Ph.D. Research Scholar at BAU, Ranchi GENETICS AND PLANT BREEDING

1. GENE EXPRESSION IN
EUKARYOTES
ANJANI KUMAR
(A/BAU/5129/2017)
Department of Genetics and Plant Breeding
FACULTY OF AGRICULTURE
BIRSA AGRICULTURAL UNIVERSITY
KANKE, RANCHI – 834006 (JHARKHAND)
DOCTORAL SEMINAR (GP-691)

2. • Deoxyribonucleic Acid
• Double helix
• Carries genetic information
• Located in the nucleus
• The monomer is a nucleotide
– A phosphate
– A ribose sugar
– A nitrogenous base
What are the bases in DNA ??
A – adenine
T – thymine
C – cytosine
G – guanine
DNA

3. Where are the genes located?
• Genes are located on
the chromosomes
• Every species has a
different number of
chromosomes
• There are two types of
chromosomes:
autosomes and sex
chromosomes

5. GENE EXPRESSION
Gene expression is the process by which the instructions
in our DNA are converted into a functional product, such
as a protein.

6. Gene expression is a multi-step process
which involves
o Transcription
o Translation

7. Central Dogma of Life

8. Transcription

17. Eukaryotic Promoter
Conserved eukaryotic promoter elements Consensus sequence
CAAT box GGCCAATCT
TATA box TATAA
GC box GGGCGG
CAP site TAC
17
Eukaryotic Promoter lies upstream of the gene. There are
several different types of promoter found in the eukaryotic
genome, with different structure and different regulatory
properties

18. PREINITIATION COMPLEX
First, an RNA polymerase along with general transcription
factors binds to the promoter region of the gene to form a
closed complex called the preinitiation complex
Preinitiation complex contains:
 Core Promoter Sequence
 Transcription Factors
 RNA Polymerase
 Activators and Repressors

19. Eukaryotic gene expression can be regulated at many stages
Chromatin accessibility
Transcription RNA processing
Post-translational eventsTranslation
Eukaryotic gene expression is regulated at 5 main levels

20.  The normal structure of the
chromatin suppresses the gene
activity, making the DNA relatively
inaccessible to transcription factors,
and thus active transcription complex
can’t occur.
 Thus chromatin remodeling is
needed ( it is a change in chromatin
conformation in which proteins of
nucleosomes are released from DNA ,
allowing DNA to be accessible for TFs
and RNA polymerase).
Chromatin conformation and Gene expression

21. Inactive chromatin remodeled into active chromatin by 2
biochemical modifications:
1. Acetylation of histone proteins by histone acetyl transferases
which loosen the association between DNA and histone.
2. Specialised protein complexes disrupt the nucleosome
structure near the gene’s promoter site.
Ways of Chromatin Remodeling

24. What is a Transcription Factor (TF)?
Any protein other than RNA Polymerase that is
required for transcription
These are of two types:
General transcription factors- Required for the binding of the
RNA pol to the core promoter and its progression to the
elongation stage are necessary for basal transcription
Regulatory transcription factors- Serve to regulate the rate of
transcription of nearby genes They influence the ability of
RNA pol to begin transcription of a particular gene. These
factors recognize cis-regulatory elements located near the
core promoter.
RNAP itself does not have the ability to recognize specific
DNA sequences such as promoters. Instead, a group of
proteins, called general transcription factors (GTFs), help
RNAP to find promoter sequences (Hampsey, 1998;
Orphanides et al, 1996)

25. The RNA-transcribing enzyme, RNA polymerase II (red), requires general transcription factors (TFII) D, A, B, F, E, and H (blue),
which themselves consist of multiple subunits, to recognize the transcription start site via the TATA box or related
sequences in the core promoter. The sum of these factors, known as the pre-initiation complex (PIC), is required for basal
transcription.
Transcription factors (green) bind to specific DNA sequences (red) via their DNA-binding domain (DBD) and modulate the
rate of transcription via their transactivation domain(s) (TAD).
A simplistic view of regulatory mechanisms of gene
transcription
DNA binding domain – DBD
Binds specific sequence of
base pairs
Transcriptional activation
domain – TAD
Interacts with basal TF
directly with RNA pol II
Protein-protein interaction
domain – PPID
Interaction with other
transcription factors

26. Review of literature suggests that TFs plays different regulatory role in
plants (stress tolerance, defense, metabolite biosynthesis etc.)
T.F spp. Function Reference
AtMYB096 Arabidopsis thaliana Drought tolerance (ABA and
JA–mediated)
Seo et al. 2009
OsWRKY11 Oryza sativa heat and drought tolerance Wu et al. 2009
ScMYBAS1 Saccharum officinarum Drought and salt tolerance Prabu and
Theertha
2011, Prabu and
Prasad 2012
AtMYB011/AtMYB0
12/ AtMYB111
Arabidopsis thaliana Phenylpropanoid pathway/
Flavonol biosynthesis
Stracke et al. 2007
AtMYB44 Arabidopsis thaliana Plant defense response
against aphid
Liu et al. 2010
AtMYB15 Arabidopsis thaliana cold stress tolerance Agarwal et al.
2006
GmMYB Glycine max salt, drought and/or cold
stress
Liao et al. 2008

27. Source: http://voices.nationalgeographic.com/2009/03/23/corn_domesticated_8700_years_ago/
Teosinte branched1
(Tb1) transcription
factor in
domestication of
maize
When Tb1 is expressed, it represses the outgrowth of lateral branches; maize plants carrying loss of function
alleles produce numerous lateral branches (tillers). During the domestication of teosinte to produce maize,
an allele was selected that altered the regulation of Tb1, increasing its expression in primary auxiliary
meristems (Doebley et al., 1997)
Why these TFs are so important....???

28. • Two TFs have been identified as playing a major role in reducing grain
shattering in domesticated rice plants.
• One of these was isolated as a quantitative trait locus (QTL) in a cross
between a shattering-type ‘Indica’ cultivar and a non-shattering type
‘Japonica’ (Konishi et al., 2006).
• This gene, qSH1, encodes a BEL1-type homeodomain protein that is
orthologous to Arabidopsis REPLUMLESS (RPL), which is involved in the
formation of an abscission zone in the Arabidopsis silique.
• The other TF affecting this trait is shattering4 (sh4), allelic to sha1 (Li et al.,
2006; Lin et al., 2007). SH4 is a member of the trihelix family of plant-specific
TFs and was isolated as a major QTL for shattering in a cross between O.
sativa and Oryza rufipogon.
Role of TF in Rice shattering

29. Fig. Phenotype of strawberry plants transformed with 35S:FvMYB10,FvMYB10 RNAi, and wild-type controls. Growth
and pigmentation of strawberry plants transformed with 35S:FvMYB10, FvMYB10 RNAi, and wild-type controls (A). Detailed phenotype of
strawberry leaves,flowers and fruit of 35S:FvMYB10,FvMYB10 RNAi, and wild-typecontrols.Linesof35S:FvMYB10 had pigmented leaves,
petioles, stigmas and petals, and mature fruit had darkred/purple skin and red flesh. The mature fruit of FvMYB10 RNAi lines had white
skin and white flesh, and the only pigmented tissue was the petioles (B).
(Kui Lin-Wang et al.2014 )
MYB
FOR ENHANCED
PIGMENTATION
Application
of TFs

30. Figure 4. Stress-tolerance assays of AaDREB1 overexpressing transgenic Arabidopsis. Ten-day-old seedlings
of AaDREB1 transgenic lines (T17, T122, T196) and the empty vector control plants (WT) were treated with either 100
mM NaCl for 12 days, 10% PEG for 10 days, or exposed to 4 °C for 20 h, then grown under normal growth conditions
for 3 days. Scale bar represents 1.5 cm
(Zong et al.2016)
DREB for salt,
drought and
cold

31. How these TFs works in plants….
(Front. Plant Sci., 09 February 2016 | http://dx.doi.org/10.3389/fpls.2016.00067)

32.  RNA polymerase II transcribes a precursor-mRNA
 We can divide eukaryotes promoter into two regions:
1. The core promoters elements. The best characterized are
 A short sequence called Inr (Initiator)
 TATA Box = TATAAAA, located at about position -30
*AT-rich DNA is easier to denature than GC-rich DNA
2. Promoter proximal elements (located upstream, ~-50 to -200 bp)
“Cat Box” = CAAT and “GC Box” GGGCGG
 Different combinations occur near different genes.
 Transcription regulatory proteins (activators and also repressors) are required.
Eukaryotes - Transcription of protein-coding genes by
RNA polymerase II

33. Assembly of eukaryotic transcriptional initiation complex
Order of binding is: IID + IIA + IIB + RNA poly. II + IIF +IIE +IIH

34. Transcription regulatory proteins = Activators
 High-level transcription is induced by binding of activator factors
to DNA sequences called enhancers.
 Enhancers are usually located upstream of the gene they control,
they modulate transcription from a distance.
 Can be several kb from the gene
 Silencer DNA elements and repressor TFs also exist

36. Enhancers
Enhancers are stretches of bases within DNA, about 50 to 150
base pairs in length; the activities of many promoters are
greatly increased by enhancers which can exert their
stimulatory actions over distances of several thousands base
pairs. It serves to increase the efficiency of transcription, so
increase the rate. It allow RNA polymerase to bind DNA till
reach the promotor.

37. How enhancers can control transcription although
they are located away from the transcription site???
Enhancers bind to transcription
factors by at Least 20 different
proteins
Form a complex
change the configuration of the
chromatin
Folding, bending or looping of DNA.

38. What happens during transcription initiation???

39. Transcriptional activators are required to turn on the expression of genes in a
eukaryotic cell (Ma et al., 2011)
A typical activator has two essential functions: DNA binding and transcriptional
activation (Ptashne, 1988)
Transcriptional activators that do not bind DNA but interact with other DNA-
binding proteins are sometimes also referred to as co-activators
Activators (gene specific transcription factors) can provide EXTRABOOST in
transcription. Activators can bind to enhancers and also permits cells to control
expression of their genes
Transcriptional Activators and Activation Mechanisms

40. During the activation process, a DNA loop may be formed as a
result of the interaction between the activator bound at the
enhancer and the transcription machinery at the core promoter
(Ptashne, 1986)
Many proteins (or complexes) have been identified that play
important roles in facilitating transcription elongation, and some
of these factors represent targets for activators (Sims et al, 2004a)
An enhanceosome model has been proposed that further
emphasizes the role of multiple activators for activation (Merika
and Thanos, 2001; Thanos and Maniatis, 1995)
To be contd….

41. Initiation
 Binding of TFIID to the TATA box or Inr is the first step of
initiation.
 The factor for RNA Polymerase II is TFIID which consist
of TBP & ~14 TAFs with a total mass ~800 Kd.
 TFIIF brings RNA Polymerase II & binds to the complex,
it initiates transcription.
 The helicase activity of the XPB subunit of TFIIH is
responsible for melting .
 This is followed by initiation of RNA synthesis at this
starting point.

42. Elongation
 Initiation is followed by promoter clearance & elongation
 RNA polymerase directs the sequential binding of riboncleotides
to the growing RNA chain in the 5' - 3' direction.
 Each ribonucleotide is inserted into the growing RNA strand
following the rules of base pairing. This process is repeated utill
the desired RNA length is synthesized…………
 Phosphorylation of the CTD is required for promoter clearance
& elongation to begin.

43. Termination
 Terminators at the end of genes; signal termination.
These work in conjunction with RNA polymerase to
loosen the association between RNA product and DNA
template. The result is that the RNA dissociate from
RNA polymerase and DNA and so stop transcription.
 The product is immature RNA or pre mRNA (Primary
transcript).

45. RNA Processing (Pre-mRNA → mRNA)
 Capping
 Addition of poly A
 Splicing
 RNA editing

46. The cap structure is added to the 5' of the
newly transcribed mRNA precursor in the
nucleus prior to processing and subsequent
transport of the mRNA molecule to the
cytoplasm.
Capping

47. Addition of cap
 Protection of mRNA from degradation
 Transport of the mRNA from nucleus to
cytoplasm
 Binding of ribosome with mRNA

49. AAUAAA5’ CA GU rich 3’
10-30 nt <30 nt
Pre-mRNA
Binding of CPSF (Cleavage and
polyadenylation specificity factor)
AAUAAA5’ CA GU rich 3’
10-30 nt <30 nt
Binding of CstF (cleavage stimulating
factor) and CF (cleavage factor)
AAUAAA5’ CA GU rich 3’
10-30 nt <30 nt
Binding of poly A polymerase (PAP)
AAUAAA5’ CAAAAAAAAAAAAA(250) 3’

51. phillip Allen Sharp is an American
geneticist and molecular biologist
who co-discovered RNA splicing. He
shared the 1993 Nobel Prize with
Richard J. Roberts for "the discovery
that genes in eukaryotes are not
contiguous strings but contain
introns, and that the splicing of
messenger RNA to delete those
introns can occur in different ways,
yielding different proteins from the
same DNA sequence".
phillip Allen Sharp Richard J. Roberts

52. Intron mediated splicing
GU-AG rule
Cut at 5’ site and form lariat
by 5’-2’ bond connecting the
intron 5’ G to the 2’ of A at the
branch site
Cut at 3’ site and join exons;
intron released as lariat
Splicing require the 5’ & 3’
splice sites and a branch site
just upstream of the 3’ splice
site

53. THE SPLICEOSOME MACHINERY
RNA splicing is carried out by a large complex called
the spliceosome
snRNP (small nuclear ribonuclear proteins)

54. The 3’OH of guanosine
acts as a nucleophile,
attacking the
Phosphate at the 5’
splice site.
The 3’ OH of the 5’
exon becomes the
nucleophile,
compeliting the
reaction.

55. Alternative Splicing
Single gene can produce multiple products by alternative
splicing

56. Definition: any process, other than splicing, that results
in a change in the sequence of a RNA transcript such
that it differs from the sequence of the DNA template
• Discovered in trypanosome mitochondria
• Also common in plant mitochondria
• Also occurs in a few chloroplast genes of higher
plants, and at least a few nuclear genes in mammals
K. Stuart L. Simpson
RNA editing

57. RNA EDITING
RNA editing is another way of altering the sequence of
an mRNA

58. Scenario of Central Dogma In
Eukaryotic Cell

59. Translation
Translation is the process by which ribosomes read the
genetic message in the mRNA and produce a protein
product according to the message's instruction

60. Ribosomes
tRNA
mRNA
 Amino acids
Initiation factors
Elongation factors
Termination factors
Aminoacyl tRNA synthetase enzymes
Energy source
60
Requirement for Translation

62. Location
 Dispersed freely in the cytosol
 Attached to the surface of ER
Ribosomes can found either:

63. Structure
A ribosome has two main constituent elements
 Protein = 25-40%
 RNA = 37-62%
Two main subunits are present i.e.,
 A larger subunit
 A smaller subunit

64. • All ribosomes have 1 mRNA binding site and 3 binding sites for tRNAs
- Remember tRNAs are going to bring the appropriate amino acids to the ribosome
- A (aminoacyl) site = Holds a tRNA that just
arrives to the ribosome
- P (peptidyl) site = Holds a tRNA that
contains the growing polypeptide chain
- E (exit) site = Holds a tRNA that has already
given up its amino acid and is getting ready
to exit the ribosome
Translation
Ribosomes

65. • The Shine–Dalgarno consensus sequence in bacterial
cells is recognized by the small unit of ribosome.
• The Kozak sequence in eukaryotic cells facilitates the
identification of the start codon.
The Initiation of Translation

66. INITIATION
1. Formation of 43S Preinitiation complex
2. Attachment of mRNA
3. Scanning for the start codon
4. Assembly of large complex
INITIATION

67. 1. Formation of 43S Preinitiation complex
Some IF bind to the 40S subunit to form
43S Preinitiation complex

68. 2. Attachment of mRNA
When the 43S complex bind to mRNA , it
scan for the initiation codon called 48S
COMPLEX

69. 4. Assembly of large complex
3. Scanning for the start codon
eIF1 & eIF1A
Enable scanning
eIF5B mediates
60S-40S joining

70. REVIEWS
eIF2
2 eIF2ternarycomplex
formation
GTP
Met-tRNAi
eIF3
E
tRNA mRNA 60S
P A
1 Ribosomerecycling
3 43Scomplex
formation
eIF2
eRF1and eRF3
ABCE1
eIF3
eIF1A
eIF1
eIF4Fcomplex
eIF4G
eIF4E
eIF4A
4 mRNAactivation
eIF5
eIF3 E
GTP
P A
40S
43Spreinitiation
complex
Post-TC
5′
m G
AUG
eIF4B
UGA
PABP
A
A
A
A
3′
ATPADP,Pi
A
A
A
AUG
A AA
5 Attachmentto mRNA
GTP
60S
E P
40S
UGA
A
eRF1
eRF3
A
A
A
A
A AUGA E P A
A 40S
A
AA AA AA
6 5′to 3′scanning
GTP
A AUGA E P A
A 40S
A
AA AA AA
7 Initiation codonrecognition,
hydrolysis of eIF2-bound GTP
andPi release
Termination
PABP
GTP
AUG
A E P A
A 40S
A
AA AA AA
48Sinitiation
complex
Elongation
eIF2 GDP
(partial loss)
GDP
AUG
A E P A
A 40S
A
AA AA AA
60S +eIF5B GTP
8 Subunitjoiningand
factor displacement
eIF2 GDP
eIF5
eIF1
80Sinitiation
complex
eIF3
60S
AUG
E P
40S
GTP
A
9 Hydrolysis of eIF5B-boundGTP
andreleaseof eIF5BandeIF1A
A
A eIF5B
A GDP
A
60S
AUG
E P
40S
A A
A
A
A
116 | FEBRUARy 2010 | vOlUME 10
Nature Reviews | Molecular CellBiology
www.nature.com/reviews/molcellbio
© 2010 Macmillan Publishers Limited. All rights reserved
GTP
Met
40S
7 A
A
A
A A
A
A
A
A
A
A
A
A
A
A
A
A
A
eIF1A
A
A
Over all process of initiation

71. ELONGATION
Peptide bond formation
Translocation

72. Peptide bond formation
Transfer of proper aminoacyl-tRNA from cytoplasm to A-site of ribosome.
eEF1A is responsible for bringing charged t RNA to the A-site.
Peptide bond formation takes place by reaction between the Peptidyl-tRNA in
the P-site and the aminoacyl-tRNA in the A-site

73. Elongation
- How are a.a. chemically-linked during
elongation?
- The amino group of one amino acid
reacts with the carboxyl group of an
adjacent amino acid
- Water is removed (dehydration) and
a peptide bond is formed
- All peptides/polypeptides/proteins have
2 ends
- N-terminus (a.a. 1) – Free NH2 group
- C-terminus (last a.a.) – Free COOH group

74. COTRANSLATIONAL TRANSLOCATION
The Cell : A molecular Approach, 5th edition

75. TERMINATION
Cleavage of the growing chain
Recycling

76. • Translation continues until it encounter termination codons; UAA, UAG
and UGA
• Release factors: Helps in releasing of free polypeptide and ribosomes
Termination

77. Termination – The process stops
- As the ribosome reads the mRNA, it eventually will arrive at one of the 3
stop codons (UAA, UAG, UGA)
- No tRNA has a corresponding anticodon  No amino acid can be
added to the chain
- This acts as a signal to the ribosome that it needs to end translation
- Protein release factors bind to the ribosome and release both
the mRNA and polypeptide (and cut the polypeptide from the last tRNA)

80. The large and small subunits undergo association and
dissociation during each cycle of translation

81. Translational Level control
RNA interference/silencing:
Shooting down mRNA
OR
Killing the Messenger

82. Definition
– RNA interference (RNAi) is an evolutionally highly conserved
process of post-transcriptional gene silencing (PTGS) by which
double stranded RNA (dsRNA) causes sequence-specific
degradation of mRNA sequences.
– It was first discovered in 1998 by Andrew Fire and Craig Mello in
the nematode worm Caenorhabditis elegans.

83. Mechanism of RNAi
dsRNA
siRNA
miRNA
shRNA

84. Dicer
• Loss of dicer→loss of silencing processing in
vitro
• Dicer homologs exist in many organisms
including C.elegans, Drosphila, yeast and
humans (Dicer is a conserved protein)
RNase III-like dsRNA-specific ribonuclease
Enzyme involved in the initiation of RNAi.
It is able to digest dsRNA into uniformly sized
small RNAs (siRNA)
Dicer family proteins are ATP-dependent
nucleases.
Rnase III enzyme acts as a dimer

85. RNAi used as a tool for treatment in cancer, HIV and several diseases
Development of virus/disease resistant plants by targeting key proteins

86. Conclusion
 This is well proven that TFs acts as master switches for major regulatory
networks.
 They regulate the co-ordinated expression of several genes in a multi-genic
pathways, thereby a single TF may be sufficient for engineering the target
traits.
 Taken together, various roles played by TFs in plants, these could be potential
source of candidate genes for modifying complex traits in crops.
 TF-based strategies appear to be very promising and could play a major role in
development of genetically engineered crops.
 This gene silencing technology is a sophisticated technology having
revolutionary capabilities could be further exploited for functional analysis of
target genes and regulation of gene expression for crop protection and
improvement.

87. Doebley J, Stec A, Hubbard L (1997) The evolution of apical dominance in maize. Nature 386: 485–
488
Jun-Mei Zong, Xiao-Wei Li, Yuan-Hang Zhou, Fa-Wei Wang, Nan Wang, Yuan-Yuan Dong, Yan-Xi
Yuan, Huan Chen, Xiu- Ming Liu, Na Yao and Hai-Yan Li (2016) The AaDREB1
Transcription Factor from the Cold-Tolerant Plant Adonis amurensis Enhances Abiotic Stress
Tolerance in Transgenic Plant. Int. J. Mol. Sci. 17(4), 611; doi:10.3390/ijms17040611
Kui Lin-Wang, TonyK.McGhie, MindyWang, YuhuiLiu, BenjaminWarren, RoyStorey,
RichardV.Espley and AndrewC.Allan (2014) Engineering the anthocyanin regulatory
complex of strawberry (Fragaria vesca) Plant science doi: 10.3389/fpls.2014.00651
KuiLin-Wang1, TonyK.McGhie2, MindyWang1, YuhuiLiu3, BenjaminWarren1, RoyStorey4,
RichardV.Espley1 and AndrewC.Allan1,5 (2014) Engineering the anthocyanin regulatory
complex of strawberry (Fragaria vesca) Plant science doi: 10.3389/fpls.2014.00651
Ma J, and Ptashne, M (1988) Converting a eukaryotic transcriptional inhibitor into an activator. Cell
55, 443-446
Merika M, and Thanos, D (2001) Enhanceosomes. Curr Opin Genet Dev 11, 205-208. Ptashne, M.
(1988). How eukaryotic transcriptional activators work. Nature 335, 683-689
Ptashne M (1986) Gene regulation by proteins acting nearby and at a distance. Nature 322, 697-701
Sims RJ, 3rd, Belotserkovskaya, R, and Reinberg, D (2004a) Elongation by RNA polymerase II: the
short and long of it. Genes Dev 18, 2437-2468
Thanos D, and Maniatis T (1995) Virus induction of human INFb gene expression requires the
assembly of an enhanceosome. Cell 53, 1091-1100
References and Further Reading

88. The Cell : A molecular Approach, 5th edition Geoffrey M. Copper and Robert E Hausman, 2009 ASM
Press Sinauer Associates inc.
Genetics: A Concepteul Approach, 4th edition, 2012 W.H Freeman and Company
Nature Reviews, Molecular Cell Biology ; 2010 Macmillan Publishers Limited. All rights reserved
116| February 2010. Volume 10
Molecular Biology of The Cell, Garland Science 6th edition
Lewin’s GENES x, Jones and Bartlett Publishers
Image source- https://images.google.com
Hampsey, M. (1998). Molecular Genetics of the RNA polymerase II general transcription
machinery. Microbiol & Mol Biol Rev 52, 465-503.
Orphanides, G., Lagrange, T., and Reinberg, D. (1996). The general transcription factors of RNA
polymerase II. Genes & Dev 10, 2657-2683.
(Front. Plant Sci., 09 February 2016 | http://dx.doi.org/10.3389/fpls.2016.00067)
Source:http://voices.nationalgeographic.com/2009/03/23/corn_domesticated_8700_years_ago

89. Thank
you