CAP-DNA COMPLEX SHOWN AS ORIENTED HETERODIMER

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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.

5. Transcription activation by pre-recruitment.

Recruitment vs. post-recruitment. Ptashne and Gann, Nature 386:569-577, 1997. These two mechanisms are summarized and examples are given.

Recruitment is the mechanism by which MelR activates transcription of the melAB promoter . Grainger et al. J. Bacteriol. 186:6938-6943, 2004. Convincing in vivo evidence for recruitment using chromatin immunoprecipitation (ChIP).

Initial evidence for pre-recruitment as a new mechanism of transcription activation. Griffith, Shah, et al., Biochem. Biophys. Res. Commun. 291:979-986, 2002. What is the key argument supporting the existence of the pre-recruitment mechanism? Are there alternative explanations and how can the model be tested?

Genetic evidence for pre-recruitment. Griffith and Wolf, J. Mol. Biol. 344:1-10, 2004. What is the genetic evidence? How good is it? What else is needed?

Target site on RNA polymerase for interaction with SoxS during binary complex formation and the physiological consequence of this interaction. Shah and Wolf, J. Mol. Biol. 343:513-532, 2004. What is the yeast two-hybrid system and how was it used to identify the protein-protein interactions between SoxS and RNA polymerase? Does data from this system assure that the same interactions occur in E. coli? What is the affinity immobilization assay? What role does induction of SoxS synthesis play in regulation of rRNA synthesis during oxidative stress? Does this make sense physiologically? In other words, does this "redeployment" increase the chance that the cell will survive the stress?