Lecture 3 The regulation of gene expression in eukaryotes. S....

Lecture 3

The regulation of gene expression in eukaryotes. S. cerevisiae Gal4 as a guide for universal principles of

transcriptional regulation

Tuesday, 6 November 2012

•The Lac operon decides to use or not to use lactose

•The λ phage deciding whether to sail or to jump in an E. coli cell

•Galactose utilization in yeast

•Making stripes in the Drosophila embryo


•Eukaryotes need to cater for the same as bacteria but also sexual reproduction, more complex and social environments

•Eukaryotes have a more complex structure more genes, MORE COMPUTATIONAL POWER


http://es.wikipedia.org/wiki/Archivo:Budding_yeast_Lifecycle.png

Saccharomyces cerevisiae




Glucose Galactose


Gal1

Gal7

Gal2

Gal10

Gal5

Saccharomyces cerevisiae Galactose utilization


Gal1Gal2Gal7Gal10

Gal4 Gal80Gal3 Gal11

There are genes that code for enzymes and genes that code for......something else. Some transcription factors but also other proteins


Gal1Gal2Gal7Gal10


Gal4 encodes a DNA binding protein


GAL1++--

Deletion analysis uncovers a 400 bp region which is sufficient for GAL1 expression, and within it a small region that is essential

Reporters facilitates the analysis of gene regulation

Reporter

ß-galactosidase

Green Fluorescent ProteinGFP

GAL1

TATA


275bp UAS GAL4

GAL1Mig1

@ 400bp

Within the 400bp required for expression, 275bp from the transcription start site there are four binding sites for Gal4 (UASGal4 for Upstream Activating Sequences for Gal4) which promote GAL1 expression

....there is also a binding site for a repressor, Mig1, which represses GAL1 expression in the presence of Glucose.


Gal1Gal2Gal7Gal10


Every gene encoding enzymes has a Gal4 binding site


Gal1



From Ptashne and Gann, Genes and signals

Mig1 is a repressor which ensures that GAL genes are only used when there is no Glucose around. It works in a different manner from bacterial repressors as it acts by recruiting a number of proteins which make the promoter inaccesible to RNA polymerase


GAL1Mig1

GAL1Mig1

GAL1Mig1

Gal80

Gal3

GAL1Mig1

Gal4


No galactose Galactose

OFF ON

Gal3 shuttles between the cytosol and the nucleus and, in the presence of Galactose, binds Gal80 releasing the activity of Gal4 and providing a rapid induction of transcriptional activity

Gal3


GAL1

GAL1

Gal80

RNA polII

GAL1Mig1

Gal11MEDIATORGal4


Gal11 is a component of the MEDIATOR complex which, through an interaction with Gal4, allows the link between Gal4 and the basal transcriptional machinery

Galactose



!

λ repressor (cI)

Gal4 is a transcriptional regulator


From Ptashne and Gann, Genes and signals!

!

λ repressor (cI)



The Gal4 protein is structurally and functionally MODULAR




LexA is a bacterial transcriptional regulator with a high affinity DNA binding site. Of course one has to provide a LexA binding site in yeast but this is no problem.




LexA is a bacterial transcriptional regulator with a high affinity DNA binding site. Of course one has to provide a LexA binding site in yeast but this is no problem.

LexA binding site


How specific is the AD of Gal4?



The modular nature of transcriptional regulators

DNA binding domainGal4, LexA, cI....

Activation domainVP16, Gal4,.......

Repression domainenR, Mig1,.....


DNA binding

Trans Activation

Transcription factors are modular


cell type or tissue specific enhancer

Yeast Gal4 works in Drosophila


lacZ

Fluorescent proteins

cell type or tissue specific enhancer


....and in fish


Summary

•The structure of an eukaryotic cells mirrors (suits) its computational capacity.

•Regulated recruitment and cooperativity are central elements of the transcriptional process that promotes functionality with a ‘small’ number of transcription factors.

•Transcription factors are modular (DNA binding + Activator or Repressor domains).

•The interactions between transcription factors and the basal transcriptional machinery are Universal.


Drosophila development is about making segments, making stripes


In bicoid (bcd) mutants, all anterior structures are missing


Bcd

Bicoid protein

bicoid encodes a transcription factor that distributes itself in a gradient across the embryo (it can do this because the embryo is a syncitium at this early stage)


http://scienceblogs.com/pharyngula/

The position of pattern landmarks is determined by the local concentration of Bicoid




hunchback(hb)

Bcd

HD AD

Bicoid (bcd) No Bicoid (bcd)

Bicoid encodes a transcription factor and the hunchback (hb) gene is a target of Bicoid which responds at a certain concentration


High a!nity Low a!nity

Bicoid gradient hunchback or syntheticbinding sites

reporter expression in embryo

The combination of Bicoid binding sites with different affinities in the promoter of hunchback, determines the pattern of response; the domain

of expression of the hunchback gene


High a!nity Low a!nity

Bicoid gradient hunchback or syntheticbinding sites

reporter expression in embryo

Synthetic High a!nity

Synthetic Low a!nity

The combination of Bicoid binding sites with different affinities in the promoter of hunchback, determines the pattern of response; the domain

of expression of the hunchback gene


Bicoid (bcd)

bcdhb bs GAL1 lacZ

HD AD

Gal4 AD

o

o

Bicoid has a DNA binding domain, homeodomain (HD) and an activation domain (AD) which can be substituted by the yeast Gal4 AD.


A cascade of transcription factors


Borok M J et al. Development 2010;137:5-13

Transcription factor gradients activate even skipped expression.


Bcd

Hb Gt Kr

eve 2


Bcd

Hb Gt Kr

eve 2

Bcd

Hb

eve 2


In summary

•The spatial localization of domains of gene expression linked to pattern formation relies on the deployment of transcription factors in specific domains.

•The deployment is mediated by transcription factors or combinations of transcription factors (activators and repressors).

•The process relies on a combination of regulated recruitment and differential affinities.

•The programmes are time chains of transcriptional regulation.

•The transcriptional mechanism is universal.


Bcd

Hb

eve 2

I lacZYA

lactose Y

lac Operon

Bcd

Hb Gt Kr

eve 2

cI cro cII O P

N

pL

pR

pRE

int

cI cro

LysisLysogeny

Positive autoregulatory loop Bistable switch Feedforward loop


Lecture 3 The regulation of gene expression in eukaryotes. S....

Documents

Transcript of Lecture 3 The regulation of gene expression in eukaryotes. S....