Transcript Ribosome stalls at trp codons, allowing 2+3 pairing Transcription

Prokaryotic Gene Regulation
Coordinate regulation of genes
involved in similar functions
Types of Control
Negative Control Product of regulatory gene
inhibits transcription
Positive Control Product of regulatory gene
enhances transcription
Operon
• Unit of coordinate gene expression
• Includes structural genes and their
adjacent regulatory elements
• We will consider
– Lac operon (inducible)
– Ara operon (inducible)
– Trp operon (repressible)
Types of Operons
Inducible
Initial condition: OFF
Inducer switches operon ON
Repressible Initial condition: ON
Repressor switches operon OFF
Regulation of the Lac Operon
Pcrp crp
Pi
I
P
O
Structural Genes
DNA Function
Pcrp
crp
Pi
I
P
O
Lac Z
Lac Y
Lac A
Lac Z
Lac Y Lac A
Protein Function
Promoter for crp gene
Gene for CAP protein
Positive regulator
Promoter for I gene
Gene for Lac Repressor
Negative regulator
Promoter for Structural Genes
Operator
Gene for B-galactosidase
Cleaves lactose
Gene for Permease
Lactose transport
Gene for Acetylase
Unknown
Transcription from the Lac Operon
Pcrp crp
Pi
I
P
Pol
O
Lac Z
Lac Y Lac A
Transcription
Z, Y, A mRNA
Translation
B-galactosidase
Permease Acetylase
RNA polymerase binds to the promoter and
produces a polycistronic mRNA from the
Lac Z, Y and A genes. All three proteins are
produced.
Regulation of the Lac Operon: Low lactose,
High glucose
Pcrp crp
crp mRNA
Inactive
CAP protein
Pi
I
I mRNA
Active
repressor
P
Pol
O
Lac Z
Lac Y Lac A
No mRNA produced
No Z, Y, A proteins produced
Transcription from Pcrp and Pi is constitutive:
always expressed in an unregulated fashion.
Active repressor binds to operator and prevents
RNA polymerase from reaching structural genes.
Regulation of the Lac Operon: High lactose,
High glucose
Pcrp crp
Pi
I
P
Pol
O
Lac Z
Lac Y Lac A
Transcription
crp mRNA
I mRNA
Z, Y, A mRNA
Translation
+
Inactive
CAP protein
Lactose
Active
repressor
B-galactosidase
Permease Acetylase
Inactive
repressor
Lactose (inducer) binds to the repressor and
inactivates it. RNA polymerase transcribes
Lac Z, Y and A at low frequency.
Regulation of the Lac Operon: High lactose,
Low glucose
Pcrp crp
Pi
I
P
Pol
O
Lac Z
Lac Y Lac A
Transcription
crp mRNA
I mRNA
Z, Y, A mRNA
Translation
+
cAMP
Inactive
CAP protein
Active
CAP protein
+
Lactose
Active
repressor
B-galactosidase
Permease Acetylase
Inactive
repressor
cAMP is produced when glucose levels are low. cAMP
activates CAP. Active CAP binds to the promoter to
increase RNA polymerase binding. RNA polymerase
transcribes Lac Z, Y and A at HIGH frequency.
Regulation of the Lac Operon: Low lactose,
Low glucose
Pcrp crp
crp mRNA
Pi
I
I mRNA
P
Pol
O
Lac Z
Lac Y Lac A
No mRNA produced
+
cAMP
Inactive
CAP protein
Active
repressor
No Z, Y, A proteins produced
Active
CAP protein
Although RNA polymerase binding is enhanced by
Active CAP, the operator is blocked by active repressor.
RNA polymerase cannot transcribe Z, Y and A.
Summary of Lac Operon
Regulation
Level of
Lactose
Low
Level of Lac Operon
Glucose
High
Off
Low
Low
Off
High
High
On at low frequency
High
Low
On at high frequency
Mutations of the Lac Operon
Pcrp CRP
Pi
I
Functional genes: I+
P
O
P+ O+
Lac Z
Z+
Lac Y Lac A
Y+ A+
I+
I-
Functional Repressor
Trans-acting
Non-functional Repressor
Trans-acting
Is
Superrepressor
(cannot bind lactose)
Trans-acting
Is > I+> I -
The diffusible product of the I+ or IS allele can
associate with an operator on the same piece of
DNA (cis) or on a separate piece of DNA (trans).
Mutations of the Lac Operon
Pcrp CRP
Pi
I
Functional genes: I+
P
O
P+ O+
Lac Z
Z+
Lac Y Lac A
Y+ A+
P+
P-
Functional Promoter
Cis-acting
Non-functional Promoter
Cis-acting
O+
Oc
Functional Operator
Cis-acting
Non-functional Operator
(Operator Constitutive)
Cis-acting
O-
Non-functional Operator
(Operator region deleted)
Cis-acting
Mutations of the Lac Operon
Pcrp CRP
Pi
I
Functional genes: I+
P
O
P+ O+
Z+
Z-
Functional B-galactosidase
Y+
YA+
A-
Functional Permease
Lac Z
Z+
Lac Y Lac A
Y+ A+
Non-functional gene for B-galactosidase
Non-functional gene for Permease
Functional Acetylase
Non-functional gene for Acetylase
A mutation in one structural gene does not affect
the production of proteins from the other
structural genes.
Lac Operon Mutations, Page 3-24
B-Galactosidase
Permease
No lactose
Lactose
No lactose
Lactose
I+P+O+Z+Y+
--
+
--
+
I-P+O+Z+Y+
I+P-O+Z+Y+
I+P+OcZ+Y+
I+P+O+Z-Y+/ I-P+O+Z+Y+
I+P+OcZ+Y-/ I+P+O+Z-Y+
I+P+OcZ-Y+/ I-P+O+Z+YIsP+O+Z+Y-/ I-P+O+Z-Y+
I+P-OcZ+Y+/ I+P+O+Z-Y-
+
--
+
--
+
--
+
--
+
+
+
+
-+
+
+
---
+
+
----
+
---
+
---
+
---
Arabinose Operon
DNA Function
Protein Function
Ara C
Codes for C protein
Positive and
Negative regulator
I
O
Ara B
Ara A
Initiator (promoter region)
Binds C protein
Operator
Binds C protein
Ara D
Structural gene
Structural gene
Structural gene
C Protein Exerts Positive and
Negative Control of the Ara Operon
Arabinose present
Arabinose absent
Summary of Ara Operon Regulation
Level of
Arabinose
Level of
Glucose
Ara Operon
Low
High
Off
C protein bound to O and I,
Inhibiting transcription
Low
Low
Off
C protein bound to O and I
High
High
On at low frequency
C protein + arabinose bound
to I, enhancing transcription
High
Low
On at high frequency
C protein + arabinose bound
to I and cAMP + CAP bound
to I, enhancing transcription
Tryptophan Operon
trpA
DNA Function
RNA/Protein Function
Trp R
Gene for
repressor
Binds to operator to inhibit
transcription
P
Promoter
O
Trp E, D,
C, B, A
Operator
5’ UTR
(Leader)
Structural
genes
Enzymes acting in pathway to
produce tryptophan.
Gene order correlates with
order of reactions in pathway.
Premature termination of
transcription when trp levels are
high
Control of Trp Operon Transcription
Trp Repressor is Inactive  Initial State: ON
Trp binding activates Repressor  Final State: OFF
tryptophan
Features of the 5’ UTR
•Contains complementary sequences that can form
hairpin structures when transcribed into RNA
•Codes for a stretch of U nucleotides that can act as
a termination signal after a hairpin structure
•Codes for several Trp codons as part of an
unstable protein product
Alternative RNA Structures from 5’ UTR
Termination signal due to
hairpin formed by 3+4 pairing
followed by string of uracils
No termination
signal formed
Formation of termination signal depends on
level of tryptophan carried by tRNA in the cell.
Attenuation
Premature Termination of Transcription
Ribosome translates
trp codons, preventing 2+3 pairing
3+4 pairing forms terminator
Antitermination
Ribosome stalls at trp codons,
allowing 2+3 pairing
Transcription continues
toward trp E, D, C. B, A
Summary of Trp Operon Regulation
Level of
Tryptophan
Trp Operon
On
Low
High
Trp repressor inactive
Lack of attenuation leads to high rate of
mRNA production
Off
Tryptophan + repressor = Active repressor
Reduction of mRNA production by attenuation