440 Setzer, Schulman, and Bumbulis
X-gal. Colony color is assessed at an empirically determined time after robust
colony growth has occurred. For analysis of the Xenopus TFIIIA–5S rRNA
gene interaction, this was done typically after 2–3 d of growth at 30°C and an
additional 2–3 d at room temperature. For the system to be exploited success-
fully, one must be able to distinguish reproducibly the color of strains contain-
ing both the expression and reporter plasmids from that of all the other control
strains (lacking either expression of the fusion protein containing the DNA-
binding domain[s] of interest or the cognate recognition site in the reporter
construct, or both). If this is not the case, it may be possible to correct the
problem by manipulation of parameters as described in Note 5. Of course, it is
also possible that the particular interaction being studied will not be amenable
to analysis with this method; among other reasons, this could result from a
low-affinity/specificity interaction or from the existence of endogenous yeast
factors that interact with the binding site introduced into the reporter plasmid,
resulting in high levels of transcriptional activity in the absence of the interac-
tion being targeted for study.
3.2. Error-Prone PCR
The DNA-binding protein or its recognition site can be subjected to random
mutagenesis using error-prone PCR. In the following protocol, we assume that
the DNA-binding protein is targeted for mutagenesis, but the procedure can be
adapted readily for mutagenesis of the recognition site.
1. Set up a 50-µL polymerase chain reaction mixture containing 10–50 ng of plas-
mid DNA containing the sequence encoding the region to be mutagenized. This
can be the expression plasmid itself (see Note 8) or another plasmid containing
the sequence of interest. In addition, add 5 mL of 10X PCR buffer lacking MgCl
2
,
long amplification primers (see Subheading 2.2., Fig. 2, and Note 6) to a final
concentration of 0.3 µM each, three deoxynucleoside triphosphates to a final con-
centration of 1 mM each, the fourth deoxynucleoside triphosphate to a final con-
centration of 0.2 mM, MgCl
2
to a final concentration of 3 mM, MnCl
2
to a final
concentration of 0.05 mM, and 1 unit Taq DNA polymerase (see Note 9).
2. Amplify using a thermal cycler for 25 cycles, with each cycle being 94°C for
1 min, 42°C for 2 min, and 72°C for 1 min. After 25 cycles, use a final extension
step of 72°C for 7 min (see Note 9).
3. Use the crude PCR product (without purification) in a yeast transformation with
linearized/gapped target plasmid as described in Subheading 3.3.
3.3. Yeast Transformation and Homologous Recombination
1. Prepare a stock of linearized or gapped target plasmid by digesting to completion
with one or two restriction endoncleases that result in ends corresponding to the
site at which integration of the mutagenized DNA fragment is to occur. As an
example, with an expression plasmid derived from pG1, this might be a double