1. Gene regulation

2. Positive vs. negative control
Positive control: A protein binds to DNA and transcription increases
mRNA 5’ 3’
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
| | |
Activator
Negative control: A protein binds to DNA and transcription decreases X
mRNA 5’ 3’
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
| | |
Repressor

3. The lac operon What is an operon?
A collection of genes that are transcribed together and often have a related function (in this case lactose metabolism).
Lactose

4. The lac operon: negative regulation
Promoter
DNA covered by RNA polymerase X
mRNA
LacI 5’ 3’
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
| | |
DNA covered by repressor
Operator
Without lactose, LacI binds to the operator, which prevents RNA polymerase from binding to the promoter

5. The lac operon: negative regulation
Promoter
DNA covered by RNA polymerase X
mRNA
LacI 5’ 3’
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
| | |
DNA covered by repressor
Operator
LacI +
Lactose
If lactose is added, it binds LacI and stabilizes the LacI conformation that doesn’t bind to the operator.

6. The lac operon: negative regulation
Promoter
DNA covered by RNA polymerase
mRNA 5’ 3’
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
| | |
DNA covered by repressor
LacI
Operator
When LacI is not bound to the operator, the promoter binding site is not occluded.

7. Keq of DNA binding
The graph to the right shows two curves that represent binding of LacI to the operator with and without lactose.
Which curve has a higher Keq for binding to DNA?
100
% DNA
bound 50
[LacI]
The red curve
Keq
Operator DNA + LacI
LacI/Operator Complex
[O]
[R]
[O-R]
[O−R] O [R]
1 R
Keq for binding is inversely related to the concentration of LacI at which 50% of the DNA is bound
Keq = =

8. Keq of DNA binding Higher Keq
The graph to the right shows two curves that represent binding of LacI to the operator with and without lactose.
Which curve represents binding of LacI without lactose?
100
Lower Keq
% DNA
bound 50
[LacI]
The red curve – LacI has a higher affinity for DNA when lactose is absent.

9. The lac operon: positive regulation
Dissociation of LacI from the operator is not sufficient to activate transcription of the lac operon, it also requires positive regulation.
CAP Binding Site
Promoter
mRNA 5’ 3’
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
| | |
Operator
Why is positive regulation necessary?
The lac promoter -10 and -35 regions poorly match the consensus sequence, and RNA polymerase binds poorly to them.

10. The lac operon: positive regulation
CAP, the activator, can only bind to the DNA when cAMP levels are high. This only occurs when glucose levels are low.
CAP Binding Site
Promoter
mRNA
CAP 5’ 3’
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
| | |
Operator
CAP +
cAMP

11. The lac operon: positive regulation
CAP, the activator, can only bind to the DNA when cAMP levels are high. cAMP levels are high when glucose levels are low.
CAP Binding Site
Promoter
mRNA
CAP 5’ 3’
CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT
GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA
| | |
Operator
CAP binding to the CAP binding site helps recruit RNA polymerase, allowing it to bind to the -35 and -10 sites that poorly match the consensus sequence.

12. Electrophoretic mobility shift assays (EMSA)
The affinity of a certain repressor for binding to its operator site is affected by the presence of molecule “A.” Given these EMSA data, would the addition of molecule A increase or decrease transcription of the gene controlled by this repressor?
[repressor]
10-12
10-11
10-10
10-9
10-8
[DNA]
Constant
without
molecule A
Without molecule A:
Keq = 109
With molecule A:
Keq = 1011
[repressor]
10-12
10-11
10-10
10-9
10-8
[DNA]
Constant
The affinity of the repressor for DNA is higher for higher when molecule A is present. The addition of molecule A would increase repressor binding to DNA, decreasing transcription.
with
molecule A

13. The lac operon Glucose CAP-Binding Site -10 Region LacZ RNA Polymerase
LacI
cAMP
Consensus Sequence
Lactose
Repressor
CAP
Activator
Promoter
Operator

14. The lac operon: a concept map
prevents LacI binding to the operator
used to metabolize
Lactose
encodes the genes needed to use
Is a
Repressor
LacI
encodes
lac Operon
LacZ
negatively
regulates
binds to a site in the DNA called the
DNA that promotes transcription of
binds
Operator
Promoter
RNA Polymerase
positively
regulates
is an
comprise
binds
Activator
-10 Region
CAP
binds
-35 Region
Binds to a site in the DNA called the
CAP
Binding Site
Mediates CAP binding to DNA
Consensus Sequence
Glucose
cAMP
Ideal -10/-35 sequences to which RNA polymerase binds best
cAMP is produced when glucose is depleted