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protein is present. The autoradiograph of a gel will contain a ladder of bands representing

the partially digested fragments. Where DNA has been protected no bands appear; this

region or hole is termed the DNA footprint. The position of the protein-binding sequence

within the DNA may be elucidated from the size of the fragments either side of the

footprint region. Footprinting is a more precise method of locating a DNA–protein

interaction than gel retardation; however, it also is unable to give any information as

to the precise interaction or the contribution of individual nucleotides.

In addition to the detection of DNA sequences that contribute to the regulation of gene

expression an ingenious way of detecting the protein transcription factors has been

developed. This is termed theyeast two-hybrid system. Transcription factors have two

domains, one for DNA binding and the other to allow binding to further proteins

(activation domain). These occur as part of the same molecule in natural transcription

factors, for example TFIID (Section 5.5.4). However they may also be formed from two

separate domains. Thus a recombinant molecule is formed encoding the protein under

studyasa fusion with the DNA-binding domain.Itcannothoweveractivate transcription.

Genes from a cDNA library are expressed as a fusion with the activator domain; this also

cannot initiate transcription. However, when the two fractions are mixed together

transcription is initiated if the domains are complementary (Fig. 6.44). This is indicated

5  3 

Fusion protein production

A and proteins (e.g. C)

Gene library production

Fusions of B and proteins (e.g. D)

CD

A B

C: Specific protein
is fused with A

A: DNA-binding domain

D: Specific protein

is fused with B

B: Activation domain

Promoter Reporter

C

D

A B

Reporter gene expression

C + D interact

A + B then allow transcription activation

5  3 

Fig. 6.44Yeast two-hybrid system (interaction trapping technique). Transcription factors have two domains,

one for DNA binding (A) and the other to allow binding to further proteins (B). Thus a recombinant molecule

is formed from a protein (C) as a fusion with the DNA-binding domain. It cannot, however, activate transcription

alone. Genes from a cDNA library (D) are expressed as a fusion with the activator domain (B) but also

cannot initiate transcription alone. When the two fractions are mixed together, transcription is initiated

if the domains are complementary and expression of a reporter gene takes place.

247 6.8 Analysing genes and gene expression