1. Regulation of Gene Expression in Bacteria

2. Introduction gene regulation Constitutive expression
the amount of gene expression can vary under different conditions
Constitutive expression
constant amounts of expression
sometimes called “housekeeping genes”
Ubiquitous expression
Expressed in all cell types (multicellular organisms)
Differential expression
Temporal & spatial expression
regulated genes expressed only when required

3. Transcriptional Regulation
Most regulation is at transcriptional level
rate of RNA synthesis increased or decreased
Transcriptional regulation involves actions of two types of regulatory proteins
Repressors  Bind to DNA & inhibit transcription
Activators  Bind to DNA & increase transcription
Negative control refers to transcriptional regulation by repressor proteins
Positive control to regulation by activator proteins

4. Transcriptional Regulation
Small effector molecules affect transcription regulation
bind to regulatory proteins not to DNA directly
increase transcription
inducer
Bind repressors & prevent repressor from binding DNA
co-activator
Bind activators & cause activator to bind DNA
Genes regulated this way are inducible
decrease transcription
co-repressor
bind repressors & cause repressor to bind DNA
inhibitor
bind activators & prevent activator from binding DNA
Genes regulated this way are repressible

5. Inducible Expression
Co-activator

6. Repressible Control

7. gene regulation gods François Jacob & André Lwoff – 1953 CSH Symposium
Jacques Monod – Paris 1961
François Jacob & André Lwoff – 1953 CSH Symposium

8. Diauxic Growth Curve Demonstrated Adaptation to Lac Metabolism

9. The lac Operon
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10. Regulatory Sequences of the Lac Operon
Stop 1 10
Operator 3

11. The Lac Operon Is Regulated both Positively & Negatively
Negative - repressor protein - LacI
Positive - activator protein – CAP or CRP
Induction of Lac operon requires 2 events
Release of repression
lactose binds to the lac repressor causing the repressor to release operator site in DNA
Activation
cAMP binds CAP protein, cAMP-CAP dimerizes & binds CAP site in DNA
Insures operon is on ONLY if
lactose is present
glucose is low

12. RNA pol cannot initiate transcription
Constitutive expression
The lac operon is now repressed

13. Lac repressor protein (violet) forms a tetramer which binds to two operator sites (red) located 93 bp apart in the DNA causing a loop to form in the DNA. As a result expression of the lac operon is turned off. This model also shows the CAP protein (dark blue) binding to the CAP site in the promoter (dark blue DNA). The -10 & -35 sequences of the promoter are indicated in green.

14. The lac operon is now induced
Translation
The lac operon is now induced
The conformation of the repressor is now altered
Repressor can no longer bind to operator
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15. The cycle of lac operon induction & repression
Repressor does not completely inhibit transcription
So very small amounts of the enzymes are made
The cycle of lac operon induction & repression

16. The lacI Gene Encodes a Repressor Protein
1950s, Jacob & Monod, & Arthur Pardee, identified mutant bacteria with abnormal lactose adaptation
defect in lacI gene
designated lacI– I = induction mutant
caused constitutive expression of lac operon (ie in absence of lactose)
The lacI– mutations mapped very close to the lac operon

17. Jacob, Monod & Pardee hypothesized 2 ways for lacI to function
This hypothesis predicts that lacI works in trans manner
This hypothesis predicts that lacI works in a cis manner
Used genetic approach to test hypotheses

18. PaJaMo Experiment Used F’ plasmids carrying part of lac operon
Introduced into mutant bacteria by conjugation
Bacteria that receive F’ have 2 copies of lacI gene
merodipoloids

19. PaJaMo Experiment 2 lacI genes in a merodiploid are alleles
lacI– on the chromosome
lacI+ on the F’ plasmid
Genes on F’ plasmid are trans to bacterial chromosome
If hypothesis 1 is correct
repressor produced from F’ plasmid can regulate the lac operon on the bacterial chromosome
If hypothesis 2 is correct
binding site on F’ plasmid cannot affect lac operon on the bacterial chromosome, because they are not physically adjacent

20. From Jacob & Monod, 1961, J Mol Biol 3:318
Wildtype
Induction mutants
From Jacob & Monod, 1961, J Mol Biol 3:318

21. Analysis of Lac Operon Mutants-
F’I-O+Z+Y+
I+O+Z-Y+
lacI

22. From Jacob & Monod, 1961, J Mol Biol 3:318

23. Analysis of Lac Operon Mutants-
Mutation is cis -
In merodiploid, LacZ constitutive, but LacY inducible
OC only controls transcription of DNA on which OC is located
O (operator) is cis-regulatory element

24. Interpreting the Data
The interaction between regulatory proteins & DNA sequences have led to two definitions
Trans-effect & trans-acting factor
Genetic regulation that can occur even though DNA segments are not physically adjacent
Mediated by genes that encode DNA-binding regulatory proteins
Example: The action of the lac repressor on the lac operon
Cis-effect & cis-acting element
A DNA sequence adjacent to the gene(s) it regulates
Mediated by sequences that are bound by regulatory proteins
Example: The lac operator

25. Genetic Implications of Trans vs Cis
mutations in trans-acting factors complemented by 2nd wt gene
mutations in cis-acting elements ARE NOT complemented by 2nd wt element
Trans interactions (complementation) indicate mutation in structural gene
Cis interactions indicate mutations in regulatory sequences

26. From Jacob & Monod, 1961, J Mol Biol 3:318
Wildtype
Induction suppression mutant – Dominant Negative
From Jacob & Monod, 1961, J Mol Biol 3:318

27. Dominant Inhibitors or Dominant Negatives
Proteins with multiple functional domains & form multimeric complexes may be altered to prevent one function, but allow the other
When mutants retain ability to form multimeric complexes, dominant inhibition may occur

28. Analysis of Lac Operon Mutants
Mutation is trans
Dominant-negative
Mutation disrupts ligand binding domain of repressor

29. Analysis of Lac Operon Mutants
Mutation disrupts DNA binding domain of repressor

30. 3 categories of dominant mutations
Figure 14.9

31. lac Operon Also Regulated By Activator Protein
catabolite repression
When exposed to both lactose & glucose
E. coli uses glucose first, & catabolite repression prevents the use of lactose
When glucose is depleted, catabolite repression is alleviated, & the lac operon is expressed
The sequential use of two sugars by a bacterium is termed diauxic growth

32. The lac Operon Is Also Regulated By an Activator Protein
Effector molecule in catabolite repression cAMP (cyclic AMP)
cAMP is produced from ATP by adenylyl cyclase
cAMP binds activator protein CAP or CRP (Catabolite Activator Protein) or (cyclic AMP receptor protein)

33. States of Lac Regulation
(b) Lactose but no cAMP

34. States of Lac Regulation
Figure 14.8

35. The trp Operon
The trp operon encodes enzymes for biosynthesis of tryptophan
trpE, trpD, trpC, trpB & trpA encode enzymes
trpR & trpL involved in regulation
trpR  Encodes trp repressor protein
Functions in repression
trpL  Encodes a short peptide called the Leader peptide
Functions in attenuation

36. RNA pol can bind to the promoter Cannot bind to the operator site
Organization of the trp operon & regulation via the trp repressor protein
Figure 14.13

37. Organization of the trp operon & regulation via the trp repressor protein
Figure 14.13

38. Another mechanism of regulation
Organization of the trp operon & regulation via the trp repressor protein
Figure 14.13

39. Attenuation occurs in bacteria because of the coupling of transcription & translation
During attenuation, transcription begins but it is terminated before entire mRNA is made
A segment of DNA, termed the attenuator, is important in facilitating this termination
In the case of the trp operon, transcription terminates shortly past the trpL region
Thus attenuation inhibits the further production of tryptophan
The trpL segment of trp operon immediately downstream from the operator site plays a critical role in attenuation
It encodes a short peptide termed the Leader peptide

40. Region 2 is complementary to regions 1 & 3
Multiple stem-loops structures are possible
The 3-4 stem loop is followed by a sequence of Uracils
It acts as an intrinsic (r-independent) terminator
These two codons provide a way to sense if there is sufficient tryptophan for translation
Sequence of the trpL mRNA produced during attenuation

41. Structure of trpL 41

42. There are three possible scenarios
Therefore, the formation of the 3-4 stem-loop causes RNA pol to terminate transcription at the end of the trpL gene
Conditions that favor the formation of the stem-loop rely on the translation of the trpL mRNA
There are three possible scenarios
1. No translation
2. Low levels of tryptophan
3. High levels of tryptophan
Refer to Figure 14.15
14-50
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43. Transcription terminates just past the trpL gene
Most stable form of mRNA occurs
Therefore no coupling of transcription & translation
Possible stem-loop structures formed from trpL mRNA under different conditions of translation
Figure 14.15
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44. 44

45. Sufficient amounts of tRNAtrp
3-4 stem-loop forms
Translation of the trpL mRNA progresses until stop codon
Transcription terminates
RNA polymerase pauses
Region 2 cannot base pair with any other region
Possible stem-loop structures formed from trpL mRNA under different conditions of translation
Figure 14.15
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46. 46

47. Region 1 is blocked
3-4 stem-loop does not form
RNA pol transcribes rest of operon
Insufficient amounts of tRNAtrp
Possible stem-loop structures formed from trpL mRNA under different conditions of translation
Figure 14.15
14-52

48. 48

49. Inducible vs Repressible Regulation
The study of many operons revealed a general trend concerning inducible versus repressible regulation
Operons involved in catabolism (ie. breakdown of a substance) are typically inducible
The substance to be broken down (or a related compound) acts as the inducer
Operons involved in anabolism (ie. biosynthesis of a substance) are typically repressible
The inhibitor or corepressor is the small molecule that is the product of the operon
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50. PaJoMo Experiment

53. Results Lactose addition has no effect because operon is already on
Induction is restored in merodiploid. Now lactose addition is required to turn operon on