1. Prokaryotic Gene Regulation
Coordinate regulation of genes involved in similar functions

2. Types of Control Negative Control
Product of regulatory gene inhibits transcription
Positive Control
Product of regulatory gene enhances transcription

3. 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)

4. Types of Operons Inducible Initial condition: OFF
Inducer switches operon ON
Repressible
Initial condition: ON
Repressor switches operon OFF

5. Regulation of the Lac Operon
Pcrp
crp Pi O I P
Lac Z
Lac Y
Lac A
DNA Function
Protein Function
Pcrp
Promoter for crp gene
crp
Gene for CAP protein
Positive regulator Pi
Promoter for I gene I
Gene for Lac Repressor
Negative regulator P
Promoter for Structural Genes O
Operator
Lac Z
Gene for B-galactosidase
Cleaves lactose
Lac Y
Gene for Permease
Lactose transport
Lac A
Gene for Acetylase
Unknown
Structural Genes

6. Transcription from the Lac Operon
Lac Z
Lac Y
Lac A P O Pi I
Pcrp
crp
RNA polymerase binds to the promoter and
produces a polycistronic mRNA from the
Lac Z, Y and A genes. All three proteins are
produced.
Pol
Z, Y, A mRNA
Transcription
B-galactosidase
Translation
Permease
Acetylase

7. Regulation of the Lac Operon: Low lactose, High glucose
Lac Z
Lac Y
Lac A P O Pi I
Pcrp
crp
Pol
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.
Active
repressor
crp mRNA
I mRNA
Inactive
CAP protein
No mRNA produced
No Z, Y, A proteins produced

8. Regulation of the Lac Operon: High lactose, High glucose
Lac Z
Lac Y
Lac A P O Pi I
Pcrp
crp
Active
repressor
crp mRNA
I mRNA
Inactive
CAP protein
Pol
Z, Y, A mRNA
Transcription
Translation
B-galactosidase
Permease
Acetylase
Lactose
Lactose (inducer) binds to the repressor and
inactivates it. RNA polymerase transcribes
Lac Z, Y and A at low frequency. +
Inactive
repressor

9. Regulation of the Lac Operon: High lactose, Low glucose
Lac Z
Lac Y
Lac A P O Pi I
Pcrp
crp
Active
repressor
crp mRNA
I mRNA
Inactive
CAP protein
Lactose +
Pol
Z, Y, A mRNA
Transcription
Translation
B-galactosidase
Permease
Acetylase +
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.
cAMP
Active
CAP protein

10. Regulation of the Lac Operon: Low lactose, Low glucose
Active
repressor
Lac Z
Lac Y
Lac A P O Pi I
Pcrp
crp
crp mRNA
I mRNA
Inactive
CAP protein
cAMP +
Although RNA polymerase binding is enhanced by
Active CAP, the operator is blocked by active repressor.
RNA polymerase cannot transcribe Z, Y and A.
Pol
No mRNA produced
No Z, Y, A proteins produced

11. CAP Protein Structure Allows Binding to DNA
Domains are regions on a protein with specific functions; motifs are characteristic structures within a domain
CAP has a DNA binding domain with a helix-turn-helix structural motif
Helices fit into the major groove on DNA

12. Summary of Lac Operon Regulation
Level of Lactose
Level of Glucose
Lac Operon
Low
High

13. Mutations of the Lac Operon
Pcrp Pi O
CRP I P
Lac Z
Lac Y
Lac A
Functional genes: I P+ O Z Y+ A+ I+
Functional Repressor
Trans-acting I-
Non-functional Repressor Is
Superrepressor
(cannot bind lactose)
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).

14. Mutations of the Lac Operon
Pcrp Pi O
CRP I P
Lac Z
Lac Y
Lac A
Functional genes: I P+ O Z Y+ A+ P+
Functional Promoter
Cis-acting P-
Non-functional Promoter O+
Functional Operator Oc
Non-functional Operator
(Operator Constitutive) O-
(Operator region deleted)

15. Mutations of the Lac Operon
Pcrp Pi O
CRP I P
Lac Z
Lac Y
Lac A
Functional genes: I P+ O Z Y+ A+ Z+
Functional B-galactosidase Z-
Non-functional gene for B-galactosidase Y+
Functional Permease Y-
Non-functional gene for Permease A+
Functional Acetylase A-
Non-functional gene for Acetylase
A mutation in one structural gene does not affect the production of proteins from the other structural genes.

16. 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+Y-
IsP+O+Z+Y-/ I-P+O+Z-Y+
I+P-OcZ+Y+/ I+P+O+Z-Y-

17. Arabinose Operon Ara C I O Ara B Ara A Ara D DNA Function
Protein Function
Ara C
Codes for C protein
Positive and Negative regulator I
Initiator (promoter region)
Binds C protein O
Operator
Ara B
Structural gene
Ara A
Ara D

18. C Protein Exerts Positive and Negative Control of the Ara Operon
Arabinose present
Arabinose absent

19. Summary of Ara Operon Regulation
Level of Arabinose
Level of Glucose
Ara Operon
Low
High

20. Tryptophan Operon Trp R P O Trp E, D, C, B, A 5’ UTR (Leader)
trpA
DNA Function
RNA/Protein Function
Trp R
Gene for repressor
Binds to operator to inhibit transcription P
Promoter O
Operator
Trp E, D, C, B, A
Structural genes
Enzymes acting in pathway to produce tryptophan.
Gene order correlates with order of reactions in pathway.
5’ UTR (Leader)
Premature termination of transcription when trp levels are high ‘

21. Control of Trp Operon Transcription
Trp Repressor is Inactive  Initial State: ON
Trp binding activates Repressor  Final State: OFF
tryptophan

22. 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

23. 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.

24. Attenuation Premature Termination of Transcription
Ribosome translates trp codons, preventing 2+3 pairing
3+4 pairing forms terminator

25. Antitermination Ribosome stalls at trp codons, allowing 2+3 pairing
Transcription continues toward trp E, D, C. B, A

26. Summary of Trp Operon Regulation
Level of Tryptophan
Trp Operon
Low
High