The need for gene regulation

1
The need for gene regulation Bacterial
genome 4,000 genes Human genome 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

2
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
3
E. COLI PROMOTER CONSENSUS SEQUENCE
n17
n5-9
TATAAT
TTGACA
(-10 region)
(-35 region)
Consensus sequence has highest affinity for RNA
polymerase - and highest frequency of
transcription initiation
4
THE TRYPTOPHAN OPERON
trp A,B,C,D,E are genes that code for enzymes of
tryptophan synthesis
5
Tryptophan absent
inactive repressor
gene on
promoter/ operator
RNA polymerase binds to promoter
Tryptophan present
active repressor
tryptophan
gene off
promoter/ operator
6
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.

7
Lactose
Galactoside permease
Outside
Cell Membrane
Inside
Lactose
b-galactosidase
Allolactose
Galactose Glucose
8
The lac operon
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
9
LAC OPERON IN REPRESSED STATE
Repressor
mRNA
No transcription
10
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
11
INDUCTION OF THE LAC OPERON
A. No lactose repressor bound to operator -
inhibits transcription
lac genes
P
O
B. lactose present release of repressor/ inducer
complex - allows transcription
RNA polymerase binds
lac genes
P
O
mRNA
12
Negative regulation (bound repressor inhibits
transcription)
A) Molecular signal causes dissociation of
regulatory protein from DNA
DNA
13
Negative regulation (bound repressor inhibits
transcription)
B) Molecular signal causes binding of regulatory
protein to DNA
DNA
14
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

15
Low glucose, low lactose
No transcription
lac genes
P
O
CAP
Lac repressor (lacI)
cAMP-CAP
High glucose, low lactose (low cAMP)
No transcription
lac genes
P
O
CAP
16
Low glucose, high lactose
Transcription
lac genes
P
O
CAP
RNA polymerase
High glucose, high lactose (low cAMP)
No transcription
lac genes
P
O
CAP
17
lac promoter is weak RNA polymerase binding site
lac promoter
TTTACA
TATGTT
-35
-10
Promoter consensus sequence
TTGACA
TATAAT
-35
-10
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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