Gene Cloning

The Analysis of the Regulation of Gene Expression 347
Deletion analysis and site-directed mutagenesis
So far, we have discussed using reporter gene assays, EMSAs and footprinting
to characterize DNA containing promoters and/or enhancer regions.
Footprinting allows us to map the precise position of binding sites for transcription
factors within a DNA fragment, but it is not a robust technique and
normally requires purified protein. If you do not have a purified system, you
need another strategy to try and map the important elements within a larger
genomic fragment. Deletion analysis and site-directed mutagenesis combined
with reporter gene assays and EMSAs provide a relatively robust strategy
to further characterize a promoter or enhancer region.
As an example of deletion analysis we will look at how you might analyze
the promoter region of a prokaryotic gene. The first task is to clone the promoter
region into a promoter probe vector to generate a reporter gene construct
as discussed in Section 11.3 and to determine the conditions in
which proper transcriptional regulation is observed in the reporter gene
assay. You would then construct a series of derivatives in which sequences
are deleted from the promoter region and the activity of these promoter
derivatives would then be determined. You could use a reporter gene assay
and/or EMSAs to study the different promoter derivatives. For example, if
you wished to determine the upstream limit of your promoter, you would
create a set of nested deletions where the 5′ limit of your promoter is successively
closer to the transcription start (Figure 11.18a) and analyze the
deletions using a reporter gene assay. Deletion of sequences that are not
important for regulation would not affect promoter activity, but deletion of
an important region will lead to deregulation. The deletions can either be
made by exploiting sites for restriction enzymes or more typically by PCR.
If using PCR you would use a common 3′ primer in all PCR reactions and
then use a set of 5′ primers to generate the series of nested deletions. The
primers would incorporate a recognition site for a restriction enzyme to
facilitate cloning (see Section 3.17).
In addition to making nested deletions you can use site-directed mutagenesis
to create specific deletions or change specific base sequences
within a promoter region (Figure 11.18b and c). This would allow you to
map more precisely any important sites within the DNA. These defined
deletions or sequence changes can be characterized in a reporter gene
assay or EMSAs.
Deletion analysis was used to investigate the nirB promoter discussed
above (Figure 11.19). Six promoter constructs were cloned in which successively
larger regions of the upstream promoter sequence was deleted. A
promoter that extends to 208 base pairs upstream from the transcription
start (–208) has normal regulation, as does a promoter that extends to –149.
Promoters that extend to –87 or –73 have slightly higher activity under
anaerobic conditions but can no longer respond to nitrite. A promoter that
only extends to –47 shows limited activation, and one that extends to –19 is