Overview of transcription, translation and

recombinant DNA technology

S. C. Kundu
Department of Biotechnology

Prokaryotic cells
without a nucleus

Eukaryotic cells
with a nucleus

Nucleus
Mitochondria
Chloroplast
Ribosomes
RER
SER
Golgi body
Cytoplasm
Vacuoles

Cytoplasm
Ribosomes
Nuclear Zone
DNA
Plasmid
Cell Membrane
Mesosome
Cell Wall
Capsule (or slime layer)
Flagellum

Macromolecules
Protein
Nucleic acids
Olygosaccharides
Lipids
Complex macromolecules

DNA consists of two strands running anti-parallel and forming

double hellical structure

RNA
RNA is a polymer of ribonucleotides that contain ribose rather than
deoxyribose sugars.
The normal base composition is made up of guanine, adenine, cytosine,
and uracil

RNA is found in nucleus and cytoplasm

Types of RNA : Messenger RNA (mRNA)

Ribosomal RNA ( rRNA)
Transfer RNA
(tRNA)

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

O
C

Side chain:
R characterises
the amino acid

N
H

carboxylic acid
group

amino group

Formation of the peptide bond

O

R1

R2

OH

OH

NH2

NH2

The molecules must be

orientated so that the
carboxylic acid group of one
can react with the amine group
of the other

R2

R1

OH

H2N
O

NH2

Two amino acid molecules;

the nature of the R group (R1
and R2) determines the amino
acid

HO

O
R2

R1

H2O

NH
O

NH2
HO

The peptide bond forms with

the elimination of a water
molecule.

Y
V
S

The levels of protein structure

The Central Dogma of Molecular Biology

Cell
DNA

Transcription

mRNA
Translation

Ribosome

Polypeptide
(protein)

This describes the flow of information from DNA into RNA (most commonly
mRNA) through transcription (copying 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).

TRANSCRIPTION AND TRANSLATION:

Prokaryotic vs Eukaryotic

COUPLED

SEPARATE COMPARTMENTS

Gene transcription in prokaryotes

Prokaryotic Gene Structure
Promoter

CDS
UTR

Terminator
UTR

Ge nomic DNA
transcription
mRNA
translation

protein

Gene is the structural and

functional unit of heridity,
which carry genetic
information from one
generation to next. In
molecular terms Gene is a
part of chromosomes
(DNA), which codes for
functional RNA or protein

Promoter is a DNA sequence usually present upstream of coding regions

where RNA polymerase binds to initiates transcription.
UTR (Untranslated sequences): 5 UTR contains ribosome binding sites
for protein synthesis; 3UTR helps in stability of RNA
CDS: coding sequences for protein synthesis
Terminator: Sequence for ending RNA synthesis

Requirement for transcription in

Gene or DNA to be transcribed, RNA polymerase, rNTPs
prokaryotes
and cellular environment

RNA transcript is complementary

to template strand and identical
to coding strand

Different genes are transcribed from different strands

Bacterium has
one RNA
polymerase to
synthesize all
three RNA:
(mRNA, rRNA,
tRNA)

RNA polymerase binds to promoter of a gene to initiate transcription

Promoter
Promoters sequences can vary tremendously.
RNA polymerase recognizes hundreds of
different promoters

13

Stages of Transcription
Chain Initiation
Chain Elongation
Chain Termination

Bacterial Transcription Initiation

Promoter

recognition by RNA polymerase

Formation of Transcription Bubble by separating DNA

strands
Bond formation between rNTPs to start RNA synthesis
Escape of transcription apparatus from promoter

How RNA Polymerase finds promoter and Initiates

Transcription
Core enzyme can synthesize RNA on a DNA template but cannot initiate
transcription.
Core polymerase loosely binds at random sites in DNA without
discriminating promoter and other sequences.
Binding of sigma in the polymerase (holoenzyme) reduced its ability to
loose binding sites, and the enzymes moves along the DNA.
When it reaches the promoter sequences, sigma factor recognize
specifically -35 sequence and binds tightly (-40 to +20 regions). Then it
unwinds DNA (17 bp) from -10 regions and adds ribonucleotide (G or A)
in the +1 site.
After the synthesis of 6-9 nucleotides long RNA without movement of
enzyme, sigma factor falls off from holoenzyme and the core enzyme
enters the elongation process [promoter escape]

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

Dissociation of sigma

Requires lower
purine [NTPs]
After RNA chain
is 6-10 NTPs long

RNA Synthesis is in the 5 to 3 Direction

DNA strand

RNA strand

Subsequent
hydrolysis of
PPi drives the
reaction forward

OH

OH

RNA has polarity (5 phosphate, 3 hydroxyl)

RNA polymerase
elongation

During Elongation
RNA polymerase
unwinds DNA ahead
of it, transcribe the
region and rewinds
the DNA at the back
and RNA comes out
of the complex.
Transcription occurs
in the Transcription
Bubble at the rate of
50 nt/sec.

12

- "
"

Elongation continues
till Core enzymes
reaches the
terminator
sequences.

Transcription Termination
Transcription ends after a terminator is transcribed
Prokaryotic Gene Structure
Promoter

CDS
UTR

Terminator
UTR

G e n o m ic DNA
transcription
m R NA
translation

protein
7

Two types of terminators in bacteria:

Rho-dependent terminators

Rho-independent terminators

Rho independent transcription termination

When a nascent RNA transcript contains a series of U residues at the 3 end
proceeded by a GC rich self complementary sequences the complementary
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 time the U-A base pairs at the 3 end of RNA
chain ( which are extremely unstable) melt releasing the RNA from the
transcription complex to terminate transcription

Rho-Independent Transcription
Termination

(depends on DNA sequence - NOT a protein factor)

Stem-loop structure

Rho independent transcription termination

The termination function of factor

The factor, a hexamer, is a ATPase

and a helicase.

Rho-Dependent Transcription Termination

(depends on a protein AND a DNA sequence)
G/C -rich site

RNAP slows down

1) Rho binds a
stretch of GC rich
sequence of
nacent RNA
upstream of the
terminator.

2) Rho acts as
hexamer, breaks
ATP and with the
energy moves
through RNA to
Rho helicase
catch DNA-RNA
catches up
hybrid and
polymerase
complex and
terminates
Elongating complex is disrupted
transcription.

Lactose operon: a regulatory gene

and 3

stuctural genes, and 2 control elements

Regulatory gene

Structural Genes

Cis-acting
elements
lacI

PlacI

lacZ

lacY

lacA

DNA

Plac Olac
m-RNA
Protein
Transacetylase
-Galactosidase
Permease
The Lac operon

1. When lactose is absent

A repressor protein is continuously synthesized. It sits on
a sequence of DNA just in the lac operon, the Operator
site
The repressor protein blocks the Promoter site where
the RNA polymerase settles before it starts transcribing
Repressor
protein

DNA

Regulator
gene

Operator
site

RNA
polymerase

Blocked

y
lac operon

2. When lactose is present

A small amount of a sugar allolactose is formed within
the bacterial cell. This fits onto the repressor protein at
another active site (allosteric site)
This causes the repressor protein to change its shape (a
conformational change). It can no longer sit on the
operator site. RNA polymerase can now reach its
promoter site and initiates transcription.

DNA
I

z
y
Promotor site

Eukaryotic Transcription
Eukaryotic transcription is much complicated
Three different polymerases:
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).
Some subunits are common to all three RNA polymerases such as TBP.

Multiple promoter types :TATA Box, Initiator elements,

CpG island for pol I),
core elements,
upstream core elements (pol I),
(A box, B Box, C Box for pol III)
Each RNA polymerase recognizes its own promoter
Many proteins (transcription factors) are involved in promoter
recognition by RNA Polymerase

Eukaryotic Gene Structure

5 - Promoter

Exon1
UTR

Intron1

Exon2
splice

splice

Terminator 3
UTR

transcription
Poly A

translation

protein
6

Eukaryote Promoter (Pol II)

Transcription by Polymerase II
Three Steps:
Intiation:
Binding of transcription factors and Pol II to promoter,
DNA strand separation and beginning of RNA synthesis.

Elongation:
Continuous process of RNA synthesis by RNA pol II.

Termination:
Ending of transcription after transcribing a poly A signal
sequence.

Transcription termination

Posttranscriptional 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
removal of non-coding sequences (introns - non
coding sequence) and joining of coding sequences
(exons) by splicing through the formation of spliceosome
(with the help of snRNPs)

Capping at 5 end of mRNA

ppp 5'N pNp
Pi

removing
phosphate group

5'

pp NpNp
GTP forming 5'-5'
triphosphate group
PPi
5'

5'

G ppp NpNp
methylating at G7
7

m GpppNpNp

2'

2'

m Gppp m N pm N p

methylating at C 2' of the

first and second
nucleotides after G

Splicing mechanism
Twice transesterification
intron

5'

5'exon

U pA

G pU

3'exon

3'

first transesterification

pG-OH
pGpA
5'

UOH

3'

G pU

second transesterification
5' pGpA
5'

U pU

3'
GOH 3'

Eukaryotic Transcription
Nuclear
pores

Cytoplasm

DNA

Transcription
RNA

RNA
Processing
mRNA G

AAAAAA

Nucleus

Export

AAAAAA

 http://www-class.unl.edu/biochem/gp2/m
_biology/animation/gene/gene_a2.html

Translation: Protein synthesis

Translation is the process of decoding a mRNA
molecule into a polypeptide chain or protein

Transcription and translation in

eukaryotic cells are separated in
space and time.
Extensive processing of primary
RNA transcripts in eukaryotic cells.

Translation
It is process of protein synthesis (assembly of amino
acids) using mRNA as template with the help of tRNA
and ribosomes (rRNA with several proteins).
Therefore, it requires the participation of multiple types
of RNAs:
messenger RNA (mRNA) carries the information from DNA that
encodes proteins
ribosomal RNA (rRNA) is a structural component of the
ribosome
transfer RNA (tRNA) carries amino acids to the ribosome for
translation

The Genetic Code

The genetic code is the way in which the nucleotide sequence in
nucleic acids specifies the amino acid sequence in proteins.
A codon is a set of 3 nucleotides that specifies a particular amino
acid.
Therefore, mRNA carries information from DNA in a three letter
genetic code.

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 to

select all 20 amino acids.

That is, there are 4 bases in RNA, so 42 (4x 4)=16;

where as for 3 lettered code the number is 43 (4x4x4)=64.

A Codon
OH
NH2

O
N

CH2

O
N

O
CH2

BACKBONE

HO

HO

Adenine

Guanine

NH2

O
P

NH

B A S E S

PHATE
SUGAR-PHOS

HO

NH2

O
N

O
CH2

N
O

OH

N
N

Adenine

Arginine

GENETIC CODE
Therefore, there is a total of 64 codons with mRNA, 61
specify particular amino acid.
The remaining three codons (UAA, UAG, & UGA) are stop
codons, which signify the end of a polypeptide chain
(protein).
This means there are more than one codon for each of
the 20 amino acids.
Besides selecting the amino acid methionine, the codon
AUG also serves as the initiator codon, which starts
the synthesis of a protein

Deciphering the Genetic code

Marshall Nirenberg, Khorana and their collegues deciphered the genetic code by
adding homopolymers such as UUU, AAA, CCC, or co-polymers such as ACA,
CAA, AAC of synthetic nucleotide triplets to cell extracts containing 20 amino
acyl tRNA, which are capable of limited translation.
In each expt. one amino acid is radioactively labeled and rest 19 are unlabeled
and the reaction mixture are passed through filter. Since, ribosomes binds to filter
if the added trinucleotide caused the labeled aminoacyl tRNA to the ribosome,
then radioactivity would be detected on the filter (positive test) otherwise label
will pass through-a negative test-bind filter

mRNA contains codons, which code for amino acids.

tRNA - Transfer RNA.

Each tRNA molecule has 2 important sites of attachment.
Anticodon binds to the codon on the mRNA molecule.
Amino acid acceptor site attaches to a particular amino acid.
During protein synthesis, the anticodon of a tRNA molecule
base pairs with the appropriate mRNA codon.

tRNA Activation

tRNA Structure
Aminoacyl tRNA synthetase

There are 20 different aminoacyl tRNA synthetases, one for each amino acid.

Ribosome
Are made up of 2 subunits, a large one and a smaller one,
each subunit contains ribosomal RNA (rRNA) & proteins.
Protein synthesis starts when the two subunits bind to
mRNA.

The ribosome has

multiple tRNA binding
sites:
P site binds the tRNA
attached to the growing
peptide chain
A site binds the tRNA
carrying the next amino
acid
E site binds the tRNA
that carried the last amino
acid

Translation has 3 Steps, each requiring

different supporting proteins
Initiation

Requires Initiation Factors

Elongation

Requires Elongation Factors

Termination

Requires Termination Factor

Initiation:
1. Binding of initiation
factors to small subunit.
Of ribosome and
attachment to mRNA
2. Binding of first tRNA
to mRNA attached to
small subunit.
3. Binding of large
subunit.

First step in elongation

(bacteria):

Binding of the second

aminoacyl-tRNA

Second step in
elongation:

Formation of the first

peptide bond

Third step in elongation:

translocation

Translation - Initiation

fMet

Large
subunit

UAC
5GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA

Small
subunit

mRNA

Translation - Elongation
Polypeptide

Arg

Met
Phe

Leu

Ser

Aminoacyl tRNA

Gly

Ribosome

UCU

CCA
5GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA

mRNA

Translation - Elongation
Polypeptide
Met
Phe

Leu

Ser
Gly

Arg

Aminoacyl tRNA

Ribosome

CCA UCU
5GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA

mRNA

Translation - Elongation
Polypeptide
Met
Phe

Leu

Ser
Gly
Arg

Ribosome

CCA UCU
5GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA

mRNA

Translation - Elongation
Polypeptide
Met
Phe

Leu

Ala

Ser
Gly

Aminoacyl tRNA

Arg

Ribosome

CGA

CCA

UCU
5GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA

mRNA

Translation - Elongation
Polypeptide
Met
Phe

Leu

Ser
Gly
Arg

CC

Ribosome

Ala

UCU CGA
5GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA

mRNA

Termination:
1. Binding of
Release Factor to
Stop Codon UGA,
UAA, UAG.
2. Disassembly

Overview of Prokaryotic Translation

Summary

http://www-class.unl.edu/biochem/gp2/
m_biology/animation/gene/gene_a3.html

Recombinant DNA
Production of a unique DNA molecule by
joining together two or more DNA fragments
not normally associated with each other
DNA fragments are usually derived from
different biological sources
A series of procedures used to recombine
DNA segments and are called Recombinant
DNA Technology

History of recombinant DNA

technology
Recombinant DNA technology is one
of the recent advances in
biotechnology, which was developed
by two scientists named Boyer and
Cohen in 1973.

DNA recombinant
technology

Basic principle of recombinant DNA

technology
One DNA molecule (called insert) is isolated from one
sources and then this DNA is inserted into another DNA
molecule called vector
Mostly bacterial plasmid is used as vector
The recombinant vector is then introduced into a host
cell where it replicates, transcribes and translates to
produce protein.

Basic steps in Recobinant DNA

Technology
1. Isolate the gene
2. Insert it in a host using a vector (plasmid)
3. Produce as many copies of the host as
possible
4. Separate and purify the product of the gene

STEP 1. DIGESTION OF DNA

SAMPLE TO ISOLATE GENE

STEP 2. DIGESTION OF
PLASMID DNA

Step 3: Inserting gene into digested

vector to create recombinant plasmid

E.co RI digested Insert

( gene)
+
E. co R1
digested
pVector

Recombinant
Plasmid
Ligase

Step 4: inserting recombinant plasmid into

host E. coli bacteria and selection
Ampicillin resistance gene (Ampr)
and target gene on bacterial plasmid
Individual colony is selected and
cultured to amplify recombinant DNA

Plasmid enters some

bacteria
Only E. coli containing plasmid
survive on Ampicillin plates

Bacterial clones

Transformation mixture is plated

on to agar plate containing
Ampicillin

Overall cloning strategy

Extracted DNA from
any organism

Applications of
Recombinant DNA
Technology

Large-scale production of human

proteins
by
genetically
engineered bacteria.
Such as : insulin, Growth
hormone, Interferons and
Blood clotting factors (VIII & IX)

Production of Human
Insulin(???
)

1) Obtaining the human insulin gene

Human insulin gene can be obtained by
making a complementary DNA (cDNA) copy
of the messenger RNA (mRNA) for human
insulin.

2)Joining the human insulin gene

into a plasmid(?? ) vector
The bacterial plasmids and the cDNA are
mixed together. The human insulin gene
(cDNA) is inserted into the plasmid through
complementary base pairing at sticky ends.

3)Introducing the recombinant

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

have taken up the correct
piece of DNA
The bacteria are spread onto nutrient agar. The
agar also contains substances such as an
antibiotic which allows growth of only the
transformed bacteria.

Purification of recombinant protein by affinity

chromatography