Gene Regulation

1
Gene Regulation

• Even though all cells (except gametes) have a
  full complement of chromosomes, certain genes
  may be heavily turned on in one cell and never
  turned on in others.
• This means that genes must be under some type of
  regulatory control
• Most of our study of this has come through
  studying prokaryotic cells (bacteria digesting
  lactose)

2

• Constitutive genes genes that encode proteins
  that are always needed
• Two ways that genes control cellular metabolism
• 1) regulating enzyme activity
• 2) regulating enzyme number
• 1961 Francois Jacob and Jacques Monod
• Studying mutant forms of E. coli discovered the
  genes bacteria use to break down the
  dissaccharide lactose
• Discovered Operons a gene complex consisting
  of several structural genes with related
  functions
• Lac operon 3 genes that produce 3 enzymes
• (lac Z, lac Y ans lac A) code for enzymes that
  digest lactose
• 1) first enzyme breaks lactose into glucose
  galactose called B-galactosidase
• 2) second enzyme converts galactose into glucose
  galactose permease,
• 3) third enzymes role is not clear

3
Fig. 18-2
Precursor
Feedback inhibition
trpE gene
Enzyme 1
trpD gene
Regulation of gene expression
trpC gene
Enzyme 2
trpB gene
Enzyme 3
trpA gene
Tryptophan
(b) Regulation of enzyme production
(a) Regulation of enzyme activity
4
Transcription of the lac operon

• 1) Promoter a region upstream from the coding
  sequences.
• Site where RNA polymerase binds to DNA
• 2) RNA polymerase transcribes a strand of mRNA
  that will form the three essential proteins
• 3) Operator switch that controls the synthesis
  of mRNA
• 4) If lactose is absent repressor protein binds
  to the operator which prevents transcription from
  occurring
• 5) Repressor genes are located just upstream from
  the promoter site
• When a cell grows in the absence of lactose the
  repressor genes almost alsways occupies the
  operator
• 6) Allosteric regulator a gene that produces
  protein that binds to a region other then the
  active site, changing its function by altering
  its shape.
• Alters the protein at the DNA binding site that
  makes it unable to recognize the operator

5
Fig. 18-3
trp operon
Promoter
Promoter
Genes of operon
DNA
trpA
trpR
trpE
trpD
trpC
trpB
Operator
Regulatory gene
Stop codon
Start codon
3?
mRNA 5?
RNA polymerase
mRNA
5?
D
A
B
C
E
Protein
Inactive repressor
Polypeptide subunits that make up enzymes for
tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon
on
DNA
No RNA made
mRNA
Protein
Active repressor
Tryptophan (corepressor)
(b) Tryptophan present, repressor active, operon
off
6
Repressible genes

• Characteristic of anabolic activities
• Build up
• These are genes that are usually turned on and
  they are only turned off under certain conditions
• Usually turned off when the products are in
  sufficient quantity
• Ex. Amino acids being put together to make
  protein

7
Ex. Of repressor operons

• Tryptophan operon in E.coli and Salmonella
• The operon consists of 5 genes clustered in a
  transcriptional unit with a promoter and an
  operator to form the amino acid tryptophan.
• It has a distant repressor gene that codes for a
  diffusible repressor protein
• This protein is inactive so it can not bind to
  the operator
• The DNA active site only becomes active when the
  final product tryptophan binds to an allosteric
  site so it is called a corepressor
• So as levels of tryptophan increase the enzyme
  binds to the operator turning it off until
  levels decrease

8
Inducible genes

• Inducible operon a repressor usually controls
  an inducible gene of operon by keeping it turned
  off
• Inducer inactivates the repressor permitting
  the gene or operon to be transcribed
• Usually involve enzymes that are part of
  catabolic pathways
• Allows the cells to conserve energy by only
  making enzymes when the substrates are present

9
Fig. 18-4
Regulatory gene
Promoter
Operator
lacI
lacZ
DNA
No RNA made
3?
mRNA
RNA polymerase
5?
Active repressor
Protein
(a) Lactose absent, repressor active, operon off
lac operon
lacZ
DNA
lacY
lacA
lacI
RNA polymerase
3?
mRNA
mRNA 5?
5?
Permease
Transacetylase
?-Galactosidase
Protein
Inactive repressor
Allolactose (inducer)
(b) Lactose present, repressor inactive, operon on
10
Positive and Negative Controls

• Negative control the DNA binding regulatory
  protein is a repressor that is turning off
  transcription
• Positive control the regulation has activator
  proteins that bind to the DNA to stimulate
  transcription turn it on
• Encourage or increase RNA polymerases affinity to
  the promoter

11
(No Transcript)
12
CAP and cAMP

• A cyclic process
• cAMP (catabolite adenosine monophosphate)
• cAMP binds to the allosteric site so when glucose
  levels decrease the amount of cAMP increases cAMP
  binds to CAP
• This complex binds to the CAP binding site near
  the operon promoter. This bends the DNAs double
  helix stregnthing the affinity of the promoter
  region for RNA polymerase so that transcription
  increases
• Only turns on fully active when lactose levels
  are high and glucose levels are low

13
Fig. 18-5
Promoter
Operator
DNA
lacI
lacZ
RNA polymerase binds and transcribes
CAP-binding site
Active CAP
cAMP
Inactive lac repressor
Inactive CAP
Allolactose
(a) Lactose present, glucose scarce (cAMP level
high) abundant lac mRNA synthesized
Promoter
Operator
DNA
lacI
lacZ
CAP-binding site
RNA polymerase less likely to bind
Inactive CAP
Inactive lac repressor
(b) Lactose present, glucose present (cAMP level
low) little lac mRNA synthesized
14

• While the Trp operon is an example of repressible
  gene regulation and the Lac operon is an example
  of inducible gene regulation, both are examples
  of negative control of genes because both operons
  are shut "off" by an active repressor.
• Gene regulation would be positive, on the other
  hand, if an activator molecule turned the operon
  "on".
• The Lac operon is also an example of a positive
  control system and is turned on by the cAMP-CAP
  complex, as the next section explains.
• E. coli can be described as a fussy eater.
• Its first choice at every meal is glucose because
  glucose supplies maximum energy for growth.
• Therefore, E. coli will only metabolize lactose
  if concentrations of glucose are low.
• For this to work, there must be a signal to tell
  the Lac operon that glucose is not available and
  to astart transcribing the genes to metabolize
  lactose.
• This signal is a small molecule called cyclic AMP
  (cAMP).
• The amount of cAMP present in a cell is inversely
  proportional to the amount of glucose present.
• As a result, the absence of glucose results in an
  increase in cAMP in the cell.
• The following describes the situation where there
  is lactose but no glucose available to the cell
• No glucose means high levels of cAMP.
• cAMP binds to a molecule known as CAP.
• CAP, when in association with cAMP, can bind to
  the promoter at the CAP binding site.
• Here, the cAMP-CAP complex stimulates
  transcription by helping RNA polymerase bind to
  the promoter.
• RNA polymerase has a weak affinity for the Lac
  promoter and will not bind without this help.
• Remember with lactose present so is allolactose.
• Allolactose binds to the repressor and prevents
  it from binding to the operator.
• Therefore, transcription and translation of the
  genes can occur.

15
Fig. 18-6
Signal
NUCLEUS
Chromatin
Chromatin modification
DNA
Gene available for transcription
Gene
Transcription
Exon
RNA
Primary transcript
Intron
RNA processing
Tail
mRNA in nucleus
Cap
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
Translation
Degradation of mRNA
Polypeptide
Protein processing
Active protein
Degradation of protein
Transport to cellular destination
Cellular function
16
Regulons

• Regulon is a group of operons that are controlled
  by a single regulator
• Ex CAP can control the metabolism of galactose,
  lactose, arinose and maltose

17
Constitutive genes

• These are genes that are continuously transcribed
• Ex. Genes involved in the production of ATP,
  repressor and activator proteins
• Not all transcribe at the same rate, the faster
  the enzyme is used up the stronger the promoters
  bind RNA polymerase and the faster the enzyme is
  produced

18
Post transcriptional controls

• Translational controls control the rate at
  which mRNA is translated
• Speed is controlled by how fast the 5 end of the
  mRNA binds to the ribosome
• The faster the mRNA can be read the more protein
  will be produced

19
Post transcriptional controls

• Posttranslational controls activate or
  inactivate enzyme(s)
• Ex. Feedback inhibition the end product binds
  to an allosteric site temporarily inactivating
  the enzyme
• This is different from repression which stops the
  formation of a new enzyme
• Feedback inhibition regulates existing enzymes

20
Molecular chaperones

• Cells contain housekeeping enzymes encoded by
  constitutive genes that can be induced when
  environmental threats or stimuli occur
• These ensure proteins are folded into their
  proper shape

21
Temporal regulation

• Genes that are induced only during certain times
  in an organisms life span

22
Tissue specific regulation

• Gene in the body that may be induced to
  transcribe by several different stimuli depending
  on its location in the body
• Hormone if it is found in a muscle cell
• Different stimuli if it is in the pancreas
• Different stimuli if in a liver cell

23
Promoters in Eukaryotes

• Transcription initatiation site a base pair
  where transcription begins
• Promoter site is a sequence of bases where RNA
  polymerase binds
• TATA box is approx. 25-35 bases upstream from
  the transcription initiation site and is where
  RNA polymerase binds to DNA for transcription to
  occur

24
Promoters in Eukaryotes cont..

• UPEs Upstream promoter elements
• 8 to 12 base sequence upstream from the RNA
  polymerase binding site (TATA box)
• The more UPEs the stronger a gene is expressed
  because transcription is more efficient
• Weaker expressed genes like constitutive genes
  may only have one UPE

25
Promoters in Eukaryotes cont

• In addition to UPEs eukaryotic cells must also
  have Enhancers
• They increase the rate of RNA synthesis that has
  been initiated at the promoter site
• An enhance can control the gene from a far
  distance from the promoter site because DNA loops
  around itself allowing them to come into contact
  with promoters or preventing the DNA to pack
  tightly allowing it to be transcribed

26
Promoters and enhancers

• Both UPEs and enhancers become active when a
  regulatory protein binds to them
• Ex. Steroid hormone interact with zinc fingered
  regulatory proteins causing a conformational
  change activating them
• This changes stimulates transcription

27
RNA interference

• Inhibition of gene expression
• Active genes (21 to 28 nucleotides) can
  permanently shut down other sections of
  chromosomes
• This is caused by being highly packed in the
  chromatin
• These areas are called Heterochromatin
• Euchromatin more loosely packed chromatin
  structure where active genes are found

28
DNA methylation

• DNA is chemically altered by enzymes that add
  methyl groups to certain cytosine nucleotides in
  DNA
• Makes the DNA inaccessible for the enzymes of
  transcription
• Ensures certain sections of DNA remain inactive

29
Gene Amplification

• Process in which a cell reproduces multiple
  copies of a gene by selective reproduction.
• This allows increased production of that gene
  product

30

• Negative control repression, repressor protein
• The binding of a specific protein (repressor
  protein) to DNA at a point that interferes with
  the action of RNA polymerase on a specific gene
  is a form of negative control of protein
  synthesis.
• This interference with RNA polymerase activity is
  termed repression (of the action of RNA
  polymerase) and the consequent to this lack of
  gene expression a gene is described as repressed.
  
• Action results in lack of activity
• This form of control of gene expression is called
  negative control because the controlling action
  results in an absence of activity.
• Contrast this with positive control.
• Positive control activation, activator protein
• Action results in activity
• In contrast to negative control, very often a
  specific gene requires the binding of a specific
  protein (an activating protein) in order to
  acheive RNA polymerase binding and gene
  expression.
• This type of control of gene expression is termed
  activation since in its absence the gene is not
  active (i.e., is not expressed).
• This type of control is also termed positive
  control in the sense that the action of the
  activating protein results in a positive action
  gene expression.