separate AD and BD fusions. Work in this area is still in the early

stages of development but may ultimately enable fluorescence microscopy screening to probe

for potential colocalization of the AD and BD proteins, which to some extent competes with

the lower-​throughput BiFC method (see Chapter 4). There are a number of variants of Y2H,

including a one-​hybrid assay designed to probe protein–​DNA interactions, which uses a

single fusion protein in which the AD subunit is linked directly to the BD subunit, and a

three-​hybrid assay to probe RNA–​protein interactions in which an RNA prey molecule links

together the AD and BD subunits. Although optimized in yeast, and thus ideal to probe

interactions of eukaryotic proteins, similar systems have now been designed to operate in

model bacterial systems.

Methods that use cell lysates for probing the interactions of biomolecules are fast but

run the risk that the spatial and temporal context of the native biomolecules is lost. The kin­

etics of binding in vivo can be influenced by several other factors that may not be present

in an in vitro assay and so can differ in some cases by several orders of magnitude. Y2H has

advantages in that the protein interactions occur inside the live cell, though these may be

affected by steric effects of the fusions used but also differences in local concentrations of

the putatively interacting proteins being different to the specific region of the cell in which

the interaction would naturally occur (as opposed to a specific region of the cell nucleus

in the case of Y2H). These issues present problems for systems biology analysis that rely sig­

nificantly on the integrity of molecular binding parameters (see Chapter 9).