CONTROLLING GENE EXPRESSION: REGULATION

1
CONTROLLING GENE EXPRESSION REGULATION
In other words, controlling transcription
Suggested reading 2nd Ch. 14, pp. 369-384 3rd Ch.
14, pp. 360-376
2
Understanding gene regulation using the
bacterial model organism
We will be using the lac operon (a series of
three genes linked together) to look at
mechanisms for controlling gene expression.
The lac operon is a prokaryotic model
for studying gene regulation
3
SO, WHAT IS AN OPERON IN BACTERIA?

• Operons are defined as genes ______ ________ and
  under ________control - regulation
• Separate genes under independent regulation are
  _____operons, even though these genes are
  regulated using ________mechanisms
• Operons generally carry genes for enzymes -
  structural genes- that _____ _________in a
  biochemical pathway
• Biochemical pathways need to be turned on when
  required and turned off when not required

4
Common ways for regulating operons or genes

• INDUCIBLE OPERONS OR GENES
• Where the operon is __________and requires an
  ________ to ________ expression (i.e.
  transcription)
• The __________operon is an example of an
  inducible operon

2nd Figure 14-2a c 3rd Figure 14-2ab
5

• REPRESSIBLE OPERONS OR GENES
• Where the operon is ___________and requires a
  signal, such as too much end product, to ____
  ____ expression
• This is a __________inhibition mode of control
• The ____________operon is an example of a
  repressible operon

2nd Figure 14-2b d 3rd Figure 14-2c d
6
The Lactose Metabolism Operon Model

• The biochemical pathway and its purpose
• The genetic components involved
• Factors controlling operon transcription
• Regulatory controls- Negative control and
• positive control mechanisms
• How the whole system works

7
The Lactose Metabolism Operon Model
Genes and transcription sequences
Both editions Figure 14.4
The biochemical pathway
Factors for transcription and regulation
Both Editions Figure 14.5
8
THE LAC OPERON AND ITS BIOCHEMICAL ROLE

• Controls the pathway for the utilization of
  lactose, a milk sugar
• Bacterial cells cannot eat lactose as is
• The cells need to break lactose into sugars in
  order to utilize it
• LACTOSE GALACTOSE GLUCOSE

9
Both editions Figure 14- 3b
10
GENETIC COMPONENTS OF THE LAC OPERON SYSTEM

• LAC OPERON ITSELF
• 3 structural genes for the 3 enzymes (lacZ, Y, A)
• Promoter DNA sequence
• Operator DNA sequence
• CAP DNA sequence
• REGULATORY GENES PHYSICALLY SEPARATE FROM THE LAC
  OPERON
• Lac I gene Lac I repressor
• CAP gene CAP activator
• (RNA polymerase RNA synthesis)

Both editions Figure 14- 3a
11

• THE STRUCTURAL GENES OF THE LAC OPERON
• Lac Z gtgtgt ß-galactosidase
• Converts tiny amounts of lactose into a proper
  signal for induction, e.g., allolactose
• Breaks down lactose into glucose and galactose
• Lac Y gtgtgt Permease
• Acts as a pore in the cell membrane to transport
  lactose into the cell
• Lac A gtgtgt Transacetylase
• Galactoside transacetylase

12
Cap
RNA polymerase
LacI
Both editions Figure 14- 3
13
Question What would happen if one or more of
the structural genes were mutated?

• Possible phenotypes would be a faulty lactose
  metabolic pathway regulation, metabolism
• Examples
• Z , Y , A gtgtgt _____________pathway
• Z-, Y-, A- gtgtgt _______________possibilities

14
Cap
RNA polymerase
LacI
Both editions Figure 14- 3
15
Regulatory Sequences in the Lac Operon

• Promoter (P) and CAP-binding sequences
• Located 5 or upstream to the three structural
  genes (Z, Y, A)
• Two important sequences
• RNA polymerase binding site for transcription
  (e.g. 35, -10 regions)
• CAP-cAMP binding site for modulating
  transcription
• ____________in P (e.g., P-) or CAP binding
  sequence would affect transcription

16
2nd Figure 12-6 3rd Figure 12-7
17
Both editions Figure 14-8
18
Cap
RNA polymerase
LacI
Both editions Figure 14- 3
19

• Operator (O) DNA sequence
• Located 5 or upstream to the __________gene
• Located 3 to the _________sequence
• An important site where the lacI repressor binds
  to ________transcription
• ____________in the O sequence would affect
  repression activity, hence preventing the
  repressor from blocking transcription,
• e.g. O-, Oc

20
Both editions Figure 14-4
21
Both editions Figure 14-9
Both editions Figure 14-10
22
Question What is the difference between the
two types of mutations structural genes
versus promoter/operator sequences?
Mutated structural genes can be saved by
introducing complete normal genes because the
functional products can move across cell space
and work
Trans-acting
Mutated promoter/operator sequences cannot be
saved by introducing normal sequences because
they cannot move across cell space and need to
be physically linked in the right order to work
Cis-acting
23
__________
______________
Both editions Figure 14-8
24
So, how is the Lac operon regulated?
Both editions Figure 14-3
25
__________ regulation
_________ regulation
2nd Figure 14-2a (modified) c 3rd Figure 14-2
a (modified) b
26

• The lac operon is on the _______an inducible
  system
• The operon is induced by lactose, the substrate
  for the lactose metabolic pathway
  gtgtgt_________________________
• gtgtgt INDUCIBLE
• The operon is also modulated by glucose, the
  product generated by the lactose metabolic
  pathway
• gtgtgt PART OF _______________ gtgtgt
  REPRESSIBLE

27

• PURPOSES OF THE TWO HALVES
• NEGATIVE CONTROL
• To determine on or off
• Transcription controlled by _____________of
• lactose
• Controlled via a repressor protein lac I, also
• a _________of lactose amounts

28

• POSITIVE CONTROL
• To determine how__________________
• Controlled by ____________of glucose
• Glucose levels/needs are monitored by modulating
  cAMP levels, e.g. glucose ___________lead to
  cAMP__________
• Controlled via an activator protein CAP
  (catabolic activator protein, encoded by the cap
  gene)

29
Negative regulation
Positive regulation
Both editions Figure 14.8
30

• REGULATORY FACTORS OF THE LAC OPERON
• Lac I gene
• Located separately from the lac operon
• Independent from the lac operon
• Forms a __________of the same subunit
• Binds lactose (allolactose or IPTG or ONPG)
• Binds the _________DNA sequence 5 to the lac
  operon
• __________transcription
• Negative control
• Can modulate blockage_________

31
Negative regulation
allolactose
Both editions Figure 14.8
32

• CAP gene
• Catabolite Activator Protein
• Located separately
• Independent
• Binds cAMP (cyclic AMP)
• Binds to a special _____on the promoter DNA
  sequence (_____sequence)
• Enhances/turns on transcription
• Can __________level of transcription by
  ____________ of binding
• Positive control
• glucose cAMP
• glucose cAMP

33
cap
Positive regulation
Both editions Figure 14.8
34
Negative regulation
Positive regulation
Both editions Figure 14.8
35

• REGULATION OF THE LAC OPERON
• NEGATIVE CONTROL
• 1. Lactose_________
• repressor lacI present and active
• binds O no transcription
• 2. Lactose_________
• lactose (converted to allolactose some) binds
  to lacI repressor
• inactivates lacI allosterically
• falls off O gtgt allows transcription if
  CAP-cAMP is bound to the promoter

36

• POSITIVE CONTROL
• 1. Glucose__________
• - cAMP increases via other pathways
• - lots to bind to CAP (CAP cAMP gtgt active)
• - CAP is now able to bind the promoter
• - various levels of transcription (low to high)
• 2. Glucose___________
• - cAMP decreases via other pathways
• - little, if any, to bind CAP (CAP, no cAMP gtgt
  inactive)
• - CAP is unable to bind the promoter
• - virtually no transcription

37

• Negative and positive together modulate or
  regulate Lac operon expression
• ________________________________________
• (Negative) Positive Effective
  Expression Outcome
• 1. Lactose Glucose cAMP
• 2. Lactose Glucose cAMP
• 3. Lactose Glucose cAMP
• 4. Lactose Glucose cAMP
• Middle of eating
• 5. Lactose Glucose cAMP

38

• So, how does one determine how a genetic system
  such as the lac operon regulate gene expression?

39

• By using powerful dissection tools like Partial
  Diploids or Merodiploids
• Bacterial cells are naturally haploid (except
  during division)
• Partial Diploids are created to assess the
  components, their roles, and modes of function
  (cis-acting versus trans-acting, for instance)
• This is done by introducing sets of genes or
  sequences on a ___________(a miniature
  chromosome)

40
LAC OPERON
LAC I
LAC OPERON
LAC I
CAP
BACTERIAL GENOME
PLASMID
(THE GENES BEING INTRODUCED CAN BE ANY
COMBINATION)
41

• Examples of findings using the partial diploid
  experimental approach
• Oc (O-) is dominant to O
• Is is dominant to I, I-
• I is dominant to I-
• P is dominant to I-
• P is dominant to P-
• CAP is dominant to CAP-
• CAP- (homozygous) is dominant to everything
• I- (homozygous) is dominant to O
• Oc (O-) is dominant to Is, I

42

• SCENARIOS WHERE THE LAC OPERON
• APPEARS TO BE OFF
• Glucose
• Lactose
• CAP-
• P-
• Is (super-repressor)
• Z-, Y-, A-

Normal system
Mutants
43

• SCENARIOS WHERE THE LAC OPERON
• APPEARS TO BE OR IS ON
• Glucose and Lactose
• (P, Z, Y, A)
• I-
• O-/Oc (constitutive)

Normal System
Mutants
44
Griffith et al 8th ed. Figure 10-8
45
Griffith et al 8th ed. Figure 10-9
46
Griffith et al 8th ed. Figure 10-10