GENE

REGULATION
 Pt. Ravishankar Shukla University
Center for Basic Sciences
Presentation on

Gene Regulation
Session: 2022-23

Presentation by- Guided by-

Meenal Meshram Dr. Jipsi Chandra
5th semester
Contents
 Prokaryotic gene regulation
1) Lac operon

2) Tryptophan operon

 Eukaryotic gene regulation

1) Chromatin structure on transcription

2) DNA methylation/gene regulation

3) Post-transcriptional gene reguation

 Riboswitches
 Gene regulation in lambda phage
PROKARYOTIC GENE
REGULATION
OPERON
 POLYCISTRONIC
 Polycistronic mRNA is a mRNA that encodes several proteins and is
characteristic of many bacterial and chloroplast mRNAs.
 Polycistronic mRNAs consist of a leader sequence which precedes the first
gene.

 MONOCISTRONIC
 Monocistronic mRNA is a mRNA that encodes only one protein and all
eukaryotic mRNAs are monocistronic
 BACTRIAL METABOLISM
 Bacteria need to respond quickly to changes in their environment

- if they have enough of a product, need to stop production

 why? waste of energy to produce more
 how? stop production of enzymes for synthesis

- if they find new food/energy source, need to utilize it quickly

 why? metabolism, growth, reproduction
 how? start production of enzymes for digestion
 TYPES OF REGULATED GENES
 CONSTITUTIVE GENES - Constitutive genes are those that are always active.
Genes for ribosomes are an example. They are constantly being transcribed because
ribosomes are constantly needed for protein synthesis.

 INDUCIBLE GENES - Inducible genes are those that have variable activity,
depending on the needs of the cell. For example, the glucose transporter proteins that
muscle cells produce in response to insulin are the product of inducible genes.

 REPRESSIBLE GENES - Repressible genes are those in which the presence of a

substance (a co-repressor) in the environment turns off the expression of those
genes (structural genes) involved in the metabolism of that substance. e.g.,
Tryptophan represses the expression of the trp genes.
 OPERON
operon, genetic regulatory system
found in bacteria and their viruses
in which genes coding for
functionally related proteins are
clustered along the DNA.

This feature allows protein

synthesis to be controlled
coordinately in response to the
needs of the cell.
ORGANIZATION OF BACTERIAL OPERON
 ORGANIZATION OF BACTERIAL OPERON
 Structural genes: code for the enzymes themselves. Lie adjacent to one another, RNA pol
moves from one structural gene to the next, transcribing all genes into a single RNA

 Promoter: RNA polymerase binding site

 single promoter controls transcription of all genes in operon
 transcribed as one unit & a single mRNA is made

 Operator: resides adjacent to or overlapping with the promoter, serves as the binding site for a
protein, called the repressor (gene regulatory protein)

 Regulatory gene: encodes the repressor protein

 Lac operon
 The lac operon is an operon, or group of genes with a single promoter (transcribed as a
single mRNA).

 The genes in the operon encode proteins that allow the bacteria to use lactose as an
energy source.

 The lactose operon (lac operon) is an operon required for the transport and metabolism
of lactose in E. coli and many other enteric bacteria.

 The gene product of lacZ is β-galactosidase which cleaves lactose, a disaccharide, into
glucose and galactose.
 FOUR SITUATIONS ARE POSSIBLE

1. When glucose is present and lactose is absent the E. coli does not produce ß-
galactosidase.

2. When glucose is present and lactose is present the E. coli does not produce ß-
galactosidase.

3. When glucose is absent and lactose is absent the [Link] does not produce B-
galactosidase.

4. When glucose is absent and lactose is present the E. coli does produce B-
galactosidase.
SUMMARY
 The Lac Operon
 It consist of:
Promoter
Operator
Structural genes
Lac Z (B-galactosidase)
Lac Y(permease)
Lac A(transacetylase)
Polycistronic m-RNA
OPERATORS ARE CIS- ACTING
REPRESSOR ARE TRANS- ACTING
Lac mutant
MUTANTS
 LACTOSE ANALOG
 Isopropyl-B-D-thiogalactoside (IPTG)

 IPTG binds to repressor and inactivates

 IPTG cannot be metabolized by E. coli

 Its concentration remains constant

 Phenyl-ß-D-galactose (phenyl-Gal) phenyl-Gal)

 It is a substrate for B-galactosidase, but does not inactivate repressor and so is not an inducer.

 Since wild type cells produce very little ß-galactosidase, they cannot grow on phenyl Gal as a carbon and
energy source.

 Mutants lacking repressor are able to grow on phenyl-Gal.

 Minimal medium containing only phenyl-Gal as a source of carbon and energy is79 selective for
repressor mutants and operator [Link], but is not a substrate for ß-galactosidase.
POSITIVE GENE REQULATION
 Tryptophan operon
 The trp operon, found in E. coli bacteria, is a group of genes that encode
biosynthetic enzymes for the amino acid tryptophan.

 The trp operon is expressed (turned "on") when tryptophan levels are low and
repressed (turned "off") when they are high.

 The trp operon is regulated by the trp repressor. When bound to tryptophan,
the trp repressor blocks expression of the operon.

 Tryptophan biosynthesis is also regulated by attenuation (a mechanism based on

coupling of transcription and translation).
General organization of theTryptophan operon of [Link]
 Regulation of the trp operon:
 Two mechanisms regulate the trp operon:

 1. Repressor/operator interaction

 2. Termination of initiated transcripts

 Repressor/operator interaction

 When tryptophan is present, tryptophan binds to trpR gene product.

 trpR protein binds to the trp operator and prevents transcription.

 Repression reduces transcription of the trp operon~70-fold.

 Termination of initiated transcripts

 Transcription also is controlled by attenuation, process of translating a short,

incomplete polypeptide.

 When cells are starved for tryptophan, trp genes are expressed maximally..
Under less severe tryptophan starvation, trp genes are expressed at lower than
maximum levels.

 Attenuation can regulate transcription level by a factor of 8 to 10, and

combined with the repression mechanism, 560-700 fold.
Leader sequence
The tryptophans are important because:
If there is plenty of tryptophan, the
ribosome won't have to wait long
for a tryptophan-carrying tRNA,
and will rapidly finish the leader
polypeptide.

If there is little tryptophan, the

ribosome will stall at the Trp
codons (waiting for a Trp-carrying
tRNA) and will be slow to finish
translation of the leader.
EUKARYOTIC GENE
REGULATION
Levels Of Gene Regulation In Eukaryotes

Transcription

Post-transcriptionally
 Influence Of Chromatin
Structure On Transcription
1. Covalent Histone Modification
2. Nucleosome Remodeling
Covalent Histone Modifications
 Structural changes in histone proteins that are associated at the time of
replication and transcription
 Mediated by chromatin modifiaction
 Types of post transcriptional modifications of histone-
I. Acetylation
II. Methylation
III. Phosphorylation
 Acetylation
 Enzyme histone acetyltransferase (HATs) adds the acetyl group to the lysine
amino acid residues in the histone tail.
 Neutrilize the positive charge.
 Reduces the binding between histones and DNA, which provides sites for
effector proteins that can change the chromatin in active state.
 More accessible for transcription
 Reversible process.
 Deacetylated by enzyme histone deacetylase (HDACs).
 Methylation
 Many lysine and arginine amino acid residues at the N-terminal histone tails
undergo methylation.
 Methylation is catalyzes by enzyme histone methyltransferases (HMTs) and
demethylation is by histone demethylases (HDMs).
 Methylation of different parts of the N-terminal tails of H3 and H4 histones is
associated with both activation and repression.
 Transcription activation – methylation of lysine at 4th position of H3
Repression – methylation of lysine at 9th poation of H3
 Fundamental role in heterochromatin formation, chromosome X-inactivation,
genomic imprinting and transcription regulation.
 Phosphorylation
 It takes place in on serines, threonines and tryosines in the N-terminal of
histone protein.
 Controlled by kinases and phosphatases.
 These modifications leads to chromatin condensation and the regulation of
charomatin structure during mitosis. Also serves as a recruiting point for DNA
damage repair protein.
Different classes of histones modifications and their role

Type of modification Residues modified Function regulated

Transcription, Repair,
Acetylation Lysine
Condensation

Methylation Lysine and Arginine Transcription, Repair

Serine, Threonine and Transcription, Repair,

Phosphorylation
Tyrosine Condensation
 Nucleosome Remodeling
 Involes-
1) Sliding
2) Histone exchange
3) Nucleosome eviction

 ATP dependent chromatin remodeling complexes (CRC) responsible for

nucleosome remodeling.
 CRCs are two types swi/snf and ISWI
DNA Methylation And Gene
Regulation
 DNA methylation occurs predominantly at CpG dinucleotides sequence.
 Enzyme DNA methyltransferase mediates the transfer of a methyl group to cytosine,
generating 5-methylcytosine.
 The regions of the genome with high number of methylated cytosine are usually
transcriptionally inactive.
 Sliencing is done by either inhibition of transcription factors through methylayed cytosine or
binding of other protein at CpG methylated site.
 There are two types of methylation process known in eukaryotic cells-

• Addition of methyl groups occurs in

Maintenance a newly synthesized strand of DNA
at positions opposite to the parent
methylation strand

De novo • Addition of methyl groups occurs at

totally new potions of daughter
methylation strand
 Regulation Of Transcription By Methylation

 DNA methylation switches off the eukaryotic gene expression, particularly

when it occur in the promoter region of upstream.
 Protein containing methyl-CpG binding domain (MBD) recognize the DNA
methylation.
 These proteins binds to 5-methyl cytosine residues and inhibit the transcription
by recruiting the protein complexes that induce the alteration of the
chromosome structure.
 Genomic Imprinting

 Epigenetic phenomenon that causes genes to be expressed in a parent-specific

manner.
 There are few imprinted genes whose expression depends on weather they are
inherited from the mother or from the father.
Ex. The gene encoding for insulin-like growth factor 2 (Igf2) is only expressed
from the allele inherited from father.
 In germline cells the imprint is erased and then re-established according to the
sex of the individual, i.e., in the developing sperm (during spermatogenesis), a
paternal imprint is established whereas in developing oocytes (during
oogenesis), a maternal imprint is established.
Post-transcriptional Gene
Regulation
Post-transcriptional Gene Regulation
 Post-transcriptional regulation may occurs at the level of translation.
 Here, regulatory proteins may bind to DNA and either promote or repress
transcription.
Ex. The response of mRNA to iron supply for translational represion
Riboswitches
 Riboswitches
 Cis-acting regulatory RNA elements reside within the transcripts of gene whose
expression they control through changes in secondary changes.
 Located in 5’ UTR of bacterial mRNA.
 Ligands sensed by riboswitches-
Magnesium ions

Nucleic acid precursors

amino acid residues
Sometimes uncharged tRNA
 Riboswitches
Composed of two domains-
 Aptamer : Acts as receptor that binds to ligand.
 Expression platform : Acts directly on gene expression in response to ligand binding.

Mechanism of working of riboswitches-

1. Aptamer binds to ligand and undergoes conformational change.

2. This causes change in secondary structure of expression platform.
3. These conformational change in expression platform alter the expression of
gene by either premature termination of transcription or inhibiting translation
initiation.
Gene Regulation In
Lambda Phage
 Bacteriophage lambda is temperate phage that infects the bacterium E. coli.
 its genome is a linear dsDNA , contains about 50 genes.
 After infecting host cells,lambda phage can follow one of two alternative processes:
lytic and lysogenic.
 Gene Function
Immediate early
gene
N Codes antitermination protein, early regulation
cro Codes cro protein, inhibitor of CI synthesis, early sysnthesis
Delayed early gene
cII Codes activator of transcription of cI and int, early regulation
cIII Codes protein that stabilizes cII, early regulation
int Codes integrase; protein required for site-specific recombination with chromosome
xis Codes excisionase; protein forms a complex with Int and function is excision of
prophage
O DNA replication
P DNA replication
Q Codes antitermination protein, allows expression of the late genes from pR’
Late genes
A to J Head and tail synthesis
S to R Lysis genes
 Lytic Process
 When DNA of a lambda phage enters a new host cell, RNA pol binds to promotor pL
and pR.
 pR – Promoter for cro and other rightward early genes.
pL – Promoter for N and other leftward early genes.
 Genes N codes for an antiterminator protein, allows the transcription of delayed early
genes. Such as cIII on the left.
 The cro gene facilitates the lytic mode.
 Genes O, P and Q are transcribed on right side.
 Q protein – codes for antiterminator protein, which modifies the RNA pol to initiate
transcription of the late genes from pR’ promoter.
 The late gene encodes protein for –
phage capsid production
DNA packaging
Host cell lysis
 Products of genes O and P are needed for DNA replication.
 Lysogenic Process
 The expression of the lytic regulators fails because gene Q is switched off due
to accumulation of cII.
 Results in the blocking of lytic pathway and switching to the lysogenic
pathway. During this switching cII stimulates the synthesis of integrase and cI
repressor.
 Integrase – catalyzes the insertion of phage DNA into the host chromosome.
cI repressor – Responsible for lysogenic cycle.
 Activation of pI, pRE and paQ promoters is critical for establishment of a
stable prophage state.
 cII
During swithing from lytic Switches expression of
to lysogenic pathway gene Q

cI
Integras Blocks lytic pathway and
represso switches to lysogenic
e pathway
r
Promoters Transcription product Location Role

Inhibit the transcription

pI Integrase Xis of xis which is needed
for excision of prophage
DNA from the
chromosome

cI gene encodes for cI

pRE cI gene cII protein responsible for
lysogenic cycle

Reduces Q synthesis and

provide mevhanism by
paQ Inhibition of Q synthesis Q protein which cII reduces late
gene expression to
enhance lysogeny
 The cI gene is also transcribed from promoter pRM, lies between cI and cro
genes.
 pRM and pR are adjacent, but not overlapping.
 It RNA pol binds to pR promoter, cro mRNA will be produced and if it binds to
pRM promoter, cI mRNA will be produced.
 Thus, cI and cro genes are never transcribed at the same time.
 Promoters pL and pR are associated with an operator OL and OR, respectively.
 OR consists of series of binding sites OR1-OR2-OR3

OL consists of the series OL1-OL2-OL3

 In each case, site 1 lies closest to the transcription start site in the promoter, and
sites 2 and 3 lie farther upsteam.
 Lysogenic Mechanism

 Synthesis of cI repressor from pRE, facilated by cII.

 The cI repressor binds to the operators O R and OL of promoters pR and pL
respectively.
 This binding shuts off the transcription of cro mRNA and leftward genes.
 Binding affinity of cI repressor to different sites of O R :-
OR1 > OR2 >OR3
 When cI repressor binds to:-
OR1 – Blocks the cro gene expression
OR2 – Activate transcription of cI gene
OR3 – Blocks over expression of cI gene
 Lysogenic Mechanism

 Thus, cI blocks the synthesis of cro and maintain its own concentration at
particular level.
 cI repressor also binds with OL of pL promoter and prevents the transcription of
leftward early genes.
 Binding of cI repressor with operatos OL and OR shuts off all transcription from
the pL and pR promoters.
 Thus, synthesis of cII and cIII proteins also stop.
 cI repressor itself activates the promoter pRM to synthesize itself.
 Regulation of cII
 cII plays key role in establishing lysogeny.
 Transcription of cII gene is inhibited by both cro and cI binding OR.
 cII are reduced by the bacterial ATP-dependent protease FtsH, encoded by the
hfl(high frequency of lysogeny).
 cIII protein is also important to establish lysogeny.
 cIII controls the rate of cII degradation by acting as an inhibitor of the FtsH
protease.
 Cro also inhibit cIII synthesis by binding to OL.
 Retroregulation of int gene expression
 Int gene transcription is activated from the pI promoter by cII.
 Int is not expressed from the pL promoter by N antitermination, even though
the N antitermination complex transcribes the int gene.
 It is because sib segment inhibit the expression of the int gene from pL but not
from pI.
 Transcription initiated from pI terminates at t1 located in the sib site. This
transcription does not act as substrate for RNaseIII.
 Transcription of int gene from pL passes through t1 and able to form an
extended sib structure, which is a substrate for RNaseIII.
 When phage first infects, integrase and cI are not initially made.
 Phage initiates gene expression that is common to both lytic and lysogenic
pathway.
 If conditions are favorable for lysogeny cI and integrase synthesis is switched
on.
 This synthesis initially required the cII and cIII function.
 Once cI has been made the functions of cII and cIII are no longer required as cI
can maintain its own synthesis.
 The stable lysogen makes sufficient cI repressor to block not only prophage
lytic cycle but also imparting immunity to lysogen against superinfection.
 The resistance of lambda lysogen against superinfection by lambda is called
immunity.
 Cro Protein Function
 Cro protein is essential for the lytic process, first one to be transcribed from the
pR promoter.
 Responsible for preventing the expression of the cI gene to form cI repressor
from pRM.
 Cro repressor binds to both OR and OL.
 Affinity of cro for OR3 is greater than its affinity for OR2 or OR1.
 Cro first binds to OR3 and inhibits RNA pol from initiating transcription from
pRM. So its first action is to prevent lysogeny.
 Then cro binds to the OR2 and OR1 with similar affinity. There in no
cooperative effect.
 Decision Process
1. Following infection, transcription from early pL and pR promoters would start the lytic
pathway by default with N antitermination of transcription and subsequent expression of
Q.
2. Protein Q, in turn, antiterminates transcription, leading to late lytic gene expression, cell
lysis and phage release.
3. To set the course for lysogeny, cII reduces Q function in two ways.
first, cII activates antisense RNA synthesis from paQ.
second, cII activates pRE for synthesis of cI, which represses pR and thus Q transcription.
4. cII continues to repress Q expression via paQ until cII transcription is repressed by cI at
pR.
5. Q shut off by cII and cI ensures the switch from the default lytic pathway to lysogeny.
 If cI action is prevented, cII inhibition of Q via paQ is not sustained because of
repression of cII by cro acting at pR and rapid cII degradation.
 Lambda phage mutants defective in cI or cII follow exclusive lytic growth,
whereas mutants defective in cro are unable to follow the lytic pathway.
 When the phage infects a population of baterial cells that are growing
vigorously, it tends to propagate lytically. When conditions are poor for
bacterial growth, phage is more likely to grow lysogenically.
 These different growth condition depends upon cII. In E. coli it is degraded by
FtsH protease.
 FtsH activity is itself regulated by the growth conditions of bateria.
 If growth condition is good, FtsH is very active, cII is destroyed efficiently, cI
repressor is not made and phage tends to grow lytically. In poor growth
condition, the opposite happens.
 Multiple Choice Question

1. Which of the following is the most appropriate definition of an operator?

a) A non-coding, regulatory DNA sequence that is bound by RNA polymerase

b) A non-coding, regulatory DNA sequence that bound by a repressor protein

c) A DNA-binding protein that regulates gene expression

d) A cluster of genes that are regulated by a single promoter

2. Which of the following statement about lac operon in [Link] is true?

a) Promoter is the binding site for the lac repressor

b) Operon is only switched on in the absence of lactose in the growth medium

c) beta-galactosidase is only produced in large quantities when the lac repressor is bound to the operator

d) lac operon mRNA is a polycistronic mRNA

 Multiple Choice Question

[Link] of the following is not part of the lac operon?

(a)I
(b)O
(c)P
(d)none of these

4. In the presence of high levels of tryptophan

(a)attenuator allows transcription of trp structural genes
(b)attenuator propogates transcription
(c)attenuator terminates transcription
(d)none of the above
 Multiple Choice Question

5. Which of the following occur in the presence of glucose?

(a)lac Z gene expression is increased
(b)cAMP increases
(c)Binding of CAP-cAMP complex to the promoter area decreases
(d)none of the above

[Link] eukaryotes, transcriptional gene control is mediated through

(a) metabolites that bind to cis-acting elements
(b) failure of trans-acting factors to bind to cis-acting elements
(c) binding of trans-acting factors to cis-acting components
(d) proteins that attach to operator sites and act as repressors
 Multiple Choice Question

[Link] most frequent type is in eukaryotes and [Link] is necessary to regulate.

(a) regulation of the promoter
(b) regulation of translation
(c) regulation of the repressor
(d) transcriptional regulation

Answers
1.(b), 2.(d), 3.(a), 4.(c), 5.(c), 6.(c), 7.(d)
References
• Watson J. D., Hopkins, N. H.,Roberts, J. W., Steitz, J. A. andWeiner, A. M.

Molecular biology of the gene, 4th edition, TheBenjamin/Cummings publishing companies,

• Stryer L

Biochemistry, 4 th edition