Overview of Transcription, Translation and Recombinant DNA Technology

7/25/2019 Overview of Transcription, Translation and Recombinant DNA Technology

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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

Overview of Transcription, Translation and Recombinant DNA Technology

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