Gene co-regulation with trans-acting factors

The galactose catabolism GAL4 homodimer protein

pathway in yeast binds to UAS elements

UAS: Upstream Activating Sequence

221.S16
 Yeast GAL system (simplified)

GAL4 GAL80

transcription (constitutive) transcription (constitutive)

translation translation

OFF
UAS

221.S16 PNAS | 2002 | vol. 99 | no. 13 | 8548-8553

 Yeast GAL system (simplified)

OFF
UAS

galactose + GAL3

-GAL3(galactose)

RNAP II ON
UAS

GAL2
GAL7
GAL10
221.S16 PNAS | 2002 | vol. 99 | no. 13 | 8548-8553
 Yeast GAL system (simplified)

221.S16
 Identification of cis-acting elements using reporter genes

cis-acting elements YFG

YFG reporter gene

typically:

proteinfusion

reporter gene
A gene whose phenotypic expression is easily monitored
• lacZ
• GFP

221.S16
 Identification of cis-acting elements using reporter genes
and deletions

cis-acting elements YFG

Expression

YFG reporter gene

100

YFG reporter gene

( ) 1

YFG reporter gene

( ) 100

YFG reporter gene

( ) 1
Both elements are required for
gene expression
221.S16
 DNase footprinting

radioactive label 5’-end of one strand

dsDNA

purified DNA binding protein

221.S16
 DNase footprinting

DNase I:
a sequence nonspecific
endodeoxyribonuclease

221.S16
 Biochemical evidence: DNase footprinting

Gel DNA length

top long

bottom short

221 S16 Krebs et al. (2011) F 19.21

 Upstream Activating Sequence (enhancer)
of the yeast GAL genes

221.S16
 Upstream Activating Sequence (enhancer)
of the yeast GAL1 and GAL10 genes

UDP-glucose-4-epimerase galactokinase

GAL4 binding to the UAS

activates GAL1 transcription
~1000-fold

221.S16
 High resolution genetic mapping

Human b-globin promoter

221.S16
 Yeast GAL4 protein structure and function

DNA binding

dimerization

N-terminal 65 aa

http://gibk26.bse.kyutech.ac.jp/jouhou/image/gallery.html

http://www.umass.edu/molvis/workshop/imgs/polyview.htm Zinc finger domains

221.S16
 Zinc finger DNA binding domain

https://youtu.be/WyU2v7HT6bw?t=3s
http://www.youtube.com/watch?v=GRL_rdB30GY&feature=relmfu

221.S16
 Mutations that affect specific functions often
map in specific protein domains

lac repressor (monomer)

221.S16 Hartwell et al. (2008) Fig 17.8c Krebs et al. (2008) Fig 26-16
 GAL4 transcription factor with function-specific domains

1-65

221.S16
 GAL4 transcription factor with function-specific domains

221.S16