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BIOB11H3 Study Guide - Midterm Guide: Lac Operon, Lac Repressor, Beta-Galactosidase


Department
Biological Sciences
Course Code
BIOB11H3
Professor
Dan Riggs
Study Guide
Midterm

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BIOB11: MOLECULAR CELLULAR AND GENETIC PRACTICAL WINTER 2018
BIOB11
Lecture 9: Gene Regulation I: Promoters and Control Circuits
CENTRAL IDEAS
An operon is a cluster of genes that are typically coordinately regulated.
Regulatory genes encode proteins that bind to operator sequences and influence the transcriptional competency of the
operon.
Inducible operons are turned on when the appropriate metabolite (inducer) is present. Conversely, repressible operons are
turned off when the appropriate metabolite (repressor) is present.
Cis refers to ‘on the same strand’. An example is a sequence of DNA in the promoter/operator that controls a linked
structural gene.
‘Trans’ refers to ‘across’. An example is a trans-acting protein or transcription factor which controls gene expression after
forming a specific protein/DNA complex.
Gene regulation can occur at four levels: transcriptional, post-transcriptional, translational and post-translational.
Recombinant DNA techniques in combination with the ability to introduce altered genes into organisms permit one to test
the functionality of cis-acting sequences, often using reporter genes.
Enhancers are DNA sequences that increase transcript production. They are often located far away from the gene they
control and are orientation independent.
LACTOSE METABOLISM IN E.COLI
Lactose disaccharide of galactose and glucose
Lactose can be an inducer of the system beta galactosidase expressed in the bacteria when it is exposed to lactose
o This enzyme converts lactose to glucose and galactose
o Glucose is the preferred carbon source of e coli.
At the 4 minute the lactose inducer is added
o Almost immediately there is an effect, beta galactosidase mRNA increases and that is translated into a protein (red
line)
o If you put them in another media that has no lactose, immediately the mRNA levels begin to fall and the protein
activity falls too
OPERON STRUCTURE
Only in prokaryotes
Operon components
o Promoter
o Operator
between the promoter and the structural
genes, it binds to a repressor protein,
o structural genes,
o regulatory gene
part of the operon but not attached, that
encodes the repressor protein that has affinity
for the operator and blocks gene expression if
it binds
o These all make up the operon
Prokaryotes have genes that are in operon structures
o There are clusters of genes that are controlled by a single promoter
o Those genes that are controlled by this one promoter are involved in a single metabolic pathway
o Ex. genes that catabolize lactose
You can turn them all on or off at the same time in the presence or absence of it
Inducer Operon: the Lac Operon
If Lac present, repressor protein binds to the lactose, changes its structure and becomes inactivated (pic 1)

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BIOB11: MOLECULAR CELLULAR AND GENETIC PRACTICAL WINTER 2018
o Now the repressor does not have the right structure to bind to the operator (and it
falls off?)
o Now it is open to be transcribed by RNA polymerase
o Now the Operon is induced and the mRNA can be
transcribed
o This is “induction of the inducable operon
Translation of mRNA yields 3 enzymes that convert lactose
glucose and galactose (pic 2)
o Now ther is an mRNA and there are 3 loading sites on
the gene that RNA polymerase operates on and this
creates the three enzymes that are going to be used
o Now you can convert lactose into glucose and galactose
what happens when the lactose supply is reduced (pic 3)
o Binding of lactose to repressor is transient (can go on and off), so as [lactose] falls,
repressor changes structure and becomes active and can bind to the operator
o Repression transcription blocked by the repressor protein
So there is no expression of the gene when lactose is low
It is called an inducer operon because lactose induces the expression of the gene
CIS VS TRANS
Cis acting promoter sequence to which a trans acting transcription factor binds
Cis on the same strand
o Ex. the DNA sequence that serves as a binding site for TF
o Ex TATA box
Trans trans-acting, like a transcription factor
o Soluble protein of the cell that can come in contact with DNA until it comes in contact with a sequence that it likes
(cis)
In gene regulation you can activator factors to encourage gene expression by recruiting RNA polymerase (positive control)
or you can have repressor proteins that come and bind to the cis sequence (negative control)
Positive and Negative Control
o Depends on the active form of the trans-acting factor (ex. repressor for negative control or activator for positive
control), and its effects upon binding to its target cis-acting sequence
BACTERIAL BIOCHEMICAL LOGIC FOR LAC=GLU+GAL
1. If glucose is available, why expand energy to make enzymes to catabolize lactose?
2. If lactose is absent, why expand energy to make enzymes to catabolize it?
Both positive and negative control involved in determining what is going to happen
Positive Control
If glucose level is low, cAMP level is high, cAMP serves as a co factor that binds to transcription factor CRP (cAMP receptor
protein, AKA CAP), and the complex binds to the promoter and activates/ up regulates the lac operon
o Glucose and cAMP levels are inversely related
THE LAC PROMOTER/OPERON
Positive control mediated by CPR and cAMP
o Promotes transcription
Negative control mediated by lac repressor that
binds to the operator
o Prevents transcription
Four Situations
Sugar availability and positive/negative control
Allolactose an isomer of lactose, is the actual inducer

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BIOB11: MOLECULAR CELLULAR AND GENETIC PRACTICAL WINTER 2018
1. Lactose high, glucose high
a. Glucose is the preferred source so we will use that instead of activating the Lac operon
b. Because the glucose is high, the cAMP is low so it cannot bind to cap
c. Cap cannot go and recruit RNA polymerase
d. Lactose is high so it is bound to the
repressor
e. So you use the glucose while you
have it, but you are not going to
repress the gene because lactose is
high, so this results in a low/slow
rate of transcription of the lac
operon
2. Lactose high, glucose low
a. The glucose is low cAMP is high, so it can bind to CAP which binds to the promoter which recruits RNA polymerase
b. Lactose being high means that the repressor cannot bind so there is no negative control
c. There is a high level of
transcription taking place
3. Lactose low, glucose high
a. Glucose is high so there is no
enxymes to catabolyze lactose
b. Since glucose is high cAMP is
low, and there is no CAP
complex so RNA polymerase is
not recruited
c. Because the lactose is low, the repressor can bind to the operator and cause repression
d. There is a low rate of transcription
4. Lactose low, glucose low
a. The operator is bound by the repressor so transcription is blocked
b. But because glucose is low you want transcription to occur if you had lactose
c. So cAMP is high so it binds to CAP and that tries to activate the promoter with RNA polymerase, but because there
is the block there, transcription is low
REPRESSIBLE OPERON: TRP OPERON
Used to make the amino acid tryptophan
Default is on, unless TRP is present in high concentrations
If TRP is present, repression occurs
o The repressor protein produced is inactive, so the operon is on, but if
tryptophan is present it binds to the repressor and causes a conformation
change that allows the repressor to bind to the operator
o When the repressor is in place, transcription is blocked
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