Effects of Arabinose on the Conformation of AraC protein.

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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.((Methods II. A set of handouts will be used by Dr. Wolf to describe: (i) the chain-termination (Sanger) method of DNA sequencing (ii) initial "large scale" DNA sequencing; (iii) first generation automated DNA sequencing; (iv) PCR and applications to cloning, e.g., "add-on" PCR; (v) mutagenesis ("QuikChange"), and gene construction ("soeing"). (v) The Gibson Assembly method of DNA cloning, both the short and complete methodology; (vi) Next generation sequencing, the Illumina method.

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.


3. 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.

Identification of the CAP subunit that interacts with RNA polymerase at the lac promoter by "oriented heterodimers".  Zhou et al., Cell 73:375, 1993. Notice the use of the previously isolated CAP mutants altered in DNA binding (E181V) and in transcription activation (T158A). How are the heterodimers prepared in vivo and in vitro?

The role of the RNA polymerase alpha subunit in CAP-dependent transcription activation at the lac promoter. Igarashi and Ishihama, Cell 65:1015, 1991. Be able to explain how reconstituted RNA polymerase is made. Draw a cartoon depicting the mechanism of CAP-dependent transcription activation of the lac promoter that takes into account the experiments in this and the previous paper.

Summary of the roles of the subunits of RNA polymerase in promoter recognition and transcriptional activation at different promoters.  Busby and Ebright, Cell 79:743, 1994.  Notice that the alpha subunit's C-terminal domain binds to a third promoter recognition element, "UP", in certain strong promoters, e.g., promoters for rRNA genes.  Also, note that sometimes the DNA binding site for a transcription activator, e.g., lambda repressor, overlaps the -35 promoter hexamer (rather than lying upstream) and that when residing there, the activator may make contact with another surface on RNA polymerase than the alpha subunit's C-terminal domain. See also the review by Busby and Ebright. J. Mol. Biol. 293:199, 1999. Lecture by Dr. Wolf.

Transcription activation at class II CAP-dependent promoters. Niu et al., Cell 87:1123-1134, 1996. How were mutants defective in transcription activation at class II promoters isolated? What is alanine scanning mutagenesis and what was learned by applying it to characterization of AR2? What other biochemical approaches were used to demonstrate that AR2 functions through protein-protein interactions with RNA polymerase and how was the target on RNAP identified? How was it determined that AR1 and AR2 affect different steps in CAP-dependent transcription activation?

Crystal structure of the CAP-alphaCTD-DNA complex. Benoff et al., Science 297:1562-1566. Structural confirmation of the protein-protein and protein-DNA contacts made by CAP and the alpha-CTD in ternary complexes as inferred from genetic and biochemical analyses.

Functional interactions between the alphaCTD of RNAP and sigma70 at UP-element and activator-dependent promoters. Chen et al., Mol. Cell. 11:1621, 2003. Summarized by Dr. Wolf.

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