Download doc NTC Mar 31

Lecture 34: March 31st
Gene Regulation I- Prokaryotic gene regulation II
Summary of where we ended last time:
 Went through experimental approach to the negative regulation of the lac operon
 We know that this inducible and that it is negative control
 So if lactose is not present in the culture medium then the Laci gene is transcribed and
is able to bind into the laci repressor protein, which is able to bind to the operator
which is a DNA sequence upstream of the promoter of the Lac Operon. Therefore the
RNA polymerase has a physical block, so no proteins are produced there
 When lactose is present the Laci protein can interact with lactose, it undergoes an
allosteric change, which makes it no longer able to bind to DNA so the operator
sequence is now free, and can be transcribed by the RNA polymerase, to be translated
into proteins utilized for the breakdown of lactose
 The function of Beta Galactosidase and Permase is known, and the function of
transacetylase is still unknown
If the permase is needed to uptake lactose, how come the organism can detect lactose?
Because there is a low level of transcriptional activity, allowing it to sense lactose.
Bacterial growth responds to the presence of difference sugars:
 A lot of primary work on understanding the lac operon was done using growth curves
 You look at how many bacteria are present in the growth medium over time, and if
the bacteria are able to grow and divide, the number will increase over time
 If you grow bacteria in a rich medium, containing glucose, you will see, no matter
what bacteria you use, you will see different phases
 Lag phase: initial slow phase, the bacteria are getting used to the medium
 Log phase: Dividing regularly at intervals of about twenty minutes, and over time
of population doubles
 Stationary Phase: At some point the medium is not as rich as it was initially
because the bacteria have been growing and producing catabolites, so growth
begins to slow, there is no more division and the cells are alive
 Death Phase: all of the nutrients have been consumed, and the number of cells
declines
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They were interested in different enzyme activations, so they started to mix various
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sugars, similar curves could also be obtained with different sugars, as long as there is
only one kind in the medium
So he tried mixing various sugars
If you combine glucose and lactose
 Growth curve becomes bi-phasic
 In phase I cells use glucose
 In phase II, glucose is exhausted, and they change to using lactose, creating a
second logarithmic phase
 So this means that bacteria can sense and choose between different sugars
 He started to study the physiology behind this situation
Showed him - Positive control of the lac operon
 Glucose is the preferred energy source, so when both glucose and lactose are
available the operons for sugars other than glucose are off
 There is always a very low transcription in the cells
 There is balance in the cells not to transcribe and translate proteins that are not
needed, but have a little bit to allow for adaptation
 The approach was done through the study of particular mutants
Cya and Crp mutants
 Decreased levels of lacZ and lacY upon induction
 In lacI-- cya and crp mutants exhibit lower levels of these enzymes
 Suggests that these two genes are also involved in the regulation of the lac operon
 However, they map outside the lac operon
 The cya gene encodes for adenylate cyclase which produces cyclic cAMP,
starting from glucoses
 Crp (cAMP, receptor protein) gene encodes for the catabolic activator protein
(CAP) that binds cAMP
 These two genes interact with each other
 Later on, cyclic AMP and its function were studied elsewhere and it was found
that cAMP levels are regulated by the amount of glucose in the cell
 They are inversely regulated (low glucose = high cAMP)
 When there is high cAMP, CAP protein can bind with it, there is an
induction of an allosteric change and this protein can bind to the lac
promoter, positively activating it
What happens on the molecular level: When CAP binds to DNA
 Structural studies have shown that when CAP binds to DNA it wraps the DNA
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around itself
The DNA is stretched and bent, exposing the nucleotide sequence much better, so
the RNA polymerase can recognize it, and bind it more efficiently
In addition to this, it has been shown that RNA polymerase and the CAP protein
can interact with each other, so RNA is also stabilized in the proximity of the
promoter
The Lac repressor and CAP proteins bind to DNA sequences with two-fold rotational
symmetry
 Certain nucleotide positions are more important than others (these are highlighted
in the figure)
 If you look at the polarities, the sequences are sort of palindromes, and in the
center of the symmetries is the dot (highlighted in red)
 They are multimers- you do not have one molecule that binds to the DNA, but
they interact with each other, they are either dimers or tertamers
 There is an advantage in this type of organization because it docks two molecules
and is stronger binding than just the single protein
 The other observation is that the AT pair is thermodynamically weaker than GC
(AT only has two hydrogen bonds so takes less energy to open up the helix)
 When we look at these two sequences that there is more AT pairs than GC pairs,
in the lac operator so it is thermodynamically easier for RNA polymerase to open
this region
 One possibility is that the bending facilitates the opening of the two DNA strands
The Lac Operon at the molecular level:
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Shows all the different binding sites upstream of the lac operon
The lacI gene: synthesizes the repressor all the time
Then you have a series of binding sites that are partially overlapping
 The site where CAP binds once it complexed with cyclic AMP
 Site where that RNA polymerases recognizes
 Then there is the operator, which binds the repressor
 The transcription start is overlapping this region
 Then there is the Shine-Dalgarno sequence which promotes mRNA translation
 There are a series of regulatory requirements that are next to each. The proximity
makes them work together
 They can work together- eg. Cap protein facilitates RNA binding
 It can be exclusive, eg. The repressor binding site stops all transcription, blocking
the RNA start site
What does this mean in terms of bacterial physiology
 Glucose present:
 cAMP is low, no cap protein complexed , no lactose is metabolized, no lac
mRNA is synthesized
 Leaky expression
 Glucose and lactose present:
 They system is balancing, there is not a lot of cAMP, but there is a bit of lactose
metabolized
 Depends on the relative concentration of glucose, how much cAMP is there
 Occasional mRNA
 No glucose, and lactose is present
 CAMP high, all of the enzymes to metabolize lactose
 In bi-phasic growth, the first plateau is when the bacteria is synthesizing all the new
enzymes
 This examples shouws that positive and negative regulation can work in the same
time
 Why use both
 Is would be simpler, but it would not be as adaptable
 More advantageous for evolution
The Ara operon:
 A lot of similar features to the lac operon
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Similarities with regulation of lac-operon
 Presence of cis regulatory sequences,
 Genes are arranged together in operating units
 Depends on regulatory genes and proteins to be expressed, these regulatory genes
and proteins undergo allosteric changes
 Also uses cAMP-CAP complex
Positive control of Ara
 The initiator of the process is AraC, situated early in the operon
 When there is arabinose present, AraC undergoes an allosteric change and binds
the DNA, activating the operon
 The cAMP-CAP complex also stimulates transcription
Negative control of Ara ( in the absence of arabinose)
 AraC has a different conformation, changes its properties and makes it behave as
a repressor
 AraC can still bind to the AraI, but it can also bind to AraO
 The result of this double interaction with the DNA is that the DNA is bent
 Steric impediment for mRNA transcription to begin
Summary:
 Activating and repressing mechanisms coexist and the can cooperate
 Regulatory proteins undergo allosteric changes upon binding affector molecules
and result in different protein properties
 Activating effector proteins bind to DNA and may change DNA conformation to
help recruit the RNA polymerase complex
 Activators may directly interact with RNA polymerase and further stabilize the
transcription initiation complex
 Repressors hinder the recruitment of RNA polymerase complex to the
transcription site by various mechanisms (steric hindrance, conformational change
of DNA etc.)
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