Gene Cloning

350 Gene Cloning
bind the transcription factor and no shift will be observed. If, however, the
mutation stops the transcription factor binding, the unlabeled fragment
will not be able to compete and you will get a shift of the labeled “wildtype”
fragment.
Q11.8. Are laboratory-based techniques, such as deletion analysis, the only
approach to identify possible binding sites for transcription factors?
11.5 Identifying Protein Factors
Techniques such as reporter gene assays and EMSAs in combination with
deletion analysis and site-directed mutagenesis can identify sites within promoter/enhancer
regions that are important for proper regulation of gene
transcription. However, often you do not know the identity of the protein
factor(s) that bind these sites. In order to fully characterize the promoter it is
vital that the transcription factors responsible for regulation are identified. In
the following section we will discuss three techniques – affinity purification,
the yeast one-hybrid screen and expression library screening – that can be
used to identify the transcription factors that bind a specific sequence.
Affinity purification
One strategy that can be used to purify and identify a transcription factor is
to use its ability to bind specifically to a DNA sequence as an aid to protein
purification (Figure 11.20). Once the site on the DNA which is bound has
been defined using deletion or site-directed mutagenesis as described
above, this DNA can be synthesized as an oligonucleotide which is then
attached to a matrix that is packed into a chromatography column. If a
crude cell extract prepared from cells that are known to contain the active
transcription factor is now passed down the column, the target factor will
bind to its DNA site and will therefore be retained on the column. The column
can be flushed with buffer to remove all proteins that are interacting
non-specifically before a different buffer is applied that results in the transcription
factor being washed from the column and collected. The transcription
factor is thus purified based on its affinity for its target DNA
sequence. The purification process can be monitored using EMSAs and
SDS PAGE, a technique designed to analyze proteins (Box 5.1) which provides
information about the subunit composition of the transcription factor,
i.e. whether it contains more than one different protein component,
and can also be used to measure the size of the protein components.
Affinity purification can be used in conjunction with more conventional
protein purification techniques such as size exclusion chromatography
and ion exchange chromatography. Once any protein factors have been
purified you can determine the amino acid sequence of the N terminus of
the protein and then design an oligonucleotide probe that can be used to