Control of Gene expression

Why the pancreatic cells produces Insulin but not Melanin and the
skin cell produces melanin but not insulin?
Human pancreas Human Skin

Insulin Melanin
Overview of Eukaryotic gene expression
Overview of Prokaryotic gene expression
Different cell types in a multicellular organism differ dramatically in
both structure and function. If we compare a nerve cell with a liver
cell they are completely different in terms of functions.

Now the question is why they are different ?

Do they have different genetic makeup?

All the cells have identical genetic makeup; the only difference lies
in their pattern of gene expression
When the nucleus of a fully differentiated frog cell is injected into
frog egg, whose nucleus has been removed is capable of producing
a normal tadpole. The tadpole contains full range of differentiated
cells, that derived from the nucleus of donor cell. Thus it can be
concluded that the donor nuclei contained all the important DNA
sequence in it.
 Different cell types synthesize different sets of RNAs and protein
• Many process of cell types are common like Histones for DNA packaging, DNA &
RNA polymerase, ribosomal proteins, proteins for cytoskeleton(Actin/myosin).
Hence these genes are transcribed and translated all types of cells.

• Some RNA and proteins are abundant in the specialized cells in which they
function. e.g Hemoglobin protein is only expressed in RBC where it carries oxygen.
The enzyme tyrosin amino transferase which breaks down tyrosin in foods is
expressed in liver but not in other tissues.
 How external signals are responsible to change the expression of the gene

During starvation or intense physical exercise, glucocorticoid hormone signals the

liver to increase the production of energy from amino acids. Hence, the enzyme
tyrosin amino transferase is produced. When the level of glucocorticoid hormone
drops down the production of tyrosin amino transferase also drops.
A cell can control the expression of a protein in the following ways:
• Controlling when and how often a given
gene is transcribed (Transcriptional control)
• Controlling the splicing and processing of
RNA transcripts (RNA processing control)
• Selecting which completed mRNA are
exported from the nucleus to the cytoplasm
and deteremining where in the cytosol they
are localized. (RNA transport and localization
control)
• Selecting which mRNA in the cytosol are
translated by ribosomes (Translational
control)
• Selective degradation of mRNA molecules
in cytosol (mRNA degradation control)
• selectively activating ,inactivating ,
degrading specific protein molecules after
translation. (protein activity control)
How sequence specific DNA binding proteins/ transcription factors
control transcription process

Gene A Gene B Gene C

5’UTR

3’UTR
TSS

ATG 3’UTR

PROMOTER of Gene B
 PROMOTER of Gene B TSS

ATG 3’UTR

Cis elements

TSS

ATG 3’UTR
RNA
pol

mRNA

To initiate the transcription process there is a need of transcription activator . Transcription

activators are proteins that binds to specific DNA sequences known as cis-regulatory
sequences. Binding of TF recruits RNA polymerase to bind the TATA box and leads to
transcription initiation.
What is a transcription factor and How it helps in gene
expression?

ATCAATG TATA
box

ATG
Promoter TSS
RNA
pol

mRNA
 How sequence specific DNA binding proteins/ transcription factors
control transcription process

TF
• Each TF makes a series of contacts with
the DNA, involving hydrogen bonds, ionic
bonds, hydrophobic interactions.

• more than 20 such contacts are

generally established in DNA –protein
interface, ensuring the specificity and
rigidity of the interactions.

• X-ray crystallographic and NMR studies

have revealed that TF contains DNA
binding domains which binds with the
major groove of DNA. The specific side
chain protruding from the DBD motif
recognize specific DNA regions.
Dimerization of TFs increase the affinity and specificity of the DNA-
protein interaction

The DNA sequence recognized by a monomer TF does not contain sufficient

information to be picked out from the background of sequences. e.g. the exact six
nucleotide DNA sequence would be expected to occur once in 4096 nucleotide (4 6).
Many TF dimers to increase the effective contact area thereby increasing the affinity
and specificity of TF binding many fold.
How to do the promoter analysis ? What is its real need?
 Candidate gene
>SL2.50ch01 SL2.50ch01:86821114..86824468 (+ strand) class=gene length=3355

ATTAACAGTTTGTTGCCAAAAATATTTCTTTTTTTTAAAATAAAAAATCCTTTATTTTTTCCATCATCTTCTTCCTT
ATCTAAGTTTTTTATTTATAATTATAATATTGCAGTGACCTTAATGTGTACAAGTATATTCGTTACATTATTTGG
GTCTCTGTAAAAACAAGTGCAGAAACACTAAAAACTATGAAGGCCTTTAATGGAACCAATGTCATCAAGAAAC
AATTTTCTTTAATTTTTGTAAAATTTACAGTTTTTGTTCTGTTGTTGGGTCTTGCCTATCGACTCATCTTGTCCAAT
TTTGAACAGTTCTCTCAGATTGAAGTGAAAAATACTGCTCTGTTTTCCGACAACACATTGCCACAGGCGCCGGT
GACCGTGAGTGAACCTCCGGTGACTGTCAATGAACCTCTGGTGACCGTTGATGAACCTACGGTGACTGTGAGT
GAATCTCTGGTGACTATTGATCTTCCAGAAAACCAAACTTTGCGAAATGGTTAGTTTATATTTGCATGAATGCT
CAGTTCTTGTAAAAATTGAATCTTGAAAGATTAATATTTTCTGTTTTTTTTTAGCTTATTGTAATTTGTTTGCGTT
GGATTTTGTGTAATCAATGACGTGGGTAATTTTCCTTCACTTCTCGTCTCTGTGTTTTGTGATTAATTTTGTATAT
TTGGTATTTGAGGATCAAGTCTTTCATCCATATTTGTAGTGAAAATCTCTTTTCTACGGAATCAAACTCTCACCA
ATCAACTGAGCTATTAAGATTTTCTAGTGGAATATATAAAATATGGATGGTGGATTAATTTTTTATGATATTATC
TTTGGCTGGTAGTCATGAATTTTTTTAGTAATAGAAAAAGGAACAACTTTGAGTACAAATAATAAGATTTTGCA
CTTTGAGCTTAGGTCACTAATGTATGAGTCCTTACAATTAACTGACTCTTTTTTTTTTCTTTTTCAGGAACATGTG
ATTTATTTATAGGAAATTGGGTTTATGATCCAAGTGGTCCAGTATACACGAACGCGACTTGCTATTCAATTGAA
TCTCATCAGAACTGTATGAAAAATGGGAGACCTGATACTGAATATCTTAACTGGAGATGGAACCCAACAGACT
GTGAGCTACCGAGATTTAGCCGTAAGAAATTCCTCAATTTGATGCGAAACAAATCATTCACCTTCATAGGCGAT
TCAATCATGCGTAATCATGTGCAGTCATTGCTTTGTATTCTCTCACAGGTACATTTCAAGCTCAAAAAGAGTTAT
ACTTGAACCAGGCAATTAGCTGCTCTACATTTCATTTGTTTTGTTTTGTTTCAGTTTGTTGTGTTAGTGAAATTG
GTAATTGCAAGCGTCATGATTTAGTTGTTTAGTCGTAACAATAATTGCTCTTAAATGGTAATGTTGATATCAAA
TCAAGTAAATACAAGCTTGTTGTGGCTGTTTGTGTATGGCATTACAATTTGTGGGGGCACTCTTGTGTTGTATA
AGCTGGTGCCTTATCTAGTTGTATCTCACTAACCATAATTAGGATCTTTTGTGTATGAGGTGGCATAGCTACAT
ATTGGATTTATCGGTAATGTCACTCTCAGTTACATGGATGTTGGTCCCGCGAGCCAGCTTGTAAAGGCTCTTTG
TAGTTATAACATTTGTTTCGAAAGTTGTTAAGAAGTTGATTTAAAGTTAAGTGTCAAACCTAACTAAGTGTACA
TTTAAGACTATTACTAAAGTTTTGCTGTTGTTCCATCTCCTTTGACCAAGAAAAATTAGCTGGCCTCATTATTAT
CTCCTTGAGGTTTCAGCAAAAATTGATGTGTAATGATATGACACTGGGGTCCAACTCAACCCCGGATATCGTA
AGTCCATATAAGCAGACGATCAGTCTAGTCTAACACCCGTCTTCACACCCAGGCCCATAATTCTTTGTGTTGTTT
AATTTTGCAAGAGATGACAGGCAGTTTTTCTAAGGACAAAACCTTAATGGTAATTGTCATAGAGTAATAAGGA
TCATTATGGTAGAAATCTTACTTACCTGTTGTGTCTTATATAGAACCCTCTTATACAAATGTCGTTTTAGATGGT
TGGCTGATCGTGCTGATTCTTTCTTTAGTACAGTACTTCTTTTACTTGGAATTGACTGACTGAATATGGAATTTT
CTTTACTATATAAAATCGAGGCTGCTAATTCGAGAATCTATATCCTAAATTTAGAATTCGATATTGTTGAATAAC
CCTTTGCTCTTTTGTACACAAGCTTCATTCGGGACATCATTGGCACGTTAAATTCATGTTGCTTCATTTCCTTGG
GAATTTTACTATACTTTTTGAGATTTTTTATTTGTAGTTGATTACGTTTCATCGTGTTGCAATGTGTTGGGTTGTC
CTATGCTGCCGAAACTTATAAGGGAAAAGATAGTTTTTGTTTCTAATCACGTTTACATTGTGAAACAGGAGGA
AGAAGCTGATGATGTGTACCATGACGAGCAATACAAAAGCAGAAGATGGTACTTTCCGCTCCACGACTTCAAT
CTTTCTGTGATTTGGTCACCTTTTCTCGCAAAATCAACTGTTTTTGAAGATGACAATGGAGCTTCAACGGACATT
ACTCAGCTTCATCTTGACAAACTGGATGATGTTTGGACTCAACAATTCGACAATTTTGATTATGTATTTGTAGCT
GGTGGCAAATGGTACCTGAAATCAACAATTTACCTTGAGAATGATACGATTGTTGGGTGTCACAACTGTCCTG
GTAAGAACATTACACAGGTGGGATTTGAATACGCCTATCGCAAAGCATTGAATACAACTTTTAAATTCATCATG
AACTCGAAGAACAAGGCGTATACATTTTTCAGAACCACTACCCCTGATCACTTTGAGAATGGTGAATGGAATA
GTGGAGGTTATTGTAACAGAACAGGACCTTTCAAAGAAGGTGAGATTGACATTGGTTATGTAGACGAGGTAA
TGCGCAAAGTTGAGCTGGAAGAATTCAAAAGAGCATCTGGATTGGGTTTGAAGGTGAAATTGTTTGACACAA
CCTTACTTTCACTGCTGAGACCAGACGGGCATCCCGGAGTCTACAGGCAATATCAGCCATTTGCTGTAGAAAA
TAAGAAGAAAAAAATTCAGAATGACTGTCTACATTGGTGTTTGCCTGGTCCAATAGACTCGTGGAACGATATA
 Identification of promoter region and Promoter analysis

Gene A Gene B Gene C

5’UTR

3’UTR
Sequence retrieval from browser
https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Csativus

Bioinformatic analysis
http://plantpan2.itps.ncku.edu.tw/

Identification of Cis-elements in promoter

Identification of TF binding sites in the promoter of a candidate
gene in PlantPAN2.0
Schematic representation of Distribution of different cis
elements in the promoter of a gene

TSS

ATG
 Identification of promoter region and Promoter analysis

Gene A Gene B Gene C

5’UTR

3’UTR
Sequence retrieval from browser https://phytozome.jgi.doe.gov/pz/port
al.html#!info?alias=Org_Csativus

Bioinformatic analysis http://plantpan2.itps.ncku.edu.tw/

Identification of Cis-elements in promoter

 Sequence retrieval from browser and promoter
analysis
Open phytozome Search any gene of an organism

Identify the promoter region

Identify if the gene is in plus strand

or minus strand

Download the promoter

sequence

Analyze the sequence in Plant

PAN database
Eukaryotic gene control region consists of promoter and many cis-regulatory sequence
Nucleosome structure promotes cooperative binding of transcription regulators
Eukaryotic transcription activators direct the modification of local chromatin structure
Transcription activators can promote transcription by releasing RNA polymerase
from promoters
How Transcription regulators switch genes on and off in
prokaryotes
 Expression of many bacterial operons is controlled by
regulation of Transcriptional elongation

This mechanism of control was discovered first in Trp operon transcription in E. Coli.

The transcription of Trp operon is repressed by trp repressor when the conc. Of
Tryptophan is high in the media. But in that scenario still very low level of expression still
occurs , which is further controlled by the process attenuation.

The first 140nt of the trp operon does not encode proteins required for tryptophan
biosynthesis. But this 140nt region consist of short peptide “Leader sequence”

5’ 1 2 3 4 UUUU 3’

Transcription
start codon

• Region one of the leader sequence consist of two successive trp codons.
• Region 3 can base pair with either 2 or 4 .
• The ribosome follows closely behind the RNA polymerase. When the 5’ of the mRNA
emerges ribosome starts translating the mRNA.
Condition 1: When there is sufficient Tryptophan amino acids in the
media.

• When there is sufficient trp, hence there is sufficient tRNA trp to

support high rate of protein synthesis.
• So the ribosome quickly translates through the region 1 to region
2 as there are available tRNA trp for decoding the two successive
Trp codons in the region 1.

• Hence there is no chance of base pairing with the region2 with

region 3 as region 2 is already occupied by ribosome.

Now , region3 can base pair with region 4 as soon as it emerges

RNA polymerase forming a stem loop followed by several uracils.
This structure signals the RNA polymerase to pause the
transcription and leads to termination.
Condition 1: When there is sufficient Tryptophan amino acids in the
media.

RNA
pol
UU 3’
1 2

4
3
tRNA trp
Condition 2: When there is insufficient Tryptophan amino
acids in the media.

When there is not sufficient Tryptophan amino acids, there is

insufficient tRNAtrp to support the protein synthesis. Hence, the
ribosome stalls at the two successive “trp codon” due to the non
availibility of tRNAtrp.

• As a consequence region 2 base pairs with region 3 as soon as it

emerges from RNA pol.
• This event prevents region 3 to base pair with region 4, as a
result 3-4 base pairing is not formed and does not cause
transcription pausing or transcription termination.
• RNA pol continues transcription.
 Region 2 and Region 3 forms
a hair pin loop

3
RNA
pol 1

Ribosome stalls in region 1

Due to non availibility of tRNAtrp

No tRNA trp RNA pol continues transcription

 How bacterial responses are controlled by two component system
At high concentration of Glutamine

No phosphorylation
Gln
Gln of Aspertic acid in
Gln
regulatory domain
Ntr-c protein
Ntr-B protein
Sensory domain
Regulatory
D
domain
DNA binding
domain
His-kinase transmitter domain
Conformational change occurs
due to binding of Glutamine
(becomes inactive)
Can’t bind to the enhancer region of
the gln A gene

Enhancer region No transcription occurs

 At low concentration of Glutamine
phosphorylation of
Gln Aspertic acid in
regulatory domain
occurs Ntr-c protein
Ntr-B protein
Sensory domain P
Regulatory
D
domain
DNA binding
domain
His-kinase transmitter
domain becomes active D
Regulatory
can bind to the enhancer domain DNA binding
domain
region of the gln A gene
Active Nt
r-C protei
n

Enhancer region transcription occurs

 What are Riboswitches and how it regulates transcription

Attenuation of transcription elongation also occurs in some operons and single

genes encoding enzymes, involved in the biosynthesis of other amino acids /
metabolites through the function of Riboswitches.

Definition: Riboswitches are sequences of RNA most commonly

found in the 5’UTR region of bacterial mRNA which fold into
complex tertiary structure called aptamers.
 How Riboswitches regulates Transcription?
When metabolite and amino acid concentration Is higher
Aptamers binds to small molecules like amino acids/ metabolites which are present in
sufficient amount leading to formation of stem loop structure . This stem loop structure
leads to premature termination of transcription.

High metabolite
conc.
RNA RNA
pol. pol.

RNA No transcription
pol.
 How Riboswitches regulates Transcription?
When metabolite and amino acid concentration Is lower
When the concentration of metabolite is lower it can’t bind to aptamers allowing the
transcription of the gene.

Lower conc. Of metabolite

RNA RNA
pol. pol.

Example: xpt-pbuX operon; which encodes enzymes for purine synthesis.

Transcription factors
Types Discover Structure Binding with DNA Structure
y

Helix- Discove Consist of Two helices connected by short They binds with DNA as dimer in the
turn- red in chain of amino acids. The Cterminal helix major groove.
Helix bacteria is known as recognition helix which binds
l TFs with major groove of DNA
protein
2

1
1
2 2

1
Homeo First Consist of three alpha helices which They bind as monomer. The
domain discover are packed tightly by hydrophobic Asparagine of helix 3 contacts with
proteins ed in interactions. The third helix is called adenine residue of DNA in major
drosoph
ila recognition helix which binds with groove.
major groove of DNA.

2
2 3 1
A sn

3
A sn
1
Types Discovery Structure Binding with DNA Structure

Leucine Leucine Zipper They bind DNA as

Zipper motif is named Dimer. Where two
proteins because the long alpha helices are
two alpha held together by
helices from leucine.
each monomer
are joined
Leucin
together to
form a short
coiled coil
Types Discovery Structure Binding with DNA Structure

Zinc It has a simple They are found in

Finger structure in which a clusters. Alpha helix of
protein zinc atom holds an each fingure contact
alpha helix and a the major groove of
beta sheet DNA.
together. Zn

Zn

Helix – Consist of a short Both homodimer and

loop- alpha helix hetero dimer is
Helix followed by a possible. Binds to
loop and a long major groove of DNA.
alpha helix.
How we can study the interaction
between transcription factor with
the DNA?
What is a transcription factor and How it helps in gene
expression?

ATCAATG TATA
box

ATG
Promoter TSS
RNA
pol

mRNA
 Types of DNA-protein interaction

DNA-protein
interaction

In vivo In vitro
1. Chromatin Immuno 1. Electrophoretic mobility shift
Assay (EMSA)
precipitation Assay (ChIP) 2. DNase I Foot printing assay
2. Trans activation assay
3. Yeast one hybrid assay
 Basic Principle of Gel Mobility Shift Assay / EMSA

Incubation of labelled
Electrophoretic
probe with TF in room
analysis Autoradiography
temperature for 30 min
5’
TF 3’
3’ Shifted
5’
5’ 5’ 3’ band
Labelling 3’

TF
3’ 5’
3’
of Probe TF 3’ 5’
5’

5’ TF
3’ 3’
5’ 5’
3’ 5’ 3’ Free
3’ 5’ probe
TF
How to label the duplex DNA ? Why labelling is necessary?

TF

TTGACC TATA
box

ATG
Promoter TSS

5’ 20-30 bp 3’
3’ 5’
Design of DNA duplex
γ P32 ATP
5’ end Labeling
T4 PNK
5’ 3’
3’ 5’

TF

5’ 3’
3’ 5’ 5’ 3’
Hot probe 3’ 5’

Cold probe
 Design of EMSA Experiment

+ + Hot probe
Protein purified
- +

Shifted band
DNA –protein comple

Autoradiography
Design of competition EMSA Experiment and super shift EMSA
It’s a competition between the hot and cold probe to bind to the protein.

- - - Cold probe
+ + + + + Hot probe

+ Protein purified
- + + +
+ Antibody
- - - -
1 2 3 4 5 6
1 2 3 4 5 6

DNA protein
complex

Free
probe

Autoradiography
Original Gel picture of EMSA and competition EMSA

Gradual increase of cold probe

The shifted band intensity

gradually decreased

The free probe band intensity

gradually increased
 How to determine the specific binding sites?
Region of mutation

- - + Mutant probe Mutant probe

+ + - Hot probe

+ Protein purified
- +
1 2 3

TF can’t bind in
the mutant site

Autoradiography
EMSA reveals the binding of OsDREB Tf in the promoter of rd29A (a dehydration
responsive gene)

Shifted band

Free probe
Chromatin immunoprecipitation
ChIP
 (Formaldehyde)

input
 ChIP PCR
sonicating

antibody

F R

input

F R
F R
F R
ChIP PCR
ChIP-on-chip / ChIP-chip
Agroinfiltration Assay
Among the 7 different cis-elements which one is most crucial for
transcriptional regulation?

TSS

ATG
 Construction of 5’ deletion promoter fragments
TSS

5’ GUS

TSS

5’ GUS

TSS

5’ GUS
 TSS
5’ GUS 1
TSS

5’ GUS 2
TSS

5’ GUS 3
Promoter
construct

Agrobacterium

1
3

2 1 3
agroinfiltration Histochemical gus assay
Site directed mutagenesis assist in identification of specific transcription
factors that are involved in transcriptional regulation
TSS

GUS Wt
TSS

GUS Mut 1
TSS

GUS
Mut 2

Wt
Mut1

Wt Mut 1
Mut2

Mut 2
Post transcriptional modifications
The schematic representation of mRNA processing and transport

Eukaryotes
Structure of prokaryotic and Eukaryotic mRNA
Initiation of RNA processing
 Mechanism of 5’ CAPPING
After the mRNA transcript is about 25-30 nucleotides long a 7-methylguanosine is
added to the 5’ end. Protects the 5’ from enzymatic degradation in the nucleus and
assists in export to the cytosol.
5’ 3’ RNA
5’ ppp N p N RNA 3’
phosphatase
pi phosphatase
cleaves the end phosphate
from the nascent RNA
pp N p N RNA
Guanyl transferase adds the GMP
residue along with a phosphate in a
GTP Guanyl transferase
inverted orientation 5’ to 5’ instead of ppi
5’to 3’
Gppp N p N RNA
Methyl transferase adds
the methyl group to the 7th Methyl transferase
position of Guanine
residue m7Gppp N p N RNA
All the three enzymes bind to the RNA pol tail phosphorylated at the ser 5 residue. As
soon as the RNA emerges from the polymerase these three protein starts adding 5’cap.
 Requirement of 5’ CAPPING

 5’ methyl cap signifies the 5’end of eukaryotic mRNA.

 The presence of 5’ methyl cap helps the cell to distinguish mRNA from other type of
RNA molecules.

 RNA pol I and III produces uncapped RNA molecules during transcription.

 5’ methyl cap have role in translation of mRNA in cytosol.

 RNA SPLICING
• Introns are removed and the exons are spliced together.
• Splice sites occur at both the 5’ and 3’ ends of introns.
• The intron is cut at the splice sites.
• Only 30-40 nucleotides at each end of an intron are required for splicing.
Self splicing where the RNA itself acts
as a Ribozyme, leads to the joinining
of two exons . Self splicing usually
occurs in lower organisms.

..
OH A
exon1 exon2

Nucleophilic attack Branch point

in the Intron – Exon
junction and breaks
the phosphodiester
bond
Need of splicing in Eukaryotes
site of splicing within Intron
Formation of Lariat
 Mechanism of Splicing in Eukaryotes

Eukaryotic introns are longer, hence the lone pair electrons can’t attack the intron-exon junction. So for a
successful nucleophilic attack two exons should come closer by folding of introns. The ribonucleoproteins helps in
folding phenomenon.

U1
..
OH A Spliceosome mediated splicing occurs in
exon1
U2
exon2 step1 higher Eukaryotes that involves U1,U2, U4,
U5, and U6 ribonucleo proteins.

U5 Branch point
U6
U4
U1 and U4 would be released
U4
U1 U5 will hold the exon1 and exon2 , now
U1
exon1 lone pair of electron can do nucleophilic
U4 exon1 attack to the intron-exon junction
step2 U5
..
U5 leading to cleavage of phosphodiester
bond
U6 exon2
A
U6 exon2 OH A
U2
U2

Nuclear exonucleases cut the lariet RNA from U1 and U4 would be released
both the 3’ and 5’ ends, thus recycling the ..
nucleotides.
OH exon1
exon1 exon2 U5

U6 exon2
The lone pair of electron of OH group of exon1 can do nucleophilic attack to the
A
U2
intron-exon junction of second exon leading to cleavage of phosphodiester bond
and ultimately leads to joining of exon1 and exon2 step3
Effect of Random mutation on splicing
Transcription termination signal
 3’POLYADENYLATION
• Primary transcript is cleaved and a poly A tails is added to the free 3’ OH.
• Up to 250 A residues may be added.
• Carried out by poly(A) polymerase
• Protects the mRNA from degradation from the 5’ end.

5’ 3’ 5’ 3’
3’ 3’
3’ 5’ 3’ 5’
CPSF cstF
AAUAAA AAUAAA
Poly Adenylation 5’
Signal 5’
A Set of protein (CPSF)recognizes the poly adenylation signal
and forms the RNA cleavage complex With cstF and other
protein. This complex having endonuclease activity cleaves
the RNA at a certain point.
3’ AAAAAAAAA Poly A
PAB PAB poymerase
AAUAAA

CPSF cstF 3’ AAAAAA

Poly A AAUAAA
poymerase
PAB(poly adenylation binding protein ) binds to poly A tail 5’
CPSF cstF
to demarcate wheter enough number of “A” were attached
or not. If it is found that enough number of A is attached it CPSF recruits poly A polymerase that only adds Adenine
signals poly A polymerase to stop further addition of “A” residues at the 3’ end of RNA.
3’POLYADENYLATION
 Transport Across the Nuclear
Membrane
Molecules of all sizes enter and leave the
nucleus via Nuclear Pore Complexes
(NPC).
• Consist of multiple copies of different
proteins called nucleoporins. From 50-100
different proteins (depending on organism)
are involved.
• Complex is extremely large, about 30
times larger than a ribosome.