BIOLOGY 1A03 Theme 3 Module 1 and 2
October 19, 2015
Gabriel Kuper
Modulating Transcription
Prokaryotes and Their Environments
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Prokaryotes require a nutrient-rich environment containing amino acids, vitamins,
nucleotides, and carbohydrates, which are needed for growth
Favorable temperatures
Gene regulation becomes important to help prokaryotes respond/adapt to their
environment
Prokaryotic Gene Regulation
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DNA that is required all of the time for normal functions are called housekeeping genes
Regulated genes can be turned on and off and on as needed
Gene Categories:
Housekeeping genes
 Constitutively expressed (always being transcribed and translated)
 Allow for the constant maintenance of general cellular activities
 Include genes that are coding for structural proteins (RNA and DNA
polymerases), as well as genes that code for ribosomal proteins
Regulated genes
 Expressed only when needed
 Enzymes that are needed to bring about changes in growth and division
Example: When E.Coli is put into an environment with glucose and lactose it will metabolize
glucose first, since glucose is it’s preferred energy source. When all of the glucose in the
bacteria’s environment has run out and only lactose is left, bacterial growth will not occur for a
short while, but then it will resume with lactose being the primary energy source. This is due to
the production of an enzyme called 𝛽 − 𝑔𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑖𝑑𝑎𝑠𝑒 being created from a regulated gene.
This means that when glucose is present, there isn’t any 𝛽 − 𝑔𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑖𝑑𝑎𝑠𝑒 𝑒𝑛𝑧𝑦𝑚𝑒 because
it is simply not needed. When there is only lactose present, the 𝛽 − 𝑔𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑖𝑑𝑎𝑠𝑒 enzyme
will split lactose into it’s two component monosaccharides; glucose and galactose.
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Francois Jacob and Jacques Monod found that the amount of 𝛽 − 𝑔𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑖𝑑𝑎𝑠𝑒
protein produced by the Ecoli cells begin to steadily increase in response to the addition
of lactose to the growth medium. They also saw that the production of 𝛽 −
𝑔𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑖𝑑𝑎𝑠𝑒 ceased once the lactose was removed.
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BIOLOGY 1A03 Theme 3 Module 1 and 2
October 19, 2015
Gabriel Kuper
Levels of Regulation
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Transcriptional control (DNA  mRNA)
o Controls the binding of proteins (RNA polymerase) to the promoter region to
either activate or inhibit transcription.
Translational control (mRNA  Protein)
o mRNA may not be immediately translated into a protein; the rate at which
translation occurs and the stability of the mRNA affects the amount of protein
produced.
Post-translational control (Protein  Activated protein)
o Polypeptide folds into a 3-dimensional shape
o There are more than a dozen post-translational modifications that regulate the
ability of the protein to become active or inactive, this is done by:
o Driving the assembly into complexes
o The binding of substrates
o Unmasking of enzymatic domains
Post-translational regulation is the fastest; this mechanism allows the cell to have a
stockpile of protein in the cell that is simply inactive. It is then just a simple modification
to turn on all of the inactive proteins. This allows for quick cellular responses to changes
in the environment.
Transcriptional regulation is the slowest, because the cell is starting from scratch. This
type of regulation is often prevalent with more drastic environmental changes that a cell
can be exposed to. For example, when glucose is depleted from E.coli environment,
growth is delayed because 𝛽 − 𝑔𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑖𝑑𝑎𝑠𝑒 must first be transcribed, translated, and
modified before it can begin metabolizing lactose.
Transcriptional regulation is the most efficient process however, because the cell does
not waste any energy or resources making an mRNA polypeptide unless it really needs
to. So, even though it is the slowest process, it is the most economical.
Note: Gene expression means the functional product of the gene is made, modified, and
activated
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Prokaryotic Transcriptional Regulation
Responses to the Environment
Environmental Cues
 Low glucose
 Presence of lactose
Transcriptional Response
 Production of 𝛽 − 𝑔𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑖𝑑𝑎𝑠𝑒cytoplasmically situated protein that cleaves
lactose into glucose and galactose
 Production of lactose permease- a transport
protein in bacterial cell membranes.
 Both are required for the metabolism of lactose
Bacterial Genes
 Clustered into operons; groups of related genes with similar functions
 Different from eukaryotes in which each gene has its own promoter and enhancers.
 Operon model discovered in 1961 by Francois Jacob and Jacques Monod
 Groups of functionally related genes are organized into transcriptional units along the
bacterial chromosome. These gene clusters are often coordinately controlled by a single
‘on-off switch’ that can control the transcription of the clustered genes.
 Coordination is mediated by an operon; which consists of a promoter, an operator (onoff switch), and the coordinated gene cluster.
 An operator is a sequence of nucleotides near the start of the operon that can be
regulated to allow or inhibit transcription.
 When the operator is not bound to any transcriptional inhibitor, the the RNA
Polymerase can attach to the promoter and transcribe the genes in the operon.
 Produces polycistronic mRNA (long mRNA that can code for many proteins)
 Bacteria can still produce separate polypeptides because the polycistronic mRNA is
punctuated with start and stop codons where each coding sequence begins and ends.
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Lac Operon
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LacI
LacO
LacZ
LacY
The regulation of beta-galactosidase and lactose permease expression is under the
control of the lac operon in E.coli cells
The operator (LacO) which is the binding site for a repressor protein that is expressed by
the LacI coding sequence.
LacY gene codes for a lactose permease protein which will imbed in the cell membrane.,
and the LacZ gene which codes for beta-galactosidase.
LacI gene controls LacY and LacZ genes because LacI is the gene that codes for a
repressor protein which can bind to the operator and inhibit transcription. This is called
negative transcriptional regulation.
– Repressor protein
– Operator
– 𝛽 − 𝑔𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑖𝑑𝑎𝑠𝑒
– Lactose permease
Negative Regulation of the Lac Operon
Lactose Operon is Negatively Regulated
 Repressor protein created from LacI (tetrameric protein) inhibits the binding of RNA
polymerase to the LacO site by twisting the operon DNA into a loop and thus
transcription is inhibited, it can only do this once all four subunits of the repressor
protein bind the lac operon DNA.
 In E.coli the presence of glucose will facilitate the constitutive expression of the
repressor protein.
Lactose Allosterically Inhibits the Repressor
 Inducer molecule
 Lactose binds to specific binding sites on the repressor protein causing a conformational
change of the repressor so it can no longer attach to the operator sequences in the lac
operon DNA
Positive Regulation of the Lac Operon
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In the absence of glucose, positive regulation of the lac operon promotes the production
of beta-galactosidase and lactose permease.
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It is the increase in cAMP levels that will contribute to the positive regulation of the lac
operon
Glucose Concentration
 High glucose
o Inhibition of the enzyme adenylyl cyclase- inactive adenylyl cyclase (which
catalyzes the production of cyclic AMP from ATP)
o Low levels of cAMP
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Low glucose
o Active adenylyl cyclase enzyme
o Accumulates high levels of cAMP
CRP/CAP: cAMP Receptor Protein or called Catabolite Activator Protein
cAMP Signals
 cAMP binds to the cAMP receptor protein as
an allosteric activator which induces a
conformational change in shape of this
transcriptional activator protein. As a result,
the CRP-cAMP complex is then able to bind to
the bacterial DNA and further transcription of
𝛽 − 𝑔𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑖𝑑𝑎𝑠𝑒 and lactose permease
 Low levels of glucose signal an increase in cAMP to activate the positive regulator (CRPcAMP) and the presence of lactose binds the repressor protein LacI to allow
transcription of the operon to occur.
 High levels of glucose signal the inhibition of the adenylyl cyclase enzyme and as a
result, low levels of cAMP. Even in the presence of lactose the CRP-cAMP complex will
not bind to the lac operon.
Note: CRP-cAMP complex is an activator protein and helps the binding of the RNA polymerase
- Levels of lac operon mRNA are highest when bacteria are actively utilizing lactose as a
nutrient source.
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