2. Classification of gene with respect to their
Expression
Constitutive ( house keeping) genes:
1- Are expressed at a fixed rate, irrespective
to the cell condition.
2- Their structure is simpler
3-eg enzymes of glycosis are synthesized by
all cells.
Controllable genes:
1- Are expressed only as needed. Their
amount may increase or decrease with
respect to their basal level in different
condition.
2- Their structure is relatively complicated
with some response elements
3. Synthesis of proteins under the influence of
genes is called as gene expression.
• In eukaryotes the regulation of gene expression is much more
complex because of the presence of nuclear membrane, which
prevents the simultaneous transcription and translation.
• In prokaryotes, the major point of regulation is the control of
transcription initiation.
• In eukaryotes the regulation of gene expression is controlled at
different points.
4. Gene regulation in prokaryotes
• In bacteria,the gene that encode the proteins
required to perform coordinated fn are
clustered into operons.
• The RNA transcribed from prokaryotic operon
is polycistronic a term implying that multiple
proteins are encoded in a single transcript.
5. Gene expression
- Expressed all the time - Expressed only in
specific cells or under
specific condition
Constitutive Regulated
6. • In both prokayotes and eukaryotes, regulatory
proteins recruit RNAP to sites within genome
to initiate transcription
• Regulatory sites are usually the binding sites
for specific DNA-binding proteins (DNABP)
8. lac operon
• E. coli usually rely on glucose as their source
of carbon and energy
• However, it can utilize lactose if it is high in
the medium
9. • No of - galactosidase increases from 10 to
several thousand when grown on lactose (by
increase in syn)
• 2 other enz ↑ together:
1. Galactosidase permease:
↑ transport of lactose across cell membrane
2. Thiogalactosidase transacetylase:
detoxification of compounds that are also
transported by permease
10. • Thus, the expression levels of a given set of
enzymes that all contribute to the adaptation to
a given change in the environment change
together
• Such a coordinated unit of gene expression is
k/as operon
11. lac operon
• 3 elements:
• Regulator gene – i – synthesizes repressor
• Operator site- o
• Structural site- z, y and a
12. lac repressor
• Exist as dimer
• Often two dimers are linked to form tetramer
• Locates operator site by unique pallindromic sequence via one-
dimensional search
• 3-D str: 2 domains-
- Small (amino-terminal) –binds DNA with helix-turn-helix motif
-Large – formation of dimer/ tetramer
14. • inducer forms complex with large domain of
repressor c modify the relation b/w the two
small domains and the ‘o’ site gets free
• Thus inhibition by ‘i’ is lost
15. Inducer
• Allolactose (not the lactose):
- glu and gal are in α- 1,6 linkage (while in α- 1,4
linkage in lactose)
- side product of reaction catalysed by -
galctosidase enzyme
- formed at low levels by few mol of enz that are
present before induction
• IPTG - used as inducer in vitro
16. pur repressor
• Represses gene involved in purine syn (sometimes
pyrimidine syn)
• 31% identical to lac repressor in 3-D str and dimeric
• Analogous to lac repressor
• Binds DNA only when bound to a small molecule
(Corepressor) [thus, behaviour opposite to lac
repressor]
• This corepresoor m/b guanine or hypoxanthine
17. • E.coli genome has >20 regulatory sites, 19
operons and 25 genes
18. Catabolite repression
• When E.coli is grown on glucose, it has very
low levels of catabolic enzymes for
metabolizing other sugars
• This inhibitory effect of glu is k/as catabolite
repression
• This occurs as Glu lowers the cAMP conc in
E.coli
19. CAP
• CAP = Catabolite Activator
Protein
• Also k/as cAMP response
protein
• Dimer of two identical subunits
• Binds at or near the start site
for transcription in lac operon
↑ cAMP
↑ CAP – cAMP
complex
↑ transcription of
lactose and
arabinose
catabolising genes
21. Trp Operon
• E. coli uses several proteins encoded by a
cluster of 5 genes to manufacture the amino
acid tryptophan
• All 5 genes are transcribed together as a unit
called an operon, which produces a single long
piece of mRNA for all the genes
• RNA polymerase binds to a promoter located
at the beginning of the first gene and proceeds
down the DNA transcribing the genes in
sequence
23. In eukaryotes
Organism
Size of
genome
(Mb)
E.coli 4.6
Yeast 12
Humans 3000
Gene regulation is much more complex as:
1. Larger genome:
2. Different cell types- gene expression is different in liver and
pancreas
3. Transcription and translation are uncoupled
25. Combinational control
• Only a few regulators may affect transcription directly in
eukaryotes ( in contrast with prokryotes)
• Each factor recruits other proteins to build up large complex
• It increases transcription on interaction with transcription
machinery
• Thus a given reg factor can have different results
• The result depends on the presence of other proteins in the
same cell
• This is k/as combinational control
26. Combinational control (cont.)
• It is crucial to multicellular organisms
• It leads to generation of different cell types in
unicellular eukaryotes like yeast.
27. Transcriptional factors
• Have several domains:
Domain Function
DNA binding
domain
bind regulatory sequence at
or near promotor
Regulatory
domain
Prevent DNA binding under
certain conditions
Activation
domain
Initiates transcription through
interaction with RNAP-II or
its associated proteins
28. • TFs can be grouped into families depending on
str of seq specific DNA binding domains.
• If binding site lies at a considerable distance
from promoter, it is k/as enhancer
• The intervening DNA can form loops that
bring the enhancer bound activator to the
promoter site
p
E
29. Activation domain has multiple
interaction partners
Transcription Factor
RNAP-II
Mediator 25-30 subunit
part of pre-initiation complex
30. Activation domains
• Less conserved than DNA binding domains
• Some common features:
1. Redundant – a part can/be deleted without loss of function
2. Modular – can ↑ transcription when paired with a variety of DNA
binding domains
3. Act synergistically – two activation domains come together create
much stronger effect
In certain cases, TFs may be repressor rather than activators
31. Nucleosomes
• Histones forms half of eukaryotic chromosome
• DNA+ Histones = chromatin
• 5 major histones: H1, 2A,2B,3,4
• The last 4 forms an octamer
• Around this octamer 200bp DNA is wrapped
• Entire str = Nucleosome
• 100 Å str visualised as beads on string in EM
33. • Studies after extensive digestion shows bead
consist of 145 bp DNA and histone octamer
• It is k/as nucleosome core particle
• Each histone has an amino terminal tail – flexible
and rich in lys and arg
• DNA b/w nucleosome = linker DNA
• DNA nucleosome 104 times
(7- fold compact) compact
34. Chromatin remodelling aids in gene
expression
• Chromatin packing make DNA less susceptible to
clevage by DNAase –I
• Regions adj to gene are more susceptible
• These are less compacted and more accessible to proteins
• k/as hypersensitive sites
• These sites are cell type specific developmentally
regulated
35. • Thus relaxing of chromatin is essential
• Yeast DNABP called GAL4 recognizes DNA at
10 sites only; however 4000 such sites are
present in its genome
• Chromatin binding makes other sites
inaccessible in eukaryotes (C.F. prokaryotes)
36. Enhancers are cell-specific
• Enhancer for CK-MM (m/s isoform) is located
b/w -1350 and -1050 bp
• Insertion of this enhancer near a gene c is
normally not expressed in m/s, l/t ↑ expression
• This is not possible for other cell types
38. Methylation of DNA correlates with
gene inactivation
Cytosine→5-methylcytosine
Methylcytosine binding proteins associates with
DNA
Other transcription factors can’t bind DNA
bcz of steric hindrance → gene inactivation
39.
40. Methylation and DNA imprinting
• In DNA Imprinting the different Methylation
patterns of DNA inherited from the sperm or egg
correlate with choice of allelic expression.
• Methylation patterns are conserved after
replication by action of hemimethylase( which
methylates only one of the 2 strands containing
the CG).
• Methylation of DNA correlates with deacetylation
of Histones.
41. Methylation of genes encoding
proteins which direct abnormally
dividing cell for apoptosis
cancer
42. In totipotent cell (e.g.
fertilized egg ) DNA
undergoes fairly
global de methylation
43. Chromatin str is modulated through
covalent modification of histone tails
• Coactivators loosen histones from DNA
• Effectiveness depends on ability to covalently modify amino
terminal tails of histones
• Lys residues are acetylated by HAT
• B/o this lys looses its charge on amino gp
• Thus, affinity to DNA is decreased
• In addition acetylated lys interact with bromodomain (acetyl lysine
binding domain of certain proteins)
44. Post transcriptional modification
• Trp operon:
• Encodes 5 genes
• 5’ end has leader seq of 162 nuc before initiation codon
• Transcript of only 1st 130 nuc occurs when trp is high
• Site of termination = attenuator
45. • When trp is low, 7000 nuc are transcripted
• When enough trp is present, a stem loop str
form in attenuator region, which l/t release of
RNAP from DNA
46. Motifs in proteins and gene expression
• A motif literally means a dominant element.
47. Common motifs in proteins that
interact with DNA & regulate
transcription
• Although their overall AA sequence &
composition uniquely identify each TF, the
domains involved in each activity can be
grouped into a few # motifs :
• Helix-turn- helix (HTH)
• Zinc finger
• Helix-loop-helix,(HLH)
• Basic region-leucine zipper (bZIP)
48. Helix-turn-helix motif proteins
• It is the domain within the protein that allows
sequence specific interaction with DNA.
• It is of about 20 amino-acids , so is a small part
of a larger protein.
• 1ST 7AA=α-helix, 4AA= non helical turn,
9AA= α-helix.
• 9AA helix: recognition helix that binds in
major groove by forming H-bonds with
exposed bases.
49. • 7AA helix : stabilizes the binding of
recognition helix through Hydrophobic
interactions.
• Both helices include AA, like valine or
leucine, that allow these hydrophobic
interactions to occur.
51. • E.g. of helix-turn-helix includes:
• Lactose repressor of E. coli
• group of developmentally important
transcription factors called homeodomain
proteins ( defect in genes for them causes
homeosis i.e. leg develops in place of antenna
in fruit fly)
52. Zinc finger proteins:
• # : specific amino acids that coordinate Zn
binding.
• it binds in the major groove of DNA in a sequence
specific manner, mediated by an α-helix formed
on one side of finger region.
• Depending on AA nature coordinating with Zn 2
subclasses are formed
• C₂H₂ class
• Cx class
54. Leucine zipper protein :
• # : periodic repeat of leucine residues in an α-
helix. These leucines form hydrophobic
interactions with a 2nd protein in which a
similar helix allows the formation of a dimer.
• so the leucine zipper refers to the protein-
protein interaction domain.
• α—helical region may continue beyond pr-pr
interaction domain, allowing binding to the
major grove of DNA.
55. • DNA contact surface of protein is basic , bcz
of arginine & lysine.
• Basic AA stabilizes DNA-protein interaction
by combining with –ve charged DNA
backbone.
• Dimer can be homo or hetero dimer .
• Homodimer bind to a site that has a dyad
symmetry (2 symmetric half sites), whereas
this symmetry is not seen with heterodimers.
56.
57. Helix-loop-helix proteins
• # : 2 amphipathic α- helical segments
separated by an intervening loop.
• E.g. myoD, myc, & max TF
• 1 helix is used for protein-protein interaction
and 2nd is used to bind major grove.
• So the dimer consists of 4 helices. DNA
binding sites are identical in homodimers &
unrelated in heterodimers.
