REPLIKASI, TRANSKRIPSI &

TRANSLASI

Dr. Marhaen Hardjo, M.Biomed, PhD

Fakultas Kedokteran Universitas Hasanuddin

Makassar 2014

1
 DNA Structure
• DNA consists of two molecules that are arranged
into a ladder-like structure called a Double Helix.

• A molecule of DNA is made up of millions of tiny

subunits called Nucleotides.

• Each nucleotide consists of:

1. Phosphate group
2. Pentose sugar
3. Nitrogenous base
 Nucleotides

Phosphate

Nitrogenous
Base

Pentose
Sugar
 Nucleotides
• The phosphate and sugar form the backbone of
the DNA molecule, whereas the bases form the
“rungs”.

• There are four types of nitrogenous bases.

 Nucleotides

A T

Adenine Thymine

C G

Cytosine Guanine
 Nucleotides
• Each base will only bond with one other
specific base.

• Adenine (A)
Form a base pair.
• Thymine (T)

• Cytosine (C)
Form a base pair.
• Guanine (G)
 DNA Structure
• Because of this complementary base pairing,
the order of the bases in one strand determines
the order of the bases in the other strand.
A T

C
G

T A

C
G

A T

G C

T A
 DNA Structure
• To crack the genetic code found in DNA we
need to look at the sequence of bases.

• The bases are arranged in triplets called

codons.

AGG-CTC-AAG-TCC-TAG
TCC-GAG-TTC-AGG-ATC
 DNA Structure
• A gene is a section of DNA that codes for a protein.

• Each unique gene has a unique sequence of bases.

• This unique sequence of bases will code for the

production of a unique protein.

• It is these proteins and combination of proteins

that give us a unique phenotype.
 DNA
Gene
Protein

Trait
Your Task
• Draw a flow chart to show
how to get from:
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 Proses biologis dalam sel
prokariot dan eukariot
A. Replikasi
B. Transkripsi
C. Translasi

15
Dogma Sentral Biologi Molekuler
Replikasi
Duplikasi DNA

Transkripsi
Sintesis RNA
Inti Sel

Membran inti sel

Sitoplasma
Translasi
Sintesis Protein
Ribosom

16
Protein
The Central Dogma

17
Alir Informasi Genetik

18
 Dari Gen ke Protein

Prokariot

Eukariot 19
Replikasi

20
 Replikasi DNA
• Mekanisme
– semi-konservatif
• Masing-masing pita DNA (yg dalam rantai ganda)
menjadi cetakan (template)
• Nukleotida-nukleotida DNA akan tersusun secara
komplementer terhadap pasangan basa
Nitrogennya
– Sintesis DNA terjadi pada orientasi 5’ --> 3’
• Nukleotida disambungkan pada gugus OH pada
atom C 3’

21
Kemungkinan Replikasi DNA

22
Kemungkinan Replikasi DNA

23
24
Replikasi DNA

25
Replikasi DNA

26
1. DNA replication is called ‘semi-conservative’.
2. Semi-conservative replication is the process in which the
original strands of DNA remain intact and act as
templates for the synthesis of duplicate strands of DNA.
3. One copy of a DNA molecule will split apart to make two
complete copies of itself. Each new DNA molecule is
made up of half of the old molecule and half of a new
molecule.

ANIMATION:
http://www.lewport.wnyric.org/jwanamaker/animations/DNA%20
Replication%20-%20long%20.html
1. UNZIPPING: The DNA molecule ‘Unzips’
as the hydrogen bonds between the base
pairs are broken. The enzyme
HELICASE causes this unzipping to occur.
2. COMPLEMENTARY
BASE PAIRING:
Complementary
nucleotides move into
position to bond with the
complementary bases on
the DNA chain.
3. FORM NEW SUGAR PHOSPHATE BACKBONE: The
nucleotides join as the sugars and phosphates bond to
form a new backbone. This process occurs due to the
enzyme DNA POLYMERASE which also checks for
mistakes as it goes.
4. This process continues along the primary chain until we
have 2 IDENTICAL STRANDS of DNA molecules
(assuming there have been no errors made).
Start at Part 2 and skip to Part 5 (end):
http://207.207.4.198/pub/flash/24/24.html
 Replikasi DNA: datangnya nukleotida

• G berpasangan dgn C
• P04 berikatan pada 3’ OH 36
 Replikasi DNA: datangnya nukleotida

3’

5’

• T berpasangan dgn A
• P04 berikatan pada 3’ OH 37
Animasi Replikasi

38
Transkripsi

39
 Transkripsi: sintesis mRNA

• single stranded RNA synthesized from ds DNA

template
– RNA Polymerase
– ribonucleotides incorporated
– ATP, CTP, GTP, UTP (instead of dTTP)
– Synthesis proceeds in 5’ --> 3’
– In Eukaryotes, splicing may occur
• removal of introns (intervening sequences)

40
 3 Tahap Transkripsi
1. INISIASI
RNA Polimerase melekat
pada Promotor DNA, pita
ganda terpisah dan sintesis
mRNA dimulai

2. ELONGASI
mRNA memanjang sejalan
dengan enzim RNA
Polimerase yang bergerak
sepanjang pita DNA
3. TERMINASI
mRNA dilepaskan dan enzim
RNA Polimerase lepas dari
pita DNA template
41
 Tahap Inisiasi
Transkripsi dimulai pada Promotor
RNA mRNA
Polimerase

promotor +1

DNA
UPSTREAM DOWNSTREAM

42
Promotor

43
 Tahap Elongasi
• RNA Pol membuka ikatan ganda
DNA (sekitar 10 – 20 basa pada
kali pertama)
• RNA Pol merangkaikan
nukleotida-nukleotida RNA sesuai
dengan pasangan basa DNA yang
terdapat pada pita DNA template
• RNA Pol menambahkan
nukleotida – nukleotida RNA pada
ujung 3’dari mRNA yang
terbentuk
• Setelah RNA Pol merangkaikan
nukleotida RNA, ikatan ganda
DNA segera terbentuk kembali
dan mRNA keluar dari lilitan DNA
template 44
 Tahap Terminasi

• Transkripsi akan terjadi sampai RNA Pol mencapai suatu

sekuens pada DNA template yang disebut Terminator
• Pada Prokariot: RNA Pol langsung berhenti ketika mencapai
Terminator
• Pada Eukariot: RNA Pol masih melanjutkan merangkaikan
nukleotida RNA sampai beberapa ratus basa sesudah
Terminator, sehingga terbentuk loop 45
Overview : Prokariot

46
Overview : Eukariot

47
Inisiasi pada
eukariotik

48
 Processing mRNA eukariot

• Ujung 5’ dimodifikasi dengan • Ujung 3’ ditambahi

menambahkan Guanosin tri nukleotida adenin 50 – 250
phosfat (poly A tail)
– Mencegah degradasi – Mencegah degradasi
– Ribosome binding site – Membantu export

49
Penghilangan intron

50
Animasi RNA splicing

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Contoh:

52
53
Animasi Transkripsi

54
“The Protein Players” - RNA polymerases, transcription factors, initiation factors,
enhancers, repressors
Prokaryotes?
Prokaryotic transcription video
Prokaryotic Transcription
DNA Sequences Important to Transcription
Prokaryotes
• Promoter –
•Pribnow Box (also called the -10 element) – TATAAT
•-35 element - TTGACA

Eukaryotes
• Promoter –(asymmetrical sequence)
• Basic core promoter –TATA box (TATAAA(A)); within 50bp upstream of
start site; found in unicellular eukaryotes
• Core promoter PLUS
•Downstream promoters
•Proximal promoter elements
• Initiator sequences

• Regulatory Elements/Response Element - Response elements are the recognition

sites of certain transcription factors Most of them are located within 1 kb from
the transcriptional start site.

• Enhancer elements -upon binding with transcription factors (activators), can

enhance transcription; located either upstream or downstream of the
transcriptional initiation site.
•Upstream enhancer elements
•Downstream enhancers
•Distal enhancer elements

• Silencers - upon binding with transcription factors (repressors), can repress

transcription.

• Insulators
Gene Regulatory Networks – control the number and type of transcripts made by a
cell.
video

Simple core
promoter

UAS = upstream activator sequence RE = regulatory elements INR = initiator sequence

DPE = downstream promoter elements
Sequences that can act as promoters (TATA is preferred)
Proteins Involved in Transcription
RNA Polymerase

General (or Basal) Transcription Factors:

TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH

Transcription Factors that Bind to Regulatory Elements

Holoenzyme or
Initiation Complex
Transcription Factors Have Common DNA Binding Motifs

• Zinc finger
• Helix-turn-helix
• Leucine zipper
RNA Polymerase
Recognizes and binds to TATA box; TBP + 10
TBP associated factors; position set
TBP bends DNA ~80o and forces
open the minor groove.
Recognizes and binds to TATA box; TBP + 10
TBP associated factors

Binds and stabilizes the TFIID complex

Recruits RNA pol II + TFIIF to the location

Two subunits - RAP38 & RAP74. Rap74 has a

helicase activity; RAP38 binds RNAPolII

Two subunits - recruits TFIIH to the

complex thereby priming the initiation
complex for promoter clearance and
elongation

complex of 9 subunits. One w/ kinase activity;

one w/ helicase activity; one is a cyclin (cdk7)
 -30 +1
TATA Inr
TAFs

Sequential
TBP
} TFIID or TBP

IIA

Binding IIB
IIF
Model for Eukaryotic RNA
polymerase II
Pol IIa

assembly of CTD of large subunit of Pol II IIE

preinitiation helicase
IIH
protein kinase

complex IIE IIF

IIB IIA Pol IIa
IIH
TATA Inr
preinitiation complex
ATP hydrolysis = PIC

IIE IIF
Polymerization of 1st few NTPs and IIB IIA Pol IIa
IIH
phosphorylation of CTD leads to TATA Inr
promoter clearance. TFIIB, TFIIE and initiation complex, DNA melted at Inr
TFIIH dissociate, PolII+IIF elongates,
and TFIID + TFIIA stays at TATA. Activated PIC
Transcription initiation in the cell often requires the local recruitment of chromatin-
modifying enzymes, including chromatin remodeling complexes and histone
acetylases - greater accessibility to the DNA present in chromatin
RNA polymerase is also assisted by DNA supercoiling
Phosphorylation of the carboxy terminal
domain (CTD) of one of the subunits of
RNA PolII;
RNA polymerase II dissociates from the
transcription factors and other protein
complexes that were required for
assembly and elongation begins

Phosphorylation also promotes the

accumulation of elongation factors –
other proteins that arrest transcription
long enough to recruiting RNA
processing enzymes
 Elongation is Coupled to RNA Processing

• Capping
• Splicing
• Polyadenylation
 RNA Capping enzymes:
• Phosphatase
• Guanyl transferase – GMP in 5’ to5’
linkage
• methyltransferase

Video of transcription and capping

CBC – cap binding complex proteins also associate and protect the cap;
Later they will direct transcript in its exit from the nucleus
RNA Splicing
How Introns Are Identified:
Consensus sequences at (5’ to 3’ direction)
•5’ splice site
•Lariate loop closure site of the intron sequence
•3’ splice site

R=A or G,Y=C or U
The Spliceosome Forms
snRNAs (U1, U2, U4, U5 and U6) and associated proteins = snRNPs

• U1 binds to the GU sequence at the 5' splice site, along with accessory
proteins/enzymes,

• U2 binds to the branch site, and ATP is hydrolyzed;

• U5/U4/U6 trimer binds, and the U5 binds exons at the 5' site, with U6
binding to U2;

• U1 is released, U5 shifts from exon to intron and the U6 binds at the

5' splice site;

• U4 is released, U6/U2 catalyzes transesterification, U5 binds exon at

3' splice site, and the 5' site is cleaved, resulting in the formation of
the lariat;

• U2/U5/U6 remain bound to the lariat, and the 3' site is cleaved and
exons are ligated using ATP hydrolysis. The spliced RNA is released and
the lariat debranches.
Rearrangements that occur during splicing
• U1 replaced by U6
• BBP (branch binding protein) and U2
• U5 complex branch forming enzymes in U6 and U2

Allows for “check and recheck” at each splice site.

Why is splicing so accurate?

Introns are small-large;

Exons are about 150bp long

Exons might be easily identified, while

introns probably couldn’t be.
 Accurate……….

As the RNA is being transcribed, SR proteins (rich in serine (S) and arginine (R)) sit down on
the exons. Along with the U proteins, demarcates the start and end of the exon.
Capping proteins or polyA binding proteins act as markers at either end of the transcript.
Other hnRNPs (heterogeneous nuclear RNPs) bind along the introns, helping to distinguish
these sequences from exons.
 ……….But Flexible

Changes in splicing patterns caused by random mutations have been an important

pathway in the evolution of genes.
3’ end splicing sequence
• AAUAAA
• Cleavage site CA – 10-30 nucleotides downstream
• Polyadenylation site – GU- or U-rich region about 30 nucleotides downstream from
the cleavage site
CPSF = Cleavage and Polyadenylation
Specificity Factor
CstF = Cleavage Stimulation Factor
 Poly A polymerase – no template
strand required

All of the A nucleotides are

derived from ATP

Poly A binding proteins remain until mRNA undergoes translation

Only very “select” RNAs can be
transported out of the nucleus
 Guided diffusion along the FG-repeats displayed by nucleoporins

Proteins bound to mature mRNA

molecules and that signal
completed splicing have nuclear
export signals as a part of their
sequence
•hNRPs “straighten out” the mature mRNA so that nuclear export signals can be “read”
•5’ cap enters the pore first
•Many of the RNA binding proteins fall off as mRNA exits the nucleus
•Initiation factors (elF-4G and elF-4E) immediately bind to the 5’ capping complex (which
falls off) and to the polyA tail, forming a loop
Test Your Knowledge – Translation

• Transcription requires only changing a DNA code of nucleotides into a similar RNA
code of nucleotides, while translation involves changing the RNA code into what?
• What are codons and what “reads” codons?
• What is “wobble” and how is it related to translation?
• What attaches amino acids to t-RNA?
• What are the “parts” of the ribosome? What function does each part perform?
• What are the A, P, and E sites of a ribosome? What binds at each of these sites?
• Does anything beside the ribosome participate in elongation of the amino acid chain?
If so, what is it and what does it do?
• What signals where translation starts and stops?
• What happens to improperly translated or proteins that don’t fold properly after
being translated?
 Transfer RNA

• anticodon- 3’ to 5’ sequence that matches the

complementary 5’ to 3’sequence (codon) on the mRNA
• Acceptor arm - Amino acid code on 3’ end
• T and D loops – provide structure for interface with
aminoacyl-tRNA synthetase

?
A different aminoacyl-tRNA synthetase enzyme for each amino acid
Large subunit does???? Small subunit does ?????
 Translation Initiation
This is the only tRNA that can bind to the small ribosomal
subunit by itself
 Protein made in 5’ to 3’ direction, with N-
terminal end made first

General Mechanism

• A site is where new codon is

translated
• P site is where the growing
peptide chain is kept and new aa
are attached
• E site is where “naked” t RNA exit
the ribosome
More Detailed View

New tRNA carrying amino acids are

accompanied by elongation factor called EF-
Tu
The tRNA-ETu occupies a hybrid binding site
(not quite in A)
Correct codon-anticodon pairing triggers ETu
to split GTP and fall off, and tRNA moves into
the A position
The delay caused by the
association/dissociation of ETu helps increase
accuracy of translation
Elongation factor G (EF-G) then binds near
the A site, forcing the tRNAs containing the
new amino acid and the growing chain into
the next (P and E) sites on the ribosome
EF-G splits GTP, changes conformation and
falls off, thus increasing the speed of
translation.
GTP exchange factors continually recharge
the GTP on both of the elongation factors.
Stop Codons = UAA, UAG, UGA
No tRNA binds to this set of codons
One of these codons at the A site attracts a
release factor
Ribosome adds a water to the last peptide,
creating the carboxyl end
Proteins Fold as They are Translated on the Ribosome
Hsp 70 works to help fold early in the lifecycle of a protein

Hsp 60 works like a quality control chamber after a protein is completely

folded
 Cap hydrolyses ATP; regulates
entry

Proteolytic core

Cap hydrolyses ATP;

regulates exit

Proteasomes are a major mechanism by which cells regulate the concentration of

particular proteins and degrade misfolded proteins.
Proteins are marked for destruction by the
addition of a small molecule called ubiquitin on
exposed lysine residues
Cells can also regulate protein degradation by activating new ubiquitin ligases via different
mechanisms.
Proteins generally “hide” degradation signals so that they are not active. However, there
are several mechanisms for exposing the degradation signals.
You have isolated an antibiotic named edeine, from a bacterial culture. Edeine inhibits
protein synthesis but has not effect on either DNA synthesis or RNA synthesis. When
added to a reticulocyte lysate, edeine stops protein synthesis after a short lag, as shown
below. By contrast, cycloheximide stops protein synthesis immediately. Analysis of the
edeine-inhibited lysate by density-gradient centrifugation showed that no polyribosomes
remained by the time protein synthesis had stopped. Instead, all the globin mRNA
accumulated in an abnormal 40S peak, which contained equimolar amounts of the small
ribosomal subunit and initiator tRNA.
A.What step in protein synthesis does edeine inhibit?
B.Why is there a lag between addition of edeine and cessation of protein synthesis? What
determines that lag?
C.Would you expect the polyribosomes to disappear if you added cycloheximide at the same
time as edeine?
Radioactivity in hemoglobin

control
Add inhibitor

edeine

cycloheximide

0 2 4 6 8
Time (min)
Translasi

117
 Translasi: Sintesis Protein
• Protein disintesis dari mRNA template
– Melibatkan Ribosom & amino acyl-tRNA
– Asam-asam amino akan dirangkaikan
membentuk polipeptida
– Dimulai pada Start Kodon yaitu AUG
• As. Amino yang pertama selalu = Met (formyl-
Methionine)
• NH2 terminus of protein
– Diakhiri pada Kodon STOP yaitu UAG, UAA, UGA
118
 Translasi
mRNA
AUG UAA
5’
3’

Protein

NH2 COOH

Amino- Carboxy-
Terminus Terminus

119
 Tabel
Kode
Genetik

120
Animasi Translasi

121
 15.1 Many Genes Encode Proteins

• The One Gene One Enzyme Hypothesis:

• Genes function by encoding enzymes, and each

gene encodes a separate enzyme.

• More specific: one gene one polypeptide

hypothesis
 Concept Check 1

Auxotrophic mutation 103 grows on minimal

medium supplemented with A, B, or C. Mutation
106 grows on medium supplemented with A and
C, but not B; and mutation 102 grows only on
medium supplemented with C. What is the order
of A, B, C in a biochemical pathway?
 Concept Check 1

Auxotrophic mutation 103 grows on minimal

medium supplemented with A, B, or C. Mutation
106 grows on medium supplemented with A and
C, but not B; and mutation 102 grows only on
medium supplemented with C. What is the order
of A, B, C in a biochemical pathway?

BAC
 Breaking the Genetic Code

• Codon: a triplet RNA code

 The Degeneracy of the Code

• Degenerate code: Amino acid may be specified by

more than one codon.

• Synonymous codons: codons that specify the same

amino acid

• Isoaccepting tRNAs: different tRNAs that accept the

same amino acid but have different anticodons

• Wobble hypothesis
 The Degeneracy of the Code

• Sense codons: encoding amino acid

• Initiation codon: AUG

• Termination codon: UAA, UAG, UGA

 Concept Check 2

Through wobble, a single can pair with

more than one .

a. codon, anticodon
b. group of three nucleotides in DNA, codon in mRNA
c. tRNA, amino acid
d. anticodon, codon
 Concept Check 2

Through wobble, a single can pair with

more than one .

a. codon, anticodon
b. group of three nucleotides in DNA, codon in mRNA
c. tRNA, amino acid
d. anticodon, codon
 15.1 Many Genes Encode Proteins

• The One Gene One Enzyme Hypothesis:

• Genes function by encoding enzymes, and each

gene encodes a separate enzyme.

• More specific: one gene one polypeptide

hypothesis
 15.2 The Genetic Code Determines How the Nucleotide
Sequence Specifies the Amino Acid Sequence of a Protein
The Reading Frame and Initiation Codons

• Reading frame: three ways in which the sequence

can be read in groups of three. Each different way of
reading encodes a different amino acid sequence.

• Nonoverlapping: A single nucleotide may not be

included in more than one codon.

• The universality of the code: near universal, with

some exceptions
15.3 Amino Acid Are Assembled into a Protein
Through the Mechanism of Translation
 The Binding of Amino Acids to Transfer
RNAs

• Aminoacyl-tRNA syntheses and tRNA charging

• The specificity between an amino acid and its

tRNA is determined by each individual aminoacyl-
tRNA synthesis. There are exactly 20 different
aminoacylt-tRNA syntheses in a cell.
 The Initiation of Translation

• Initiation factors IF-3, initiator tRNA with N-

formylmethionine attached to form fmet-tRNA

• Energy molecule: GTP

 The Initiation of Translation

• The Shine–Dalgarno consensus sequence in bacterial

cells is recognized by the small unit of ribosome.

• The Kozak sequence in eukaryotic cells facilitates the

identification of the start codon.
 Elongation

• Exit site E

• Peptidyl site P

• Aminoacyl site A

• Elongation factors: Tu, Ts, and G

 Concept Check 3

In elongation, the creation of peptide bonds

between amino acids is catalyzed by .

a. rRNA
b. protein in the small subunit
c. protein in the large subunit
d. tRNA
 Concept Check 3

In elongation, the creation of peptide bonds

between amino acids is catalyzed by .

a. rRNA
b. protein in the small subunit
c. protein in the large subunit
d. tRNA
 Termination

• Termination codons: UAA, UAG, and UGA

• Release factors
 15.4 Additional Properties of RNA and
Ribosomes Affect Protein Synthesis

• The three-dimensional structure of the ribosome

• Polyribosomes:
– An mRNA with several ribosomes attached
 15.4 Additional Properties of RNA and
Ribosomes Affect Protein Synthesis

• Messenger RNA surveillance:

• Detect and deal with errors in mRNA

• Nonsense – mediated mRNA decay: eliminating

mRNA containing premature termination codons

• The posttranslational modifications of proteins

160