You are on page 1of 25

MOLECULAR BIOLOGY

Dr. Cynthia Haseltine

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?

B. What are the advantages of controlling gene expression

at transcription?

Gene regulation
Allows prokaryotic cells to:
To conserve energy
To respond to changes in the environment

Most prokaryotic control is at the level of transcription

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

Diauxic growth of E. coli

Genes of the lac Operon


Genes are grouped:
lacZ = -galactosidase
lacY = galactoside permease
lacA = galactoside transacetylase
All 3 genes are transcribed together producing 1 mRNA, a

polycistronic message that starts from a single promoter

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

The -galactosidase reaction

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.

How could you use these plates to isolate CAP mutants?

(e) You find that you obtain two classes of mutants with this screen. The first class
of mutants is CAP mutants.

What do you think the second class could be?

17

Lac operon control


Figure 18.6

Involves
regulatory
proteins:
Lac repressor
CAP
(activator)

18

Control sequences

Figure 18.8

How could you identify binding sites experimentally?


Lac repressor binds operator
Blocks RNAP binding to promoter
CAP binds CAP site
Facilitates RNAP binding to promoter

19

CAP binding to DNA


Lac promoter is weak
-35 is not optimal
No UP-element

CAP binds CAP site

and CTD of RNAP


Helps RNAP bind to

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

Found in CAP and lac repressor

Figure 18.11

21

Lac repressor binding to DNA


Can bind DNA as a tetramer
To two of three possible operator sites
Requires DNA looping

Figures 8.7 & 18.12

22

Allosteric regulation of repressor

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

Allosteric regulation of activator


CAP regulated by cAMP
Acts as a glucose sensor (cAMP is high when glucose is low)
Allows CAP to bind to CAP site

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.