Transcript Negative regulation

The need for gene regulation
Bacterial genome
Human genome
4,000 genes
100,000 genes
•Not all expressed at any one time
•May need very high levels e.g.
translation elongation factors
•May need very low levels e.g. some
DNA repair enzymes
•Expression needs to vary with time
and cell type - otherwise every cell
would be the same and there would
be no organisms except microbes
TERMINOLOGY
Housekeeping genes:
required all the time
Constitutive expression:
gene constantly switched on
Inducible gene:
gene switched on only in defined
conditions
Repressible gene:
gene switched off in defined
conditions
E. COLI PROMOTER
CONSENSUS SEQUENCE
TTGACA
(-35 region)
n17
TATAAT n5-9
(-10 region)
Consensus sequence has highest
affinity for RNA polymerase and highest frequency of
transcription initiation
THE TRYPTOPHAN OPERON
promoter
E
D
C
B
A
Operator (binding site for repressor)
trp A,B,C,D,E are genes that code
for enzymes of tryptophan synthesis
Tryptophan absent inactive
repressor
gene on
promoter/
operator
start site
RNA polymerase
binds to promoter
Tryptophan present
tryptophan
active
repressor
promoter/
operator
gene off
start site
NEGATIVE REGULATION
OF THE TRP OPERON
• Operator sequence lies within
the trp promoter sequence
• In absence of tryptophan the
repressor protein is unable to
bind to the operator hence RNA
polymerase can bind to the
promoter and initiate transcription
• In presence of tryptophan, 2
molecules of tryptophan bind to
the repressor protein allowing it
to bind to the operator. This
prevents RNA polymerase from
binding to the promoter, hence
transcription is blocked.
Lactose
Outside
Galactoside
permease
Cell
Membrane
Inside
Lactose
b-galactosidase
Galactose
+
Glucose
Allolactose
The lac operon
Pi I
P
O
Z
Y
A
Pi : promoter for I gene
P : promoter for lac Z, A, Y genes
O : operator for lac Z, A, Y genes
Z : b-galactosidase
Y : galactoside permease
A : thiogalactoside transacetylase
LAC OPERON IN REPRESSED STATE
Repressor
mRNA
DNA PI
I
P O Z
Y
No transcription
A
lac operon is subject to
negative regulation
• In absence of lactose the lac operon is
repressed - only a few copies of b-gal
present in the cell.
• In presence of lactose the lac operon is
induced.
• Mutations in I gene (upstream of lac
operon) or operator cause constitutive
expression of lac operon gene products.
• If I gene is defective, can introduce
correct copy of I gene on another DNA
molecule and recover gene repression.
i.e. I gene product is a diffusible
product that represses the lac
operon
INDUCTION OF THE LAC OPERON
A. No lactose: repressor bound to
operator - inhibits transcription
P
O
lac genes
B. lactose present: release of repressor/
inducer complex - allows transcription
RNA polymerase binds
P
O
lac genes
mRNA
Negative regulation
(bound repressor
inhibits transcription)
A) Molecular signal causes dissociation
of regulatory protein from DNA
DNA
Negative regulation
(bound repressor
inhibits transcription)
B) Molecular signal causes binding
of regulatory protein to DNA
DNA
POSITIVE REGULATION
OF LAC OPERON
• Glucose is preferred carbon source
• If glucose is present, lac operon is
repressed - even if lactose is present
•This is catabolite repression
• Repressive effect mediated via cAMP
and a protein called catabolite gene
activator protein or CAP
• In absence of glucose CAP binds to
site near promoter and enhances
transcription 50 fold
Low glucose, low lactose
CAP
cAMP-CAP
No transcription
P O lac genes
Lac repressor (lacI)
High glucose, low lactose (low cAMP)
No transcription
CAP
P O lac genes
Low glucose, high lactose
Transcription
CAP
P O lac genes
RNA polymerase
High glucose, high lactose (low cAMP)
No transcription
CAP
P O lac genes
lac promoter is weak RNA
polymerase binding site
lac promoter
TTTACA
TATGTT
-35
-10
Promoter consensus sequence
TTGACA
-35
TATAAT
-10
Summary of Lecture 2
• Negative regulation - active form of
repressor protein binds to operator,
inhibiting transcription
• Trp operon (tryptophan biosynthesis)
repressor protein binds to trp operator
when tryptophan is present
• Lac operon - repressor protein binds to
lac operon when lactose is absent
• Lac operon also subject to positive
regulation - in absence of glucose CAP
binds upstream from lac operon greatly
enhancing transcription