Yeast Two-Hybrid System

Vladimir Svetlov svetlov at oncology.wisc.edu
Mon Oct 20 17:48:36 EST 1997


In article <34476967.F7DB6ABF at ikp.unibe.ch>, Zen <zen.lu at ikp.unibe.ch> wrote:

> Can someone please tell me how exactly does the yeast two-hybid system
> work? And what are the possible applications?

Yeast two-hybrid systems make use of a fact that one can have an active
transcription activator hold together by non-covalent bonds. In order to
make such an activator, one fuses a (potential) interactor "A" to a
sequence-specific DNA-binding domain [=DBD] (Gal4- or Lexa-derived) and
another interactor ("B") to a polymerase-specific activation domain [=AD]
(say, Gal4- or VP16-derived). By themselves DBD and AD do not interact.
When co-expressed, these two fusions associate through their "A" and "B"
modules and such heteromeric activator stimulates transcription from a
reporter gene, preceded with TATA box and binding site(s) for the DBD. No
interaction - no activation. LacZ is often used as a reporter. Can be used
for mapping the interaction domains or library screenings. 
As for comprehensive analysis of possible applications, Stanley Fields and
Roger Brent (among others) did a pretty good job comparing two-hybrid
system(s) to other methods of protein-protein interactions analysis. I
believe that there is even a monograph on the subject by the former, and a
number of reviews. Fields, Brent, Golemis + Medline = references.
Good luck.
V.



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