Protein Synthesis and DNA Replication: Processes and Mechanisms

Learning objectives

By the end of this session, you should be able to:
Describe, using diagrams the processes involved in
Transcription
Translation
Replication of DNA

Protein synthesis: Transcription

Occurs in the nucleus of eukaryotes and cytoplasm of prokaryotes
Produces pre-mRNA which is then modified in post-transcriptional modification to form mRNA
Starts with INITIATION, DNA unwinds in part.
One strand the acts as the template, the other is non-coding- BUT this can vary from gene to gene
and both strands of DNA are needed to recruited the RNA polymerase and associated
proteins

Initiation of Transcription

Transcription is catalysed by the enzyme RNA polymerase, which attaches to and moves along
the DNA molecule until it recognises a promoter sequence, which is made up of a few hundred
base pairs.
In eukaryotes, the promoter sequence includes base sequences of 5'-TATAAA-3' s known as the
TATA box.
Transcription starts when six general transcription factors or proteins come together at the
promoter region of a gene. Transcription factors control the rate of transcription
Next one or more transcriptional activator proteins bind to an enhancer region on the DNA
(these are not in the promoter region)
When the activators have bound to the enhancer DNA, they attract a mediator complex of
proteins which attract RNA polymerase (aka Pol ll) and transcription can begin.

Transcription complex diagram

Enhancer sequences
Transcriptional
start site
-ONA
Promoter
Transcriptional activator proteins
General
transcription
factors
Enhancer
sequence
DNA-
Mediator
complex
Promoter
5
3
ANA transcript
ANA polymerase
complex (Pol /1)

Elongation in Transcription

Occurs in a transcription, where the two strands of DNA are separated and the RNA
nucleosides come and base pair with the template strand.
The template strand on the DNA is read in a 3' to 5' direction
Triphosphate ribonucleosides, join to form the strand of pre-MRNA
The O in the OH on the 3C forms a phosphodiester bond with the innermost phosphate group
on the nucleoside
Breaking of the bond releases the other two phosphate groups and provides energy for the
reaction to occur

Termination of Transcription

Elongation continues until the RNA polymerase encounters a stop sequence.
Now, the mRNA needs to be stabilised so that it does not become damaged as it
leaves the nucleus and travels through the cytoplasm to the ribosomes.
At the 3' end of the strand, a series of 250 adenine nucleotides are added in a
process called polyadenylation. This stabilises the mRNA as it makes its way
through the cytoplasm to the ribosomes
At the 5' end of the new strand, a methylated guanine cap is added which is
essential for translation to occur

Post -transcriptional modification

In addition to the polyadenylation and capping, mRNA also has to undergo splicing to
remove non-coding regions of the new strand (introns) copied for the DNA
1,2,3
ENZYMES CATALYSE
HYDROLYSIS
CONDENSATION-
EXON
INTROS
GRON
2
IN TROU
EXON,
3
INTROS
1
T
EXON
EXON
EXON
3,2,1,
EXPRESSED
SPLICEOSOME
CONSISTS OF
ENZYMES AND
RNA.
SMALL NUCLEAR
RNA.
ARE REMOVED
+
EXONS SPUCEO
BACK TOGETHER .
3,1,2
Multiple polypeptides from one strand of DNA
2,1,3
I

Translation

Translates the strand of mRNA into a polypeptide
chain
Happens on the ribosomes
Involves ribosome, mRNA and tRNA
Ribosome has a large and small subunit and large
subunit has three sites:
· A aminoacyl
· P=peptidyl
· E = Exit site
E
P
A
LARGE
SUBUNIT
- SMALL
SUBUNIT_
E = EXIT SITE .
P = PEPTIDYL SITE - HOLDS THE
GROWING CHAIN .
A = AMINOACYL SITE - HOLDS THE
INCOMING + RNA + AMINO ACID.
1

Stages of translation

Transfer RNA consists of 70-90 nucleotides and has a trefoil shape
With an amino acid binding site at its 3' end (base sequence CCA)
An anticodon corresponding to a specific amino acid
a.
3'end
5' end A
C
Amino acid
attachment
site
CCA
-Amino acid
attachment
site
IG
GACAC
ÜĞÜG
Anticodon loop
Anticodon
Anticodon
b.

Charging of tRNA

Aminoacyl tRNA synthetases connect a
specific amino acid to the amino acid
binding site on the tRNA by catalysing the
formation of a covalent bond
Once an amino acid is added the tRNA is said
to be charged
The codon at which translation starts is the initiation
codon, AUG which codes for methionine
Val
Gin
lle
Aminoacyl
tRNA
synthetase
Free amino acids
Ile
Each aminoacyl
tRNA synthetase
binds to one
uncharged tRNA
and its
CAC
GUU
UAG
Uncharged tRNAs
Gin
Val
GUU
CAC
lle
The enzyme attaches
the amino acid to the
3' end of the tRNA.
U AG
Charged tRNA
corresponding
amino acid.
UAG

Initiation of Translation

Requires some protein initiation factors which attach to the
5' cap on the mRNA
These recruit a small subunit of the ribosome, and a charged
tRNA, anticodon UAC, with methionine attached
The formed initiation complex moves along the mRNA until
it encounters an AUG, establishing a reading frame
Now a large subunit joins the complex, the initiation factors
are released and the next phase - elongation - begins
Initiation
factor
tRNA
SVANMet
Small
ribosomal
subunit
UÁČ
AUGGUAAGA
mRNA
Large ribosomal
subunit
Met
E
SP
A
UAC
AUGGUAAGA
ofis

Elongation in Translation

* The charged MET tRNA is situated on the P site
The next charged tRNA with its amino acid comes down and
attaches to the A site
: A peptide bond is formed between the two amino acids
The ribosome now shifts along, the tRNA that was carrying the MET is now
uncharged and goes to the E site and leaves the ribosome
The tRNA on the A site is now on the P site, freeing the A site for the next charged
tRNA
This whole process is helped by proteins called elongation factors which are bound
Val
www Met
E
A
CAU
JACI
AUGGUAAGA
Peptide bond
Met
E
UAC CAU
AUGGUAAGA
Met
Va
E
A
AUGĞÜÃAĞA
Met-Val -Arg
E
CAUUCU
AUGGUAAGA
to GTP and break the triphosphate bond, releasing the energy to allow the polypeptide chain to elongate

Termination of Translation

Elongation occurs until a stop codon is reached
This doesn't code for a tRNA
At this point, a protein release factor binds to the
A site and releases the polypeptide chain
Phe
Ala
Met
Gly
Val
Arg
Thr
E
site
P
A
site
Release
factor
UGG
AGAACQUAA
5'
3'
mRNA

Replication of DNA - the enzymes

Semi-conservative replication- produces a helix with one new and one original strand
The DNA unwinds at the replication fork, with each strand becoming a template strand and
the new strand that will form is a daughter strand
DNA helicase separates the parent strands by breaking H bonds between the complementary
base pair
A single-strand binding protein attaches to the strands and stops them re-annealing
Topoisomerase moves up the helix, away from the fork relieving the stress of DNA unwinding
DNA polymerase catalyses the formation of the new strand.

DNA replication- the process

DNA replication can't start with isolated DNA nucleotides therefore for both strands,
RNA primase produces a short strand or primer to start the process
DNA polymerase then takes over and adds nucleotides to the 3' end of the replicating strand.
DNA polymerase can only add nucleotides to the 5' end of a strand
Triphosphate nucleosides are added with the inner-most bond inter phosphate bond
broken and the end two phosphates released. This provides the energy for the joining of the
nucleotides
The sugar-phosphate backbone is formed by phosphodiester bonds

DNA replication continued

In the leading strand, replication is continuous
In the lag strand, it is discontinuous and produces fragments ( Okasaki fragemnts) which are joined
by DNA ligase.
Both daughter strands are synthesised at the same time, using the same polymerase,
so the lagging strand needs to loop round to be replicated.
Errors are reduced as the polymerase proofreads the forming strands

DNA replication diagram

RNA
RNA
primase primer
5'
Template
strand
3'
13
5
3×
5'
RNA primase lays
down an RNA
primer.
3
DNA
polymerase
5'
3
3'
5'
DNA polymerase
extends the RNA
primer.
3'
RNA
primer
removed
DNA
polymerase
(5'
3'
5'
3
5
5'
A different DNA
polymerase
removes the
primer and
replaces it with
DNA.
3'
19
DNA
ligase
5
3
3
5'
DNA ligase
forms a bond
joining the two
DNA fragments.
3
5'
10
3'
3'
5'
Leading
strand
As each new primer for an Okazaki fragment is
synthesized, the lagging strand forms a loop that
persists until the new lagging strand encounters
the previous Okazaki fragment.
3'
5'
Trombone model
Lagging
strand
5
3'

How do we know it is semi-conservative replication

Experiments by Meselson and Stahl determined the
method of DNA replication
Growing E coli on a medium with ammonium chloride
as a nitrogen source, where N is N15
Extracting DNA and centrifuging in a medium that
supports different densities
Using those bacteria and adding N14 source of N
Quantity of DNA that is original after n replications = 1/2"
3
CONSERVATIVE
SEMI CONSERVATIVE DISPERSIVE
9
100000000
-
15
Nº5
0000000
-D
-
Nº5
1 + "~
0
1 asaga = 100000g
000000
H
->
2
1 0000000
ID
N'4/N14
NIF /N'S
N14/ 15
12
Nº/N'+
N14/ N15
1
,
1
1
1
/ M
1
NIX /Nº4
N14/N15
D
N14 /N15

Semi-conservative replication proof

Above show why this proved replication was semi-conservative
Work out the quantity of original DNA after 256 replications

Learning outcomes

Describe, using diagrams the processes involved in
Transcription
** Translation
Replication of DNA