23.7 In Vivo Experimental Methods for Interactions

325

where tau equals left brace 4 l 0 k Subscript normal a Baseline k Subscript normal d Baseline plus left bracket k Subscript normal a Baseline left parenthesis l 0 minus r 0 right parenthesis minus k Subscript normal d Baseline right bracket squared right brace Superscript negative 1 divided by 2τ = {4l0kakd + [ka(l0 −r0) −kd]²}^−1/2 and upper Y equals left parenthesis k Subscript normal d Baseline plus k Subscript normal a Baseline left bracket l 0 plus r 0 right bracket minus 1 divided by tau right parenthesis divided by left parenthesis k Subscript normal d Baseline plus k Subscript normal a Baseline left bracket l 0 plus r 0 right bracket plus 1 divided by tau right parenthesisY = (kd + ka[l0 + r0] −1/τ)/

(kd + ka[l0 + r0] + 1/τ), subscripts 0 denoting the initial concentrations of R and L,

and the temporal evolution of the activated form is then found from

r left parenthesis t right parenthesis equals r 0 exp left bracket ln left parenthesis StartFraction 1 minus upper Y e Superscript negative t divided by tau Baseline Over 1 minus upper Y EndFraction right parenthesis minus StartFraction t Over tau EndFraction right bracket periodr(t) = r0 exp

[

ln

(1 −Ye−t/τ

1 −Y

)

−^t

τ

]

.

(23.19)

23.7 In Vivo Experimental Methods for Interactions

Several methods have been developed involving manipulations on living cells.

Although sometimes called in vivo, they cannot be called noninvasive. The cell

is assaulted quite violently: Either it is given unnatural, but not lethal reagents, or

it is killed and swiftly analysed before decay sets it, the interactions present at the

moment of death being assumed to remain until they have been measured.

23.7.1

The Yeast Two-Hybrid Assay

Suppose that it is desired to investigate whether protein A interacts with protein B.

The general concept behind this type of assay is to link A to another protein C,

and B to a fourth protein D. C and D are chosen such that if they are complexed

together (via the association of A and B), they can activate some other process (e.g.,

gene expression) in yeast. In that case, C could be the DNA-binding domain of a

transcription factor, and D could trigger the activation of RNA polymerase. The name

“hybrid” refers to the need to make hybrid proteins (i.e., the fusion proteins A-C and

B-D). If A indeed associates with B, when A-C binds to the promoter site of the

reporter gene, B-D will be recruited and transcription of the reporter gene will begin.

The advantage of the technique is that the interaction takes place in vivo.

Many variants of the basic approach can be conceived and some have been real-

ized; for example, A could be anchored to the cell membrane, and D (to which B is

fused) could activate some other physiological process if B becomes bound to the

membrane.

Disadvantages of the technique include the following: the cumbersome prepara-

tions needed (i.e., making the fusion proteins by genetic engineering); the possible,

or even likely, modification of the affinities of A and B for each other, and of C and D

for their native binding partners, through the unnatural fusion protein constructs; and

the fact that the interactions take place in the nucleus, which may not be the native

environment for the A–B interaction. It is also restrictive that interactions are tested

in pairs only, although this does not seem to be a problem in principle; transcription

factors requiring three or more proteins to activate transcription could be used.