Strategies for regulation of gene expression in prokaryotes and eukaryotes for biology, biochemistry and medical undergraduates

1. Regulation
of
Gene Expression
R.C. Gupta
Professor and Head
Dept. of Biochemistry
National Institute of Medical Sciences
Jaipur, India

2. Expression of gene
means synthesis of
the product encoded
by the gene

3. A large number of genes
are present in DNA
Most of them encode proteins;
some encode tRNA and rRNA

4. Proteins encoded by different genes are
not required all the time
A gene should be expressed only when
the protein encoded by it is required
This is possible only if gene expression
is regulated

5. Living organisms employ a number of
strategies to regulate gene expression
Regulation increases viability and
adaptability of the organism
Regulation can occur at different stages
of gene expression

6. Gene expression can be
regulated at the level of:
• Transcription
• Post-transcriptional modification
• Translation
• RNA degradation

7. Gene
hnRNA
mRNA (in nucleus)
mRNA (in cytosol)
Protein
Control point
Control point
Control point
Control point
Points for regulation of gene expression

8. Gene expression is regulated in pro-
karyotes as well as eukaryotes
Regulation is different in prokaryotes
and eukaryotes

9. First insights into regulation of
gene expression were provided
by Jacob and Monod in 1961
Jacob
Monod
Regulation in prokaryotes
They studied the regulation of
genes encoding key enzymes
involved in metabolism of lactose

10. Jacob and Monod conducted their study in
E.coli
b-Galactosidase
The enzymes required to metabolize
lactose in E.coli are:
Galactoside transacetylase
Galactoside permease

11. b-Galactosidase is encoded by z gene
Galactoside permease is encoded by
y gene
Galactoside transacetylase is encoded
by a gene

12. The z, y and a genes are structural
(protein encoding) genes
A regulatory (i) gene
A promoter (p) site
An operator (o) site
These are preceded by:
These genes are present in a cluster

13. Regulatory
gene
Control
sites
Structural
genes
i p o z y a
This cluster is called the lac operon

14. Operon
In bacteria, related genes are often
present in a cluster on the chromosome
They are trans-
cribed together
They are preceded by
a common promoter
The cluster of related genes is known as
an operon

15. E.coli can use lactose as well as other
sources of energy
Enzymes that metabolize lactose are not
required, and hence not synthesized, in
the absence of lactose
Entry of lactose in the cell induces
the synthesis of these enzymes

16. The i gene encodes a repressor subunit
Its expression is constitutive
The repressor subunits are synthesized
continuously
Four subunits combine to form the
repressor tetramer

17. i gene
i gene
mRNA
Repressor
subunits
Repressor
tetramer

18. Repressor tetramer attaches to the
operator site
This prevents the transcription of z, y
and a genes by RNA polymerase
The enzymes encoded by these genes
are not synthesized

20. When lactose enters the cell, it is
converted into the inducer, allo-lactose
One allo-lactose molecule binds to each
repressor subunit
This prevents the formation of repressor
tetramer

21. The operator site remains free
RNA polymerase can move down-
stream
RNA polymerase transcribes z, y and a
genes

22. The polycystronic mRNA is translated
b-Galactosidase, galactoside permease
and galactoside transacetylase are
synthesized

24. Lac operon has a positive regulator
also
When the bacterial cell is starved of
energy, concentration of cAMP rises
cAMP forms a complex with catabolite
gene activator protein (CAP)

25. CAP-cAMP complex binds to promoter
site
This facilitates the binding of RNA
polymerase holoenzyme to promoter site
When energy is abundant, cAMP
concentration falls, and CAP-cAMP
complex is not formed

27. In eukaryotes, gene expression is far
more complex
Regulation can occur at several stages
Regulation in eukaryotes

28. The regulatory strategies in
eukaryotes include:
Gene amplification
Gene re-arrangement
Regulation of transcription
Alternative splicing
RNA editing
EMB-RCG

29. Gene amplification means formation
of multiple copies of a gene
Gene amplification

30. Synthesis of proteins can be increased
by amplifying the genes encoding them
Amplification may be spontaneous or
secondary to exogenous signals e.g.
drugs

31. Amplification of genes encoding tissue
proteins has been seen in Drosophilia
This occurs during growing age to
permit rapid formation of tissues

32. Amplification of dihydrofolate reductase
gene has been seen in cancer cells on
administration of amethopterin
Amethopterin is a competitive inhibitor of
dihydrofolate reductase

33. Human beings can form millions of
different antibodies
Antibody diversity arises from gene re-
arrangement
Light chains and heavy chains are made
from different segment
Gene re-arrangement

34. Genes for different segments are
located at different sites on the DNA
These genes are present in small
clusters
Each cluster contains a small number
of genes

35. The gene segments are joined in different
combinations in different B cells
This creates a huge diversity of antibodies
having different antigen specificities

38. Regulation of transcription
Transcription is the major control point in
eukaryotes
Control of transcription can control the
overall process of protein synthesis

39. The regulatory mechanisms
determine:
When a gene should
be transcribed


How often it should
be transcribed
In which tissues it
should be transcribed

40. Some regulatory sequences are present
in DNA itself
These intra-DNA sequences which
affect transcription are known as cis-
acting elements

41. The cis-acting elements include:
Enhancer elements
Silencer elements
Hormone response elements
Heat shock elements
Promoter elements
EMB-RCG

42. Some extra-DNA factors influence the
cis-acting elements
The extra-DNA factors that act on the
cis-acting elements are called trans-
acting factors
These include inducers, repressors and
a number of protein factors

43. cis-Acting element
trans-Acting factor
Genomic DNA
Initiation of transcription

44. Enhancer elements are cis-acting
elements
These may be located far away from the
gene that they influence
They may be upstream or downstream
from the gene they influence
Enhancer elements

45. Binding of trans-acting factors to the
enhancer element increases the
transcription of the gene
By looping of DNA, enhancer element
and the trans-acting factors are brought
close to the gene

47. Enhancer element and the trans-acting
factor facilitate binding of basal trans-
cription apparatus to the TATA box
This increases the transcription of the
gene
Enhancer elements are poor in
specificity

48. Silencer elements are also located at a
distance from the gene they influence
They may be upstream or downstream
of the gene they influence
Silencer elements

49. Silencer elements suppress transcription
of the genes that they influence
Tissue-specific expression of genes may
be a function of enhancer elements and
silencer elements

50. Some hormones produce their actions
by acting on DNA
Examples are steroid hormones, thyroid
hormones, calcitriol, retinoic acid etc
They bind to their receptors forming
hormone-receptor complexes
Hormone response elements

51. The gene influenced by the hormone is
preceded by a hormone-response
element
The receptor-hormone complex binds to
the hormone-response element
This increases the transcription of the
downstream structural gene

52. Hormone-response element (HRE) is the
binding site for hormone-receptor complex

53. Heat shock elements also increase the
expression of some genes
They act in response to heat stress and
other types of stress
Heat shock elements

54. Promoters respond to inducers and
repressors
Inducers increase genes expression
Repressors decrease gene expression
Promoters

55. Stability of mRNA also affects the rate of
gene expression
Long-lived mRNAs can be translated
many times over
This increases the rate of protein
synthesis
mRNA stability

56. The primary transcript (hnRNA) can be
spliced in different ways
By joining different exons, different
proteins can be formed
Alternative splicing

57. hnRNA
mRNA 1 mRNA 2
Alternative
splicing
EXON1 INTRON1 EXON2 INTRON2 EXON3 INTRON3 EXON4
EXON1 EXON3 EXON4EXON1 EXON2 EXON4

58. hnRNA of tropomyosin is spliced in
different ways in different tissues
This results in the formation of different
tropomyosins in different tissues

59. hnRNA of troponin T is also spliced in
different ways in different tissues
Membrane-bound form and secreted
form of IgM are formed by alternative
splicing of the same hnRNA

60. mRNA may be edited before translation
Editing may be different in different
tissues
Different proteins may be synthesized in
different tissues from the same mRNA
RNA editing

61. An example is synthesis of apo B in liver
and in intestine from the same gene
The gene is transcribed to form identical
mRNAs in liver and intestine
The mRNA is translated as such in liver
but is edited before translation in
intestine

62. The product of apo B gene in liver is
apo B-100, made up of 4536 amino acid
residues
The product of apo B gene in intestine is
apo B-48, made up of 2152 amino acid
residues

63. Amino acid sequence of apo B-48 is
identical with the first 2152 amino acid
residues of apo B-100
In the intestinal cells, the mRNA for apo
B is deaminated at position 6666

64. The base at position 6666 is cytosine
Deamination converts it into uracil
The codon CAA (glutamine) is changed
to UAA (stop signal)
Premature termination of translation
yields a protein 48% in size as
compared to apo B-100

65. Proteins affecting transcription by inter-
acting with DNA possess some distinct
structural motifs
These motifs help them to bind to
specific DNA sequences with high affinity
Protein-DNA interaction

66. Helix-turn-helix motif
Zinc finger motif
Leucine zipper motif
Structural motifs of DNA-
interacting proteins include:

67. Proteins having helix-turn-helix motif
consist of two identical monomers
The DNA recognition domain of each
monomer binds to five base pairs of
DNA in a major groove
An example is catabolite gene activator
protein (CAP) of E.coli
Helix-turn-helix motif

69. DNA binding proteins having zinc finger
motif have two to nine zinc fingers
Zinc fingers are finger-like projections in
the polypeptide
These are formed by binding of zinc to
four cysteine residues or two cysteine
and two histidine residues
Zinc finger motif

71. Each zinc finger binds five base pairs of
DNA in a major groove
Examples are steroid hormone receptors,
thyroid hormone receptors, calcitriol
receptor etc

72. Proteins having leucine zipper motif
consist of two identical monomers
Leucine residues are present at every
7th position in the monomers
The leucine residues are stacked one
above the other in the monomer helices
Leucine zipper motif

73. Leucine residues are hydrophobic
Leucine residues of two monomers
attract each other to form a zipper-like
structure
An example is cAMP response element
binding protein (CREB)

74. HOOC
COOH
NH2
NH2
Leucine
Zipper
Leucine zipper
motif
Monomer