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Lecture 9

BIOB11H3 Lecture Notes - Lecture 9: Green Fluorescent Protein, Recombinant Dna, Transgene

Biological Sciences
Course Code
Dan Riggs

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Lactose (disaccharide) metabolism in E.coli
E.coli use lactose as a carbon source. When the bacterium finds itself in the presence of lactose, it
induces the lac operon and ultimately one of the genes from the lac operon encodes an enzyme called
beta-galactosidase gets expressed. Then the beta-galactosidase cleaves the lactose into sugar molecules
(glucose + galactose). The bacterium evolved this system because it loves glucose as its favorite carbon
source. If the cell does not have glucose in its environment, but has lactose it can get what it likes to eat
by following this pathway.
Lactose= dissacharide of calactose + glucose
The kinetics of this induction process is dramatically rapid. In Fig 12-1, the cells are growing fine. When
the investigator added lactose (inducer) at 4 min mark, the lac operon is activated/ induced. As you can
see the blue line was shooting up dramatically which represents the beta-galactosidase. There was a
rapid response to that inducer. About a minute or so, the RNA gets translated into protein. You will see
the beta-galactosidase protein in red line. It goes up dramatically too. As time goes on, the investigator
removes the inducer. After some time the bacterium uses up all the lactose. The mRNA level suddenly
crashes. The level beta-galactosidase protein will also go down because that protein would be turned
over after certain amount of time. There is rapid induction response to the inducer; rapid sensation of
mRNA synthesis and levels and protein levels when the inducer is removed.
Operon Structure
An operon is a collection of components that is responsible for organizing a response to a particular
stimulus. (ex: induction by lactose)
Fig 12-2
All of the operon components are orange/sometimes in yellow. Operon components consist of
promotor, operator, structural gene, and regulatory gene. Regulatory gene encoding a repressor protein
is going to interact with operator sequence and it is going to determine in part whether or not that
particular group of genes get expressed. During the evolution of operons, things were organized such
that there are several genes that are involved in coordinating the same pathway, same response and
they are all coordinately transcribed together. You have a single stimulus and generate several proteins
for the response.
Inducible Operon: The lac operon
Structural genes are Z, Y, and A. Promoter and operator as P and O. There is your inducer. Ecoli is
coming in contact with lactose and the repressor is normally active. As soon as it is synthesized, the
repressor binds to the operator sequence. If lactose is present, the repressor interacts with it and forms
a complex. If Lac present, repressor bound and changes its structure such that it is inactivated. Operon
induced, mRNA transcribed. This is idutio.
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Multiple enzymes, RNA polymerase, are transcribing the mRNA. Eventually ribosomes associate with
various initiation sites. Ultimately, 3 enzymes are produced and they take the substrate lactose and
convert it into final products (glucose and galactose). The lactose levels will drop. The binding of lactose
to repressor is transient, so as [lactose] falls (it is not able to find its binding partner readily), repressor
becomes active. Repressor is now able to bind to the operator. When transcription is blocked,
repression takes place.
Cis s. Tas: Cis-acting promoter sequence to which a trans-atig tasiptio fato ids
Trans: trans-acting or soluble or can defuse (eg. A transcription factor)
Cis: o the sae stad (eg. DNA seuee that serves as a binding site for a TF)
TATA box is an example of a cis acting element. The sigma factor or TPB (in eukaryotes) will recognize
that specific sequence and bind to the TATA box.
Positive vs. negative control: depends on the active form of the trans-acting factor (eg. Repressor), and
its effect upon binding to its target cis-acting sequence.
Bacterial biochemical logic for Lac=Glu + Gal
1. If glucose is available, why expend energy to make energy to catabolize lactose?
2. If lactose is absent, why expend energy to make enzymes to catabolize it?
Both positive and negative control involved.
Positive control: If glucose level is low, cAMP level is high (inverse relationship); cAMP binds to,
transcription factor, CRP (cAMP receptor protein), and the complex activates the lac operon.
The LAC promoter/operator
Positive control can dictate how well RNA polymerase can load in its binding site.
Negative control has cis acting repressors and serves as a road block for RNA polymerases.
Fig 12-11
Four situations: sugar availability and positive/negative control
Allolactoses: an isomer of lactose, is the actual inducer
Lactose is not an actual inducer.
Lactose is high and glucose is high.
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Bacterium has its favorite carbon source, glucose. Why go through the trouble to activate the lac operon
even though lactose is high. Since lactose is high, we have lots of allolactose that is bound up to the
repressor that made it inactive. If you have lactose in your environment, you want to inactivate the
repressor so that you can get transcription if it is possible to do so.
Whe the leel of gluose is high, the leel of AMP is lo. You at fo a atie CAP ople,
because the cAMP is low. So cat go to the CAP site actively recruit RNA polymerase. There is a low rate
in transcription in this process because there is no road block.
That glucose is running out, but lactose level is high. Alloactose is present, binds to the repressor, takes
it off the operator to get high levels of transcription. Since glucose is low, cAMP is high. Since cAMP is
high it can activate CAP and go to the CAP binding site. Actively recruit RNA polymerase, where RNA
polymerases load one after another, where high transcription is taking place.
The glucose level is low and the lactose level is high. Lactose level is low, no lactos to bind the lactose
epesso. No the epesso potei ids the opeato ad its saig that it doest akes sese to
transcribe these genes because there is no substrate to work on. It got its favorite carbon source and as
a consequence of that, positive control is not implemented. Glucose level is high, cAMP is low. Therefore
it cannot from active CAP complex and at actively recruit RNA polymerase. Whatever RNA
polymerase does get recruited it gets blocked. There is a very low rate of transcription.
Lactose low, glucose low. For negative control, no lactose means that the repressor is going to bind to
the opeato. Doest ake sese to transcribe the lac operon if there is no lactose to convert into
glucose. You wanted to be able to transcribe this if you could but as the glucose level is low, cAMP is
high. You can form this CAP complex to come and recruit lot of molecules of RNA polymerase. Since
there is no lactose, the road block is in place and you get a very low rate of transcription.
Fig. 12-3
Repressible Operon: TRP Operon
Used to make the amino acid tryptophan.
Default is ON (you need it), unless TRP is present. If tryptophan is present, why go to the trouble of
making it? If TRP is present, it interacts with the inactive repressor it makes an active repressor that
binds to the operator sequence. TRP acts as a corepressor.
Repressed state: no TRP production
As TRP used, [TRP] falls. Thus no co-repressor present and repressor no longer functions. Depression
(reactivation) occurs. mRNA processed and translation yields 5 enzymes that convert precursors to
Fig 12-33
Eukaryotes: An overview of levels of control of gene expression
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