Word of the day…

armamentarium (noun): the aggregate of equipment,

methods, and techniques available to perform one’s job.

 Pick up the worksheet from the front of the class.

 Get your clickers ready.
 This is a good time to form groups!
Gene Regulation
Learning Objectives
 Explain using examples of how gene expression may be
regulated at the transcriptional and post-transcriptional
levels.
 Contrast the general mechanisms of positive and negative
transcriptional regulation
 Describe what is meant by basal level (constitutive)
transcription and how the cell regulates this transcription.
 Explain how promoter binding strength and operator
location (upstream vs. downstream) affects the expression
of a positively or negatively regulated operon.
 Identify the structural components of a bacterial operon,
and define the roles of each component in operon
regulation
The “logic” of gene regulation…
 When working on gene regulation or operon
questions, especially those involving unknown gene
systems, it is often useful to consider the “logic” of
regulating that gene or group of genes and the
protein they make.
 In other words, ask yourself when (i.e. under what
conditions) would the cell want to make the
proteins, and when would they NOT want to make
them.
 Cells don’t waste energy and resources making
proteins that are not needed.
Activity time!
 Work on Q1 of the worksheet, and then we will
discuss it.

P Gene 1 Gene 2 T
Basal Transcription
 Basal transcription means a minimal amount of
(but not zero) transcription.
 Even when gene expression is turned off, there is
usually a very small amount (basal) transcription.
 Absolute zero transcription can only be achieved by
removing or destroying a promoter so that no RNA
Polymerase (or more accurately, the transcription
factors) can bind.
Regulatory Proteins and Operators
 A regulatory protein is a protein that regulates
(controls) the expression of a gene or operon in
some fashion.
 An operator is the DNA region to which a
regulatory protein binds. Note that sometimes the
operator is the promoter itself, but often they are
different sequences or have only partial overlap.
Extra Practice Question
Do you think the genes that code for regulatory
proteins can be part of the operons they control?
Why or why not?

Gene 1 Regulatory gene

Extra Practice Question
 No, it wouldn’t make sense since having the
regulatory gene inside the operon it controls means
it would control its own regulation (chicken and egg
sort of problem).
 As far as I know, there are no examples of a
regulatory protein being part of the operon it
controls (but, knowing life, there probably is an
exception out there somewhere…).
Negative vs. Positive Regulation

Positive Regulation Negative Regulation

• A regulatory protein binds
to a region near the
promoter (the operator
region) and increases
transcription.
• The regulatory protein is
called an activator protein
• An example of this is MalT.
• A regulatory protein binds
to a region near the
promoter (the operator
region) and decreases
transcription.
• The regulatory protein is
called a repressor protein
• An example of this is LacI.

This is really important to understand and know!!!

Positive Regulation
Negative Regulation
Worksheet on Canvas to try…
Lac Operon
Lac Operon
Learning Objectives
 Explain the logic of the function and regulation of the lac
operon relative to the availability of lactose.
 Draw a correct representation of the lac operon that
includes the lacZYA genes, the promoter and operator
regions, the lacI gene with its promoter.
 Describe “strong” vs. “weak” promoters as related to the lac
operon.
 Explain how the presence of lactose results in the induction
of the lac operon.
Lac Operon
Learning Objectives
 Explain the mechanisms of repression/blocking of
transcription of lac operon by the negative regulator LacI.
 Predict the effects of mutations in the DNA sequences of
the lac operon and the genes and promoters for lacI.
 Use the lac operon example to explain other examples of
negative regulation in operons.
Some Terminology…
 When we discuss genes expression and regulation
we are often discussing both genes and proteins.
 In order to help distinguish them, we usually write
gene names in lower case and italics, while protein
names are partially capitalized.

lacZ = the gene that codes for the LacZ enzyme.

Operon Examples: Lac and Mal
 Two nice examples of environmentally regulated
genes are the Lac operon and Mal operon.
 Both code for enzymes involved in the breakdown
of sugars that can be used for carbon and energy.
 Therefore the genes only need to be expressed IF
the sugars are available to the cell (and if glucose is
not available…but that is a story for another time).

Lactose Maltose
 Operon Examples: Lac and Mal

Bacteria culture growing in a flask…

glucose lactose maltose

mal operon = “off” mal operon = “off” mal operon = “ON”
lac operon = “off” lac operon = “ON” lac operon = “off”
Inducers
 Because lactose and maltose are the signals that
turn on their relevant operons, we say that their
presence “induces” the operon expression.
 In other words, lactose and maltose are inducer
molecules.
Extra Post-Class Notes
 Note that for us in Biol 112, inducers are small
molecules that the proteins will act on (break down
for energy and food) but note that this isn’t true for
all inducer molecules.
The Lactose (lac) Operon
 The lac operon contains a set of genes that allow
bacteria to break down lactose into glucose and
galactose, both of which can be used for energy
and carbon.
The Lactose (lac) Operon
 The Lac operon codes for three proteins:
• LacZ: a b-galactosidase enzyme that cleaves
lactose in half.
• LacY: a permease transport protein that allows
lactose to enter the cell.
• LacA: a transacetylase whose function is still not
understood and seems unnecessary! We can
safely ignore LacA.
 The Lactose (lac) Operon

E. coli

b-Galactosidase (LacZ)
Permease (LacY)
Glucose

Used for
growth
Lactose
Galactose
Plasma
membrane
 The Lactose (lac) Operon
Operator shown in blue
(overlaps the promoter)

+1 Site
5’ 3’
P lacZ lacY lacA T
3’ 5’

RBS ORF 1 RBS ORF 2 RBS ORF 3

5’ 3’

LacZ LacY LacA

From here on, we will pretend that the lacA gene does not exist.
Extra Post-Class Notes
 The operator of the lac operon partially overlaps
with the promoter sequence, and exists at the
downstream end of the promoter.
 Note that it is possible to remove the part of the
operator to stop the repressor from binding while
keeping the promoter intact (that is important for
the Lac operon worksheet).
The Lactose (lac) Operon
The LacI Repressor
 A regulatory protein called LacI regulates the
expression of the lac operon via negative regulation
(i.e. it is a repressor).
 The gene that encodes LacI is upstream of the
operon.
 LacI is constitutively expressed (all the time).
The LacI Repressor

P lacI T P O lacZ lacY T

LacI Protein LacI binds to the operator of the lac

(repressor) operon, blocking the recruitment of
the RNA polymerase.
lac Operon – without lactose

P lacI T P O lacZ lacY T

LacI binds to the

operator of the Lac
LacI Protein operon, blocking the
(repressor)
RNA recruitment of the
Pol RNA polymerase by
the sigma factor (not
shown).
lac Operon – with lactose

P lacI T P O lacZ lacY T

RNA
Pol
Lactose binds to LacI,
LacI Protein
changing its shape. This
(repressor)
prevents it from binding to
the operator, allowing the
RNA Pol to be recruited.

The Lac operon has a strong

promoter!
Activity time!
 Complete the rest of the worksheet
 Hint: ask yourself the following questions when
working on Q3:
• Given the mutation and the conditions (with or without
lactose):
• What will bind to the DNA and where?
• If something binds to the DNA, what does it do? In
other words, does it block something, does it move
and make something and for how long, etc.
Activity time!
 Work on the rest of the worksheet.
+1 +1

P1 lacI T1 P2 O lacZ lacY T2

ORF1 ORF2 ORF3

R1 R2 R3

LacI LacZ LacY

From the worksheet…
Given what we have just talked about, do you think
the promoter for the lac operon is a strong promoter
or a weak promoter? Explain.

P lacI T P O lacZ lacY T

LacI Protein
(repressor)
 In the absence of the regulatory
protein, the promoter is likely to be
_______ for a positively regulated
gene, and to be _______ for a
negatively regulated gene.

A. weak/weak
B. weak/strong
C. strong/weak
D.strong/strong
E. Not enough information is provided.
Extra Post-Class Notes
 The operator of the Lac operon partially overlaps
with the promoter sequence and exists at the
downstream end of the promoter.
 Note that it is possible to remove the part of the
operator to stop the repressor from binding while
keeping the promoter intact (that is important for
the Lac operon worksheet).
Extra Post-Class Notes
 The fact that basal levels of transcription always
happen is a critical point for gene expression.
 Due to simple chemical equilibrium, any repressor
will eventually detach from the operator just long
enough for a RNA Pol to slip by and transcribe the
operon.
 This allows a very small amount of LacY to always
be present in the cell membrane to allow lactose to
enter (a “just in case” situation).
Extra Post-Class Notes
 There is always some gene expression happening
(basal transcription), with a couple of notable
exceptions:
1) If a mutation destroys a promoter, there is no
where for the transcription factors to bind to and
recruit RNA Pol.
2) In eukaryotes with packaged DNA, only
unpackaged DNA can be transcribed. Any genes
in packaged DNA are not transcribed.
 Please do try to work on the lac operon activity
before the next class as it will help you out when
trying the practice questions.