2/17/16
BIOLOGY 207 - Dr. Locke
Lecture#6 - Further regulation of the lac operon
Required readings and problems:
Reading: Open Genetics, Chapter 12
Problems: Chapter 12
Optional
Griffiths (2008) 9th Ed. Readings: pp 364-367
Problems: 9th Ed. Ch. 6: 1-7, 25, 28, 50
Campbell (2008) 8th Ed. Readings: Concept 18.1
Concepts:
How is the lac operon regulated? (continued)
1. Negative regulation of the lac operon is described using genetics involving the Ffactor (a genetic tool).
2. The lac I mutations define a trans-acting locus.
3. The Oc mutations in the lac operon define a cis-acting locus.
4. The lac operon has both positive and negative regulation.
Animations: http://bcs.whfreeman.com/iga9e/default.asp
Biol207 Dr. Locke section
Lecture#6
Fall'11
page 1
2/17/16
The next step in lac operon genetics needs a brief explanation of "heterozygous
diploid strains"
F-factor / Fertility factor
(pp 195-6, Griffiths 9th)
F factor is an E. coli episome - capable of being either either
a free plasmid or integrated into bacterial chromosome
+
If F factor is present, then the strain is an F strain (bacterial sex)
The F factor can contain some E. coli genes, therefore making the cell a partial diploid.
(merozygote)
- can be transferred from one cell to another
mutant
-
lac host with
F factor containing a lac
+
- +
= lac / F
lac
+
Advantage: We can:
-
-> examine lac mutants in partial diploid strains with other lac mutations
-> analyze of the mechanisms of regulation using a diploid (partial) state.
Biol207 Dr. Locke section
Lecture#6
Fall'11
page 2
2/17/16
“Complementation tests” are possible
Determine if mutations are cis
or trans acting.
cis =
trans =
trans-acting locus
A gene able to affect the activity of other genes on a different DNA molecule usually
through a diffusible product (e.g. polypeptide/protein)
Example: the I locus affecting lac expression in E.coli.
Biol207 Dr. Locke section
Lecture#6
Fall'11
page 3
2/17/16
I+ is dominant over I- (acts in trans position)
Fig
strain:
I+Z-Y+/F I-Z+Y-
Z inducible; Y is inducible
I+ is a wild type repressor, which binds to both lac gene operators and makes them
inducible
I- is a mutation that affects the DNA binding region and therefore there is no repressor
binding and thus transcription occurs even without an inducer.
IS is dominant to both I+ and I- in trans
Fig
Strain
S
+
+
I Z Y
is un-inducible.
IS mutant gene product can't bind inducer (mutation in the inducer binding region) so
that the repressor remains bound to operator even in the presence of inducer.
Repressor protein:
Biol207 Dr. Locke section
Lecture#6
Fall'11
page 4
2/17/16
I gene repressor product –> two functional domains
- Mutate each independently
I+ Normal
I- mutation
DNA
Inducer
IS mutation
DNA
DNA
Inducer
Inducer
Summary of I mutant classes:
- Dominant in trans therefore it is a diffusible product
- the I gene encodes a repressor with two independent domains.
Biol207 Dr. Locke section
Lecture#6
Fall'11
page 5
2/17/16
Operator mutants - OC mutants - Fig
cis-acting locus
- a genetic region affecting the activity of genes on that same DNA molecule
- Such a locus usually does not code for a protein but instead acts as a binding site for
trans-acting proteins.
Jacob and Monod proposed the
"operator element" in the lac operon.
- If mutated this operator element should be dominant in cis, in that it only affects the
genes on the same chromosome (directly adjacent to it).
- It will not be dominant in trans.
How were these mutants obtained?
J & M selected for mutants that constitutively expressed lac in cells with two I+ genes.
(2 I+ genes prevents the recovery of I- constitutive mutants).
This led to the recovery of OC - operator constitutive mutants.
Genetic mapping - positioned the OC mutants to between I locus and Z locus.
Biol207 Dr. Locke section
Lecture#6
Fall'11
page 6
2/17/16
OC is dominant in the cis position
Fig
- no diffusible product.
cis dominant - the ability of locus to influence the expression of one or more adjacent
loci on the same chromosome, as occurs in lac operator mutants of E.coli
Level of constitutive expression of Operator mutations:
OC mutants may not be maximally expressed,
but are still constitutively expressed
- no inducer - 10-20% of max
- with inducer - 100% of max
Note: extent of mutation affects the level of expression
Fig
Biol207 Dr. Locke section
Lecture#6
Fall'11
page 7
2/17/16
Summary of "negative" control aspect of the Lac operon
Negative control:
Repressor protein acts to prevent transcription
Inducer binds to repressor to alter conformation so it no longer binds to the operator
sequence and transcription can take place.
Prokaryotic gene expression summary:
Fig
Activation - inducer promotes transcription
Repression – inducer lifts inhibition of transcription
Biol207 Dr. Locke section
Lecture#6
Fall'11
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