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Eukaryotic Transcriptional Regulation, Part II

Oct 27, 2000

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Announcements:

• Bio312 students rock! Congratulations on a really high rate of success! Here's the plan for the coming week: (I'll distribute protocols on Monday morning)
  • Between now and Mon: make a "streak plate" of E. coli carrying your construct, so that you obtain individual colonies from which to start a pure culture
  • Mon: inoculate a 5 ml LB-amp culture with a single colony of E. coli carrying your construct
  • Tues: use your 5 ml culture to inoculate a 50 ml LB-amp culture that will grow overnight
  • Wed: harvest the cells from your 50 ml culture, do a large-scale, extra-clean DNA prep--high enough quality for use in in vitro transcription/translation reactions
• Don't forget--Monday is the discussion of the Jacob reading, so homework #7 is in class on Monday
• Homework #6 (chapter 9) is due at noon today

Learning objectives for Fri 10/27

Students should be able to…

1. Briefly describe how one might go about isolating a protein that binds to a particular "control region" sequence of DNA
2. In general terms, describe the initiation of eukaryotic transcription, and the levels at which this might be regulated by activators or repressors. (You need not memorize the names of the individual protein complexes involved in this process.)
3. Describe three general ways in which activators or repressors might themselves be regulated.
4. (If we have time to get to this) Explain how the yeast two-hybrid system can be used to identify protein-protein interactions. Describe how this system was developed from the study of a yeast transcriptional activator, Gal4.

Outline:

• Review/summary from Wed
• "trans" elements: Isolating and identifying proteins that bind to control regions
• How activators might activate, and repressors might repress: the complexity of the euk system
• What regulates the regulators?
• (If time permits) An aside…the yeast two-hybrid system

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