1. Introduction to Genetic Analysis
PowerPoint Slides for
Introduction to Genetic Analysis
Ninth Edition
Anthony J. F. Griffiths, Susan R. Wessler, Richard C. Lewontin, and Sean B. Carroll
CHAPTER 10
Regulation of Gene Expression in Bacteria and Their Viruses
Copyright 2008 © W H Freeman and Company

2. Regulation of Gene Expression in Bacteria
Functionally related genes are clustered in operons (Jacob and Monod)
An operon = single promoter, a shared operator region, + bunch of related operon (structural) genes
Any gene is a DNA region that may be transcribed into mRNA
Operon genes are transcribed together into polycistronic mRNA (poly=many, cistron=gene)
Promoters and operators are examples of regulatory regions that are not transcribed into RNA
Strong selective pressure for avoiding making unnecessary proteins (don’t want to slow cellular replication, lose evolutionary foothold)
Positive control mechanisms stimulate gene expression
Negative control mechanisms inhibit gene expression
Short prokaryotic mRNA half lives mean transcriptional control very effective.

3. Regulatory proteins control operon transcription:
What is the job of an activator?
What is the job of a repressor?
Where do these regulatory proteins come from?

4. We say that operons are either under POSITIVE or NEGATIVE CONTROL
Repressors and activators are PROTEINS encoded by CONSTITUTIVE (= always on) REGULATORY genes elsewhere in the genome.
We say that operons are either under POSITIVE or NEGATIVE CONTROL
NEGATIVE:
POSITIVE:
Uses a repressor protein
Uses an activator protein

5. Why are regulatory genes constitutive?
Think of activator and repressor proteins as smoke detectors
Always present in your house
But only sound the alarm when they sense smoke
What kind of smoke are they sensing?
Some kind of metabolite
An end-product of an anabolic pathway
A beginning substrate of a catabolic pathway

6. Operons are either REPRESSIBLE or INDUCIBLE
Involves binding a co-repressor (metabolite/end-product) to repress/turn OFF operon transcription
Involves binding an inducer (metabolite/starting substrate) to induce/turn ON operon transcription

7. 3 combinations found in nature:
REPRESSIBLE
INDUCIBLE
NEGATIVE
This operon uses a repressor that binds a co-repressor to repress txn.
This operon uses a repressor that binds an inducer to induce txn.
POSITIVE
Not found.
This operon uses an activator that binds an inducer to induce txn.

8. Concept Checks: True or False?
Structural genes are transcribed into mRNA, but regulatory genes aren’t
Structural genes encode proteins involved in carrying out metabolic reactions; regulator genes encode proteins involved in controlling the transcription of structural genes
Regulatory genes are considered part of the operon
Transcription of constitutive genes may be positively or negatively controlled

9. Concept Checks: True or False?
In a repressible operon, transcription is on until it is turned off
In an inducible operon, transcription is off until it is turned on

10. Example #1: The Lac operon of E
Example #1: The Lac operon of E. coli makes the three enzymes needed for lactose breakdown:
LacZ encodes Beta-galactosidase
breaks up lactose into glucose and galactose (galactose also converted to glucose for metabolism)
Isomerizes lactose into allolactose inducer (presence of lactose means presence of allolactose)
LacY encodes permease
For lactose transport across cell membrane
LacA encodes transacetylase
Poorly understood function

11. The Lac operon has 2 control circuits:
NEGATIVE INDUCIBLE
Uses a repressor that binds an inducer (allolactose) to induce operon txn.
Car ignition
Requires presence of lactose
POSITIVE INDUCIBLE
Uses an activator (CAP) that binds an inducer (cyclic AMP) to induce operon txn.
Gas pedal
Requires absence of (preferred) glucose

12. So how does it work? First we’ll need allolactose to start the car:

13. LAC OPERON NEGATIVE INDUCIBLE CONTROL CIRCUIT:
In the absence of lactose, an active repressor protein binds to the operator and blocks transcription by RNA Polymerase:

14. When lactose is present in the cell, allolactose, an
When lactose is present in the cell, allolactose, an isomer of lactose, binds to the repressor.
This inactivates the repressor, because it can no longer bind the operator. Now RNA Polymerase can transcribe the Lac operon:
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

15. But RNA Polymerase has low affinity for the Lac operon promoter…
But RNA Polymerase has low affinity for the Lac operon promoter…. so even though the Lac operon is turned on by the presence of lactose, it is transcribed at low levels (like your car merely starting to roll forward after the ignition key is turned).

16. So once the car is turned on, how do we step on the gas?

17. LAC OPERON POSITIVE INDUCIBLE CONTROL CIRCUIT
If glucose levels are low (along with overall ATP energy
levels), then cAMP
is high.
cAMP binds to CAP (a.k.a. CRP) which activates Lac operon
transcription.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

18. P+
wt promoter region; can initiate transcription P-
defective promoter O+
wt operator region can bind repressor Oc
constitutive operator cannot bind repressor - operon always on I+
wt regulatory gene - codes for (diffusable) repressor protein I-
inactive repressor cannot bind operator IS
super-repressor always represses (cannot bind inducer)
Z+, Y+, A+
wt structural operon genes (producing diffusable protein)
Z-, Y-, A-
no operon proteins made

19. Cis-acting: Trans-acting: DNA elements
Only affect whatever is on the same piece of DNA
E.g. lacOC, lacP-
Trans-acting:
Structural genes
Involve diffusable proteins
Proteins may affect own piece of operon DNA and/or the other piece of operon DNA in the same (partial diploid) cell
E.g. lacI+, lacIS, lacZ+, lacY+, lacA+

20. Structure of IPTG
Figure 10-7

21. O = cis-acting Z+ = trans-acting
Growing in the absence of glucose, with or without IPTG synthetic inducer:
Haploid or Partial Diploid Genotype
Z produced?
(no inducer)
(with inducer)
Y produced?
O+Z+Y+ - +
O+Z-Y+
O+Z+Y+ / F’ O+Z-Y+
OcZ+Y+
OcZ+Y-
O+Z-Y+ / F’ OcZ+Y-
O = cis-acting Z+ = trans-acting
Oc = constitutive (always on) Z+ = trans-dominant to Z-
O+ = inducible

22. Lac Operon Animations
Lac operon animations from BioCoach:
I+P+O+Z+Y+ wild type animation:
I+P+OCZ+Y+ animation:
I- P+O+Z+Y+ animation:
IS P+O+Z+Y+ animation:

23. Example #2: The Trp operon of E
Example #2: The Trp operon of E. coli encodes five enzymes needed to catalyze the synthesis of Tryptophan:
TrpE gene product
TrpD gene product
TrpC gene product
TrpB gene product
TrpA gene product

24. The Trp operon has 2 control mechanisms
NEGATIVE REPRESSIBLE OPERON
Uses a repressor that binds a co-repressor (end product Trp) to repress operon transcription by 70-fold
Requires presence of Trp
ATTENUATION
Acts / represses on top of above mechanism by another 8 to 10-fold
Involves premature txn termination
Requires high Trp levels

25. TRP OPERON NEGATIVE REPRESSIBLE CONTROL CIRCUIT: By itself, the operon is on. RNA polymerase can bind to the promotor and moves freely through the operator to transcribe the genes:

26. When co-repressor (end-product) Trp is present, it binds to the repressor. This activates the repressor, causing it to bind the operator to block Trp operon transcription:
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

27. TRP OPERON ATTENUATION MECHANISM: involves 3 alternate ways to fold mRNA:
(Making a baby protein)
Pairing of 1 and 2
causes ribosomes
to load onto mRNA
right after RNA Pol.
(COUPLING txn +
tln)

28. Models for attenuation in the trp operon of E.coli
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

29. Models for attenuation in the trp operon of E.coli
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

30. Lac Operon and Trp Operon Animations
Lac and Trp Operon Control by McGraw-Hill:
Trp Operon Negative Repressible Control and Attenuation:

31. Arabinose Operon Control System
AraC protein acts as either an activator or a repressor
Operon genes encode enzymes needed for arabinose breakdown
Ara operon induced by
araC/arabinose complex binding to araI region (in presence of arabinose) – araC acts as an activator
CAP/cAMP complex binding to araO region (in absence of glucose)
Operon repressed by
araC protein binding to both araI and araO at same time – araC acts as a repressor
Looping prevents binding of RNA Polymerase/operon txn 31

32. AraC serves as an activator and as a repressor 32

33. Regulation of gene expression in Lambda Phage - overview
Recall lysogeny (integrated prophage) vs. lytic cycle: 33

34. The lambda phage which infects E
The lambda phage which infects E. coli demonstrates the cycles of a temperate phage.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings 34

35. How does Lambda choose between lysogeny and lytic cycle?35

36. Phage  genome is organized for coordinate control36

37. Most of the Lambda genes are necessary for lytic cycle
Most of the Lambda genes are necessary for lytic cycle. These genes will need to be turned ON for Lambda to do the lytic cycle, while the genes associated with lysogenesis must be off. Only a few are needed for lysogenic cycle. These genes will need to be ON to for Lambda to do the lysogenenic cycle, while the genes associated with lysis will need to be turned off. 37

38. What happens upon infection of E. coli by Lambda?
The PR promoter transcribes CRO (Control of Repressor and Other)
The PL promoter transcribes N
N is a “transcription antiterminator” protein that allows read-through transcription of CII and CIII
CIII protein hangs around long enough to protect CII from degradation (otherwise CII protein is very unstable)
An epic protein battle begins… which proteins will gain the upper hand?
If CII is high, then CI and integrase (int) will be produced, and Lambda will do lysogeny
If CRO dominates, then CI and int will not be produced, lysis genes will get turned on instead, and lambda will do lysis 38

39. When times are bad: Lysogeny
Starving E. coli cells are low on glucose, high on cAMP
CII is unstable, but high cAMP inhibits the host enzymes that would normally rapidly degrade CII
CII is able to turn on CI (“lambda repressor”) and int
CI lambda repressor shuts off all genes except itself
Lambda integrates/becomes prophage, and lysogeny is established. 39

40. Shuts off all lysis genes
Starving E. coli
infection
Low glucose
High cAMP
CRO N
stabilizes
Allows production
protects
CIII
CII
turns on
int
CI (Lambda Repressor)
Shuts off all lysis genes
Lambda integrates into E.coli DNA (lysogeny) 40

41. When times are good: Lytic Cycle
Low cAMP means CII is degraded; cannot turn on CI lambda repressor and int
On top of that, CRO represses CI
No CI means genes associated with lysis are no longer repressed
Lytic genes go into overdrive
Phage enters lytic cycle 41

42. Lambda enters lytic cycle
Happy E. coli
infection
High glucose
Low cAMP
CRO N
Now degrading
represses
CII
So no more
int
CI (Lambda Repressor)
CI absence de-represses/activates
all lysis genes
Lambda enters lytic cycle 42

43. Solved problems 1-3, and problems 1, 2, 4, 15
CH 10 Problems
Solved problems 1-3, and problems 1, 2, 4, 15 43

44. More Operon Concept Checks: True or False?
In a negative repressible operon, the regulatory protein is synthesized as an active repressor
In a positive inducible operon, the regulatory protein is synthesized as an active activator 44

45. Sample Lac Operon Problems (NO premature peeking at the YouTube Answers in the links, or it won’t do you any good!!
Z protein produced?
Y protein produced?
-inducer inducer
-inducer inducer
IsP+O+Z+Y+
ISP+OCZ+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-O+Z-Y+
ISP-O+Z-Y+/I+P+O+Z+Y+ 45