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Overview of Transcription, Translation andRecombinant DNA Technology
BY
. .
Department of Biotechnology
Indian Institute of Technology Kharagpur
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Eukaryotic cells
with a nucleus
Prokaryotic cells
without a nucleus
Nucleus
Mitochondria
Cytoplasm
Ribosomes
Chloroplast Ribosomes
RER
SER
Nuclear Zone DNA
Plasmid
Cell Membrane
Cytoplasm
Vacuoles
Cell Wall
Capsule (or slime layer) Flagellum
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MacromoleculesProtein
Nucleic acids
olygosaccharides
Lipids
Complex macromolecules
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Nucleic acids
Deoxyribonucleic acid (a polymer of deoxyribonucleotides)
Ribonucleic acid (a polymer of ribonucleotides)
,phosphate
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DNA RNA
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DNA RNA
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phosphate
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DNA consists of two strands running anti-parallel andforming double hellical structure
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RNA
RNA is a polymer ribonucleotides that contains ribose ratherthan deoxyribose sugars. The normal base composition ismade up of guanine, adenine, cytosine, and uracil
Types of RNA : Messenger RNA (mRNA)
Ribosomal RNA rRNA
Transfer RNA (tRNA)
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DNA vs. RNA
DNA
Double Helix RNA
Deoxyribose sugar
Ribose sugar
Thymine (A-T)
Thymine!
do the work
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Proteins
Proteins are made up of one or more polypeptide. Each
polypeptide is a chain of co-valently bonded amino acids
The general molecular formula of an amino acid is RCH(NH2)COOH
OOH carboxylic acid
C
C
R HSide chain:
Namine group
R characterisesthe amino acid
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Formation of the e tide bond
Two amino acid molecules;O O
the nature of the R group (R1and R2) determines the aminoacid
NH2
R1
OH
NH2
R2OH
The molecules must be
orientated so that the
O
R1
R2
can react wi th the amine group
of the otherNH2O
2
OH
O
R1NH
R2
OH2
The peptide bond forms with
the elimination of a water
molecule; it is another
NH2O
OH
example of a condensation
reaction
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S G
A
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The Central Dogma of Molecular Biology
e
DNATranscription
mRNA
Translation Ribosome
Polypeptide
(protein)
This describes the flow of information from DNA into RNA (most commonly
mRNA throu h transcri tion co in the same code from one molecule to
another), and then expressing the code into a functional molecule by
translation (converting from a nucleic acid code into an amino acid code).
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TRANSCRIPTION AND TRANSLATION:
SEPARATE COMPARTMENTSCOUPLED
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Gene is the structural
Gene transcription in prokaryotes
Prom oter C D S T erm inator
Genomic DNA
U T R U T R
heridity which carry
genetic information
from one generation totranscription
m R N A
translation
next. In molecular
terms Gene is a part ofchromosomes (DNA)
7
p ro tein
functional RNA or
protein
romoter s a sequence usua y present upstream o co ng reg ons
where RNA polymerase binds to init iates transcription.
for protein synthesis; 3UTR helps in stabili ty of RNA
CDS: coding sequences for protein synthesis
Terminator: Sequence for ending RNA synthesis
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Requirement for transcription in prokaryotesGene or DNA to be transcribed, RNA polymerase, rNTPS
an ce u ar env ronmen
RNA transcript is complementary
to coding strand
Different genes are transcribed from different strands
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RNA polymerase
to synthesize all
three RNA:
(mRNA, rRNA,
tRNA)
RNA polymerase binds to promoter of a gene to initiate transcription
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P r o m o t e r
P r o m o t e r s s e q u e n c e s c a n v a r y t r e m e n d o u s l y .
R N A p o l y m e r a s e r e c o g n i z e s h u n d r e d s o f
d i f f e r e n t p r o m o t e r s
1 3
P r o k a r o t i c r o m o t e r
5 '
3 '
3 '
5 '- 5 0 - 4 0 - 3 0 - 2 0 - 1 0 1 1 0
-
s t a r t - 1 0 r e g i o n
T A T A A T
r e g i o n
T T G A C AA A C T G T
A T A T T A
( P r i b n o w b o x )
C o n s e n s u s s e q u e n c e
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Stages of Transcription
Chain Elongation
Chain Termination
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Bacterial Transcri tion Initiation
Formation of Transcription Bubble by unwinding DNA
s ran s
Addition and Bond creation between rNTPs to start RNAsynthesis
clearance)
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How RNA Polymerase finds promoter and Initiates
Transcription
Core enzyme has the ability to synthesize RNA on a DNA template but
cannot transcription at proper site
random sites in DNA without discriminating promoter and othersequences.
n ng o s gma n ro uce a ma or c anges n e po ymerase an e
holoenzyme drastically reduced abil ity to recognize loose binding sites,
and the enzymes moves along the DNA by directly displaced by another.
When it reaches the promoter sequences, sigma factor recognize
specifically -35 sequence and binds tightly.
The holoenzyme occupies -50 to +20 regions of DNA and unwinds DNA(17 bp) from -10 regions and adds ribonucleotide (G or A) in the +1 site.
-
enzyme, sigma factor falls off from holoenzyme and the core enzyme
enters the elongation process
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Finding and binding
the promoter
initiation
Closed complex
formation
RNAP bound -40 to
+20
Open complex
formation
RNAP unwinds from
-10 to +2
Binding of 1st NTP
Requires high
purine [NTP]
Addition of next NTPs
Requires lower
purine [NTPs]
Dissociation of si ma
After RNA chain
is 6-10 NTPs lon
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RNA Synthesis is in the 5 to 3 Direction
RNA strand DNA strand
hydrolysis of
PPi drives the
reaction forward
OH
OH
RNA has polarity (5 phosphate, 3 hydroxyl)
A
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RNApolymeraseDuring Elongation
RNA ol meraseunwinds DNA ahead
of it, transcribe the
region and rewinds
the DNA at the back
and RNA comes outof the complex.
Transcription occurs
in the Transcription
50 nt/sec.
til l Core enzymesreaches the
terminator se uences.
"-12"
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Transcri tion TerminationTranscription ends after a terminator is transcribed
P r o k a r y o t i c G e n e S t r u c t u r eP r o m o t e r C D S T e r m i n a t o r
G e n o m i c D N A
U T R U T R
7
t r a n s c r i p t i o n
m R N A
p r o t e i n
t r a n s l a t i o n
Two types of terminators in bacteria:
Rho-dependent terminators
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Rho independent transcription termination
contains a series of U residues at the 3 end proceedded by a GC rich self
complementary sequences, the complementory sequences base pair with one
another, forming a stem loop structure.
This stem loop structures interacts with the surface of RNA polymerase
causing it to pause. During this t ime the rU-dA base pairs at the 3 end of
RNA chain ( which are extremely unstable) melt releasing the RNA from the
R h o - In d e p e n d e n t T r a n s c r i p t i o n
ranscr p on comp ex o erm na e ranscr p on
( d e p e n d s o n D N A s e q u e n c e - N O T a p r o t e i n f a c t o r )
S te m - lo o p s t ru c t u r e
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Rho independent transcription termination
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Rho-Dependent Transcription Termination 1) Rho binds a(depends on a protein AND a DNA sequence) 72 nt long
sequence of
nacent RNA
G/C -rich site
upstream of the
terminator.
2) Rho acts as
RNAP slows down
hexamer, after
binding to RNA
breaks ATP by its
Rhohelicase
and with this
energy moves
through RNA to
catches upca c -
hybrid and
Unwinds by its
hellicase activit
Elongating complex is disrupted
and terminates
transcription.
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The factor, a hexamer, is aATPaseand a helicase.
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Regulation of transcription in prokaryotes
ene regu a on as een we s u e n . co . oug ere are oof genes are present but they are not all expressed all the time. it is
determined by the growth status of the cell, metabolic condit ion etc.
As an example, When a bacterial cell encounters a potential foodsource it will manufacture the enzymes necessary to metabolize that
.
In 1959 Jacques Monod and Fracois Jacob looked at the abil ity of E. coli
In the presence of the sugar lactose, E. coli makes an enzyme called
beta galactosidase to breaks down the sugar lactose so the E. coli can
digest it for food but not in the absence of lactose
It is the LAC Z gene in E coli that codes for the enzyme beta
galactosidase and this gene is present in lac operon ( cluster of genes
transcribed by same promoter as polycistronic mRNA)
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Lactose operon: a regulatory gene and
s uc ura genes, an con ro e emen s
Structural GenesCis-actingelements
lacI lacZ lacY lacA DNA
PlacI P Olac
m-RNA
-Galactosidase Transacetylase
Protein
Permease
The LAC operon
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.
a sequence of DNA just in front of the lac operon, theOperator site
the RNA polymerase settles before it starts transcribing
Repressor
protein
RNA
polymeraseBlocked
z y aDNA
I O
egu ator
gene lac operon
perator
site
2007 Paul Billiet ODWS
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. A small amount of a su ar allolactose is formed within
the bacterial cell. This fits onto the repressor protein at
another active site (allosteric site)
conformational change). It can no longer sit on theoperator site. RNA polymerase can now reach its
Promotor site
z y aI O
2007 Paul Billiet ODWS
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Eukaryotic Transcription
Eukaryotic Transcription is Horribly Complicated
Three different ol merases:
RNA polymerase I: synthesizes rRNA in the nucleolus.RNA polymerase II: synthesizes mRNA in the nucleoplasm.
RNA polymerase III: synthesizes tRNA, 5S rRNA, small RNAs in the nucleoplasm
All eukaryotic RNA polymerases have 12-16 subunits (aggregates of >500 kD).
.
Multiple promoter types :TATA Box, Initiator elements, CpG island for
ol I , core elements, u stream core elements
pol I), A box, B Box, C Box for pol III)
Each RNA polymerase recognizes its own promoter
Many proteins (transcription factor) are involved in promoter recognition
by RNA Polymerase
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5 - P r o m o t e r E x o n 1 I n t r o n 1 E x o n 2 T e r m i n a t o r 3
U T R s p l i c e s p l i c e U T R
t r a n s c r i p t i o n
P o l y A
6
t r a n s l a ti o n
p r o t e i n
Eukaryote Promoter (Pol II)
Transcription by Polymerase II
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Transcription by Polymerase II
Three Steps:
n a on:
Binding of transcription factors and Pol II to promoter,
.
Elongation:
Continuous Process of RNA synthesis by RNA pol II.
Termination:
Ending of transcription after transcribing a polyA signalsequence.
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Transcription
Initiation: Assembly
Of the initiationmac nery
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Post transcriptional modification of Pre-mRNA
In eukaryotes, the primary transcript (pre mRNA) must be
modified by:
addition of a 5 cap
addition of a 3 poly-A tail
- n rons- non co ng
sequence) and joining of coding sequences(Exons) by
splicing through the formation of spliceoome ( with the helpof snRNPs)
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Capping at 5 end of mRNA
ppp 5'NpNp
removing
pp 5'NpNp
GTP' '
phosphate groupPi
PPi
G5'
ppp5'
NpNp
-
triphosphate group
methylating at G7
m7GpppNpNp
methylating at C2' of the
first and second
nucleotides after G7 2' 2'
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View the iActivity
For this chapter!
Mature
RNA
S li i h i
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Splicing mechanism
U-rich small nuclear
of proteins calledsnRNP forms
spliceosome and
help in the splicing
rocess.
lariat
lt ti li i
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alternative splicing
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CytoplasmDNA
TranscriptionRNA
RNAProcessing
NucleusExport
G AAAAAAG AAAAAAmRNA
Translation is the process of decoding a mRNA
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Translation is the process of decoding a mRNA
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Transcription and translation in
eukaryotic cells are separated in
space and time.
Extensive processing of primary
RNA transcripts in eukaryotic cells.
Translation
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Translation or protein synthesis requires theparticipation of multiple types of RNA:
messenger RNA (mRNA) carries the information
from DNA that encodes proteins
ribosomal RNA (rRNA) is a structural
com onent of the ribosometransfer RNA (tRNA) carries amino acids to the
The Genetic Code
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e Ge et c Code
The genetic code is the way in which the nucleotide sequence in nucleicacids specifies the amino acid sequence in proteins.
.
Therefore, mRNA carries information from DNA in a three letter geneticcode.
A three-letter code is used because there are 20 different amino acids
that are used to make proteins.
If a two-letter code were used there would not be enough codons toselect all 20 amino acids.
That is, there are 4 bases in RNA, so 42 (4x 4)=16; where as 43(4x4x4)=64.
A Codon
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A Codon
P
O
HO ONH2
N
N
N AdenineAdenine
O H
O2
PO
HO O
CH2 NH2N
NH
N
NO
GuanineGuanine
HO Ar inineP
O
HO
O
O
CH2
2
N
N
N
N
AdenineAdenine
HOH
GENETIC CODE
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GENETIC CODE
There is a total of 64 codons with mRNA, 61 specify aparticular amino acid.
The remaining three codons (UAA, UAG, & UGA) are stop
codons, which signify the end of a polypeptide chain
pro e n .
This means there are more than one codon for each of the 20amino acids.
also serves as the initiator codon, which starts thesynthesis of a protein
mRNA contains codons which code for amino acids.
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tRNA - Transfer RNA.
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Each tRNA molecule folds as cloverleaf structure and has 2 important
sites of attachment.One site, called the anticodon, binds to the codon on the mRNA
molecule.
e o er s e a ac es o a par cu ar am no ac .
During protein synthesis, the anticodon of a tRNA molecule base pairswith the appropriate mRNA codon.
tRNA Structure
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ere are eren anm noacy syn e ases, one or eac am no ac .
Ribosome
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, ,each subunit contains ribosomal RNA (rRNA) & proteins.
Protein synthesis starts when the two subunits bind to mRNA.
Translation has 3 Steps, Each Requiring
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p , q g
eren uppor ng ro e ns
Initiation Re uires Initiation Factors
Requires Elongation Factors
Termination
Requires Termination Factor
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1. Bindin of initiation
factors to small subunit.
. n ng o rst t an
mRNA to small subunit.
3. Binding of large subunit.
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1. Binding of next
us ng s a
A site.E P A
2. Peptide Bond
formation between 2E P A
.
3. Translocation ofE P A
r osome.
E P A
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Termination:
.
Release Factor to
Stop Codon UGA,
, .
2. Disassembly
Overview of Prokaryotic Translation
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Overview of Prokaryotic Translation
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-
fMet
ELarge
subunit
UAC
- -- -- -- -- -- -- -- -- -
Smallsubunit
... ...
mRNA
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-Arg
Aminoacyl tRNAPhe Leu
Met
Ser
Gly
ERibosome
CCA
- -- -- -- -- -- -- -- -- - ... ...
mRNA
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-
Phe Leu
Met
Ser
Gly Arg
Aminoacyl tRNA
ERibosome
UCUCCA
- -- -- -- -- -- -- -- -- - ... ...
mRNA
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-Met
Polypeptide
Arg
PheLeu Ser
Gly
ERibosome
CCA UCU
- -- -- -- -- -- -- -- -- - ... ...
mRNA
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-Met
Polypeptide
Aminoacyl tRNA
Ala
Arg
PheLeu Ser
Gly
ERibosome
CCAUCU
- -- -- -- -- -- -- -- -- - ... ...
mRNA
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-Met
Polypeptide
Arg
PheLeu Ser
Gly
Ala
ERibosome
UCU CGA
- -- -- -- -- -- -- -- -- - ... ...
mRNA
Summary
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Recombinant DNA
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Production of a unique DNA molecule by
joining together two or more DNA
fragments not normally associated witheac o er
DNA fragments are usually derived from
A series of procedures used to recombine
Recombinant DNA Technology
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technology
Recombinant DNA technology is oneof the recent advances in
biotechnology, which was developedy two sc ent sts name oyer an
Cohen in 1973.
Basic steps in Recobinant DNA
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1. Isolate the gene
2. Insert it in a host using a vector (plasmid)
3. Produce as many copies of the host as
ossible4. Separate and purify the product of the gene
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Ligase
Outline of recombinant DNA Technology
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Applications of
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Recombinant DNA
Large-scale production of human
engineered bacteria.
Such as : insulin, Growthhormone, Interferons and
Blood clotting factors (VIII & IX)
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Insulin(???)
Human insulin gene can be obtained by
making a complementary DNA (cDNA) copyof the messen er RNA mRNA for humaninsulin.
2 Joinin the human insulin ene
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into a plasmid( ) vector
The bacterial plasmids and the cDNA are
mixed together. The human insulin gene
complementary base pairing at sticky ends.
3 Introducin the recombinant
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DNA plasmids into bacteria
The bacteria E.coli is used as the host cell. If E.coli and the recombinant plasmids are mixed
together in a test-tube.
4)Selecting the bacteria which
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ave a en up e correcpiece of DNA
.agar also contains substances such as an
antibiotic which allows growth of only the.
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RNA Structure
More commonly, RNA is single stranded and can form complex and
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unusual shapes.
Identification of promoter by DNase 1 footprinting technique
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1 A DNA fragment is labeled at
one end with 32P (red dot).
.
digested with DNase I in the
presence and absence of RNApolymerase holoenzyme.
3. A low concentration of DNase I
is used so that on average each
DNA molecule is cleaved just once
.4. The two samples of DNA then
are separated from protein,
denatured to separate the strands,
an e ec rop orese . e resu ng
gel is analyzed by autoradiography,5 The set of DNA fragments left
after DNase I di estion reveals the
promoter
Eukaryotic gene regulation
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Fig. 18.17, Model of glucocorticoid steroid hormone regulation.
h //
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http://www-class.unl.edu/biochem/ 2/m biolo /ani
mation/gene/gene_a2.html
Deciphering the Genetic code
Marshall Nirenberg, Khorana and their collegous deciphered the genetic code by
adding homopolymers such as UUU, AAA, CCC, or co-polymers such as ACA,
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CAA, AAC of synthetic nucleotide triplets to cell extracts containing 20 amino
acyl tRNA which are capable of limited translation
n eac ex arc one am no ac s ra oac ve y a e e an res are un a e e
and the reaction mixture are passed through fil ter. Since ribosomes binds to fi lter,
if the added trinucleotide caused the labelled aminoacyl trNA to attach to theribosome then radioactivit would be detected on the filter ositive test
otherwise label will pass thrrough-a negative test.
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http://www-c ass.un .e u oc em gp m_ o ogy a
nimation/gene/gene_a3.html