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June 5, 2014
What to do when you arrive
Take a quarter sheet of paper
Todays class
Transcriptional control in prokaryotes
TRANSCRIPTIONAL
CONTROL IN
PROKARYOTES
CLASS ACTIVITY
#1.
A. Why is it important to control gene expression in
prokaryotes?
at transcription?
Gene regulation
Allows prokaryotic cells to:
To conserve energy
To respond to changes in the environment
initiation
Mediated by regulatory proteins
Regulatory proteins
Bind to DNA near
sequences they
regulate
Can have positive or
negative effects
Activators - recruit
Repressors - block
Figure 18.1
Regulatory proteins
May have allosteric
effects on RNAP
Example: to allow
isomerization
May have allosteric
effects on DNA
Figure 18.2
Example: to position
promoter sites
Distant interactions
DNA looping allows proteins to interact
DNA-bending proteins facilitate looping
Figures
18.3 & 18.4
OPERONS
Note: Each cistron, or gene, has its own ribosome binding site and can be
transcribed by separate ribosomes that bind independently of each other
Figure 18.5
CLASS ACTIVITY
15
#2. When glucose is present, the lac genes are not fully expressed, even in the
presence of inducer. This is called catabolite repression.
(a) Why does it make biological sense to have the lactose operon under negative
control by Lac repressor? Why does it make biological sense to have the lactose
operon controlled by catabolite repression?
(b) It is commonly stated that lactose induces the lac operon. However,
allolactose, which is a product of basal galactosidase activity on lactose, is the
actual inducer molecule.
Devise an experiment to prove this.
16
#2 (continued)
(d) CAP is necessary to turn on several sugar operons (including the arabinose,
lactose, maltose, and galactose operons). Cells with mutations in CAP cannot
efficiently metabolize any of these sugars. On plates that contain a sugar and
tetrazolium (an indicator dye), colonies are white if that sugar is metabolized and
red if it is not. This kind of plate is often used to screen for cells that cannot
metabolize a particular sugar.
(e) You find that you obtain two classes of mutants with this screen. The first class
of mutants is CAP mutants.
17
Involves
regulatory
proteins:
Lac repressor
CAP
(activator)
18
Control sequences
Figure 18.8
19
promoter
Figures
18.9 & 18.10
20
Helix-turn-helix
Common DNA-binding motif
One -helix associates with major groove
One -helix associates with backbone
Figure 18.11
21
22
Recall:
What is allosteric
regulation?
Figure 18.13
Lac repressor regulated by allolactose
Acts as a lactose sensor
Prevents lac repressor from binding operator
23
Figure 18.14
CLASS ACTIVITY
25
#3. An operon in E. coli is controlled by a repressor that binds at two operator sites (O1
and O2). In the presence of the appropriate inducer, a transcription rate of 100 is
observed, but in the absence of inducer, the transcription rate falls to 5. If either of the two
sites is mutated so that the repressor cannot bind, then the transcription rate is observed
to be 100. Additionally, if base pairs are inserted between the two sites, the level of
transcription is found to vary with the size of the insert. Briefly explain this data.