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(Continued from page 2)
Models for repressor-operator
interaction. Summarized by Dr. Wolf, using handouts.
CAP protein mutants
altered in DNA binding. Ebright et al., Nature
311:232,
1984. Concentrate on the genetic selection employed and the
properties of the mutants rather than on the model building speculations.
Why did the authors map their mutations genetically, rather than
just sequencing them?
Additional techniques
of molecular genetics. A set of handouts will be used by Dr.
Wolf to describe: (i) bacteriophage M13, its life cycle and use
as a cloning vector, including the "blue-white screen" based on
alpha complementation of lacZ; (ii) dideoxy (Sanger) and
automated DNA sequencing; (iii) oligonucleotide-directed mutagenesis
with M13; (iv) PCR and applications to cloning ("add-on" PCR), mutagenesis
("QuikChange"), and gene construction ("soeing").
The basis for DNA binding specificity by CAP using the "loss
of contact" approach. Ebright et al., Proc.
Natl. Acad. Sci. 84:6083, 1987.
Be prepared to relate the in vitro data in this paper for
wild type CAP and the mutant CAP' proteins to the in vivo
properties of the wild type and mutant proteins.
-
Positive
control of an inducible system: the arabinose operon (covered if time permits).
Initial genetic evidence of positive control. Englesburg
et al., J. Bacteriol.
90:946, 1965. Pay particular attention to the complementation
tests and to the Discussion. Be able to compare negative and positive
control. We won't discuss the genetic mapping data.
Evidence for DNA looping in the ara operon.
Dunn et al., Proc. Natl. Acad. Sci. 81:5017,
1984. Be able to explain how the deletion mutants were prepared
and characterized. What is the evidence for DNA looping?
The DNA loop model studied by in vivo footprinting.
Martin et al., Proc. Natl. Acad. Sci.
83:3654, 1986. Be able to describe the steps in in
vivo footprinting. What is an advantage of in vivo footprinting
compared to in vitro footprinting with DNase I?
The model of N. Lee for activation of araBAD
transcription by AraC protein. Lee et al., Proc.
Natl. Acad. Sci. 250:8814,
1987. How does this model
differ from Schleif's and how does Lee reconcile her data with his?
Are there other explanations?
Details of DNA looping. Lobell and Schleif, Science
250:528,
1990. Are the Lee and Schleif
groups reconciled on the mechanism of action of AraC protein? What
remains unknown?
Evidence
for the "light switch" mechanism specifying whether AraC
binds in "trans" (repressing condition) or "cis"
(inducing condition). Saviola
et al., J. Mol. Biol. 278:539-548,
1998. Paper to be covered if time permits.
4. Protein-protein
interactions in the lac operon: the mechanism of transcription activation
by CAP.
Isolation of CAP mutants defective
in transcription activation. Zhou et al., Proc.
Natl. Acad. Sci. 90:6081,
1993. Pay particular attention
to the genetic selection and the assays for DNA binding and DNA
bending.
(Continued on page 4)
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