1. Nilansu Das Dept. of Microbiology Surendranath College
Operons
Nilansu Das
Dept. of Microbiology
Surendranath College

2. Group A: Cellular and Molecular Biology
Paper III
Group A: Cellular and Molecular Biology
Unit I
1. DNA Replication: (10)
DNA-Replication-Meselson-Stahl experiment as evidence for semiconservative
replication; Mechanism of replication-Rolling-circle model & Theta (8) structure
(bidirectional)
2. Transcription in prokaryotes: (15)
Mechanisms (Initiation, elongation, termination); promoter structures, subunits of bacterial polymerases, functions and domains responsible for activity, elongationprocess, mechanism of termination, -dependent and independent termination; lac, trp, ara operons.
3. Mechanism of translation in prokaryotes: (15)
Description of ribosomal cycle including phenomena of initiation, elongation, termination; description of factors involved in these processes; genetic code; tRNA: clover-leaf structure & function; rRNA: structure and function; role of aminoacyl tRNA synthetases.
Non-ribosomal peptide synthesis: cyclic peptide antibiotics e.g. Gramicidin etc.

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

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

5. Operon Unit of coordinate gene expression
Includes structural genes and their adjacent regulatory elements
Lac operon (inducible)
Ara operon (inducible)
Trp operon (repressible)

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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14. Summary of Lac Operon Regulation
Level of Lactose
Level of Glucose
Lac Operon
Low
High
Off
Off
On at low frequency
On at high frequency

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+ 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).

16. 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)

17. 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.

18. Lac Operon Mutations I+P+O+Z+Y+ I-P+O+Z+Y+ -- + -- + I+P-O+Z+Y+
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-

19. 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

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

21. 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
Off C protein bound to O and I
On at low frequency C protein + arabinose bound to I, enhancing transcription
On at high frequency C protein + arabinose bound to I and cAMP + CAP bound to I, enhancing transcription

22. Trp Operon

25. 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 ‘

28. The Regulatory protein of Trp Operon

29. Control of Trp Operon: ON

30. Control of Trp Operon: OFF

32. Attenuation The Observations
A novel means of controlling gene expression discovered by Charles Yanofsky and his colleagues while studying trp operon
The Observations
Mutants with deletions between the operator and the gene for the first enzyme (trpE) showed increased production of trp mRNA
5’ end of the trp mRNA revealed the presence of a leader sequence of 162 nucleotides before the initiation codon of trpE.
Mutations that enhanced trp mRNA level mapped in this leader region
Nonmutants (wt) produced a transcript consisting of only the first 130 nucleotides of the leader when the tryptophan level was high
However, 7000 nucleotide full length trp mRNA including the entire leader sequence was produced when tryptophan was scare

33. 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

34. 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.

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

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

38. Summary of Trp Operon Regulation
Level of Tryptophan
Trp Operon
Low
High
On Trp repressor inactive Lack of attenuation leads to high rate of mRNA production
Off Tryptophan + repressor = Active repressor Reduction of mRNA production by attenuation

39. Thank You