INDRI SAFITRI

PURNOMO SURYOHUDOYO
DEPT. OF MEDICAL BIOCHEMISTRY
SCHOOL OF MEDICINE
AIRLANGGA UNIVERSITY

REPLIKASI

REPLICATION
1. BEGINS AT SPECIAL LOCATIONS CALLED : ORIGIN OF REPLICATION(O)
2. O CONTAINS SEVERAL PROTEINS NEEDED FOR THE START
OF REPLICATION FORMING AN ORIGIN OF REPLICATION COMPLEX (ORC)
3. PROKARIOTES :
USUALLY ONLY I O REPLICON
EUKARIOTES :
SEVERAL (MANY) O SEVERAL (MANY) REPLICONS
4. REPLICON :
A SEGMENT OF DNA, REPLICATING FROM ONE ORIGIN OF REPLICATION

PROKARIOTES :

EUKARIOTES :

Steps of DNA replication

strand splitting at origin of replication

Procaryots: one origin of replication on

ONE REPLICON
start of DNA replication in both directions

Eucaryots:

many replication origins

MANY REPLICONS

further DNA replication

end of DNA replication

strand separation

RNA Primer

O : Origin
OK : Okasaki pieces

Ld : Leading strand
Lg : Lagging strand

REPLICATION
LEADING & LAGGING STRAND

REPLICATION

DNA Pol I.
Remove RNA primer &
Replaces it with DNA
DNA ligase
causes gap

RNA primer
Remains at 5 end of new strand
3 end of old strand

Processes in DNA
replication
primase

3
5
5
3
helicase

Okasaki-fragment

Lagging strand
RNA PRIMER SYNTHETIZED
BY PRIMASE
CHAIN EXTENDED BY
DNA POL III
DNA POL I REPLACES
RNA BY DNA
LEAVING A GAP
GAP CLOSED BY
DNA LIGASE

ACTION OF
RNA PRIMASE
DNA POLYMERASE III
DNA POLYMERASE I
DNA - LIGASE

REMOVAL OF RNA PRIMER

Prokariotic chromosomes are circular
Removal of primer on 5end will cause
no problem primer can be removed
starting from 3end

RNA PRIMER

Eukariotic chromosomes are linear a special mechanism

is needed to remove primer

REMOVAL & REPLACEMENT OF 5 END RNA PRIMER (EUKARIOTES)

1. REMOVAL & REPLACENT OF 5 END RNA PRIMER IN EUKARIOTES IS A
COMPLICATED PROCESS INVOLVING MANY ENZYMES. THE MOST
IMPORTANT OF WHICH IS AN ENZYME CALLED : TELOMERASE
(TELOMERE = ENDS OF CHROMOSOMES)
2. WITHOUT TELOMERASE DNA WILL SHORTEN AT EACH ROUND OF
REPLICATION
3. TELOMERASE IS ONLY PRESENT IN CELLS OF THE EARLY EMBRYO OR IN
GERM CELLS (SPERMS & EGGS) OR THEIR PRECURSOR CELLS
4. IN SOMATIC CELLS TELOMERASE IS NOT PRESENT DNA SHORTENS
AT EACH ROUND OF REPLICATION
5. SOMATIC CELLS WILL STOP REPLICATING WHEN DNA SHORTEN UP TILL
A CERTAIN LENGTH CELLS DIE

Telomer replication

chromosome shortening
for each replication cycle

cell aging

MECHANISM OF REMOVAL OF RNA PRIMER AT 5END (TETRAHYMENA)

(MURRAY, 1985)
Centromere

Telomere

Primer RNA

ADDITION OF POLY-dLA
UNITS (C4 A2) BY TELOMERASE

NEW PRIMER RNA FORMED

DNA STRAND SYNTHETIZED BY DNA POL II

REPLACEMENT OF ORIGINAL PRIMER

RNA BY DNA (DNA POL I)

REPLACEMENT COMPLETED

REMOVAL OF NEWLY FORMED RNA PRIMER

(BY RNA-ASE d ?)

REMOVAL OF EXCESS STRAND (BY END-NUCLEASED

ORIGINAL LENGTH RESTORED

GENE EXPRESSION ON PROKARIOTES

DNA (GENE)
TRANSCRIPTION

P : PROMOTER
T : TERMINATOR

(m) RNA
UTR : UNTRANSLATED

TRANSLATION

REGION

PROTEIN
PROKARIOTIC mRNA ARE
POLYCYSTRONIC
(CODES FOR MORE THAN
ONE PROTEIN)

GENE EXPRESSION IN EUKARIOTES

TRANSCRIPTION

TRANSCRIPTION STARTS
ADDITION OF CAP

SPLICING

TRANSLATION

PROTEIN
(MONOCYSTRONIC)

EUKARIOTIC GENES ARE SPLITTED GENES

PEMATANGAN mRNA (Eukariot)

hn RNA (Eukariot)

PEMATANGAN mRNA (Eukariot)

Capping

PEMATANGAN mRNA (Eukariot)

Penambahan poli A

PEMATANGAN mRNA (Eukariot)

Splicing

PEMATANGAN mRNA (Eukariot)

SPLICING REACTION

(1)

SPLICING REACTION (2)

mRNA yang siap dipakai untuk template translasi

TRANSLATION
1. INVOLVES :
A. mRNA SUPPLIES AMINO ACID CODE
B. tRNA AMINO ACID CARRIER
CONTAINS : ANTICODON
= NUCLEOTIDE SEQUENCE COMPLEMENTARY TO
THE
A. ACID CODE
C. RIBOSOME MACHINERY FOR TRANSLATION rRNA +
2. OCCURS IN 3 STAGES
PROTEINS
INITIATION
ELONGATION
TERMINATION
3. ALSO NEEDS TRANSLATION FACTORS :
PROTEINS ACTING AT EACH STAGE :

INITIATION FACTORS (IF, eIF)

ELONGATION FACTORS (EF, eEF)

TERMINATION FACTORS (TF, eTF)

TRANSLATION

t RNA ala

ANTICODON WOBBLE
(1)

ANTICODON WOBBLE (2)

RIBOSOMES

(1)

1. CONSIST OF 2 SUBUNITS, EACH CONTAINING rRNA & PROTEINS

2. PROKARIOTES
RIBOSOME : 70S (S=SVEDBERG UNIT)
LARGE SUBUNIT : 50S
23S rRNA, 5 SrRNA
+ 34 PROTEINS
SMALL SUBUNIT : 30S
16SrRNA
+ 21 PROTEINS
3. EUKARIOTES
RIBOSOME : 80S
LARGE SUBUNIT : 60S
28S rRNA, 5,8SrRNA, 5SrRNA
+ 40 PROTEINS
SMALL SUBUNIT : 40S
18SrRNA
+ 30 PROTEINS

RIBOSOMES

(2)

TRANSLATION

PENGENDALIAN
EKSPRESI GEN

CONTROL OF GENE EXPRESSION

CONTROL OF GENE EXPRESSION CAN THEORETICALLY BE EXERSIZED
ON DIFFERENT LEVELS
1. TRANSCRIPTIONAL CONTROL
2. POST TRANSCRIPTIONAL CONTROL : REGULATION OF
mRNA STABILITY
3. TRANSLATIONAL CONTROL
GENE EXPRESSION IS MOSTLY, BUT NOT EXCLUSIVELY EXCERSIZED AT
TRANSCRIPTIONAL LEVEL
THERE ARE DIFFERENCES IN THE WAY OF GENE EXPRESSION IS
REGULATED BETWEEN PROKARYOTES & EUKARIOTES
MUCH MORE IS KNOWN OF GENE EXPRESSION CONTROL IN
PROKARIOTES THAN IN EUKARIOTES.

PENGENDALIAN PADA
PROKARIOTA

Pengendalian negatif

Represi

Pengendalian positif

Induksi
Aktivasi

PENGENDALIAN EKSPRESI GEN (PROKARIOTA)

TRYPTOPHAN OPERON

INDUKSI
(PROKARIOTA)

LAC OPERON

AKTIVASI
(PROKARIOTA)

ACTIVATION
Catabolite Activator Protein
LAC OPERON
Glucose
Lactose
Transcription :
(Repressor bound
to operator)
Glucose
Lactose
Transcription : +
- Inactivated repressor cannot
bind to DNA
- Weak LAC promoter
Glucose
Lactose
Transcription :
CAP c-AMP bound to
activator site

cAMP is produced when glucose

is not available

REGULATORY SEQUENCES IN EUKARIOTES

E.E.

UPE

C.P.
I.S.
U.P.E
E.E.

:
:
:
:

CP
(IS)

Core Promoter
Initiating Sequences
Upper Promoter Elements
Enhancer Elements

GENE

TRANSCRIPTION CONTROL IN EUKARYOTES

1. Basically control elements in eukaryotic gene transcription consists of 2
components : a DNA component called CIS elements and a protein
component called Trans Elements
2. CIS elements : CP (Core Promoter)
UPE (Upstream Promoter Elements)
EE (Enhancer Elements)
these are DNA sequences onto which certain proteins (called transcription
factors) are bound they are located adjacent (CP), close to (UPE) or some
distance away (EE) gene
3. Trans elements includes
Transcription factor bound to DNA (CIS element)
Auxilliary factors not bound to DNA :
Coactivators
Basal factors
4. Transcription factors usually act as activators (induce transcription) but
some act as inhibitors, prevent transcription

TRANSCRIPTION CONTROL IN EUKARYOTES

BASAL TRANSCRIPTION COMPLEX

RNA Pol II
: RNA Polymerase II
TBP
: Tata box Binding Protein
Basal Factors : A, B, F, E, H

Assembly of basal transcription complex only results

in Low Transcription Rate
High transcription rate needs transcription factor (S)
bound to either UPE or EE

TRANSCRIPTION CONTROL IN EUKARYOTES (UPE)

FULL TRANSCRIPTION COMPLEX

By binding of a transcription factor (TF) : SpI bound to GC box
giving high rate of transcription
GC box is a sequence located in UPE near core promoter (CP)
BF
SP1

: Basal Factors
: Specificity Factor 1

CONTROL ELEMENTS IN HUMAN METALLOTHIONEIN GENE

CS : Cap Site
TATA : Tata Box
GC : GC Box

MRE : Metal Response Element

CRE : Corticosteroid Response Element
BLE : Basal Level Enhancer

I.E
: Initiating Elements
UPE : Upstream Promoter Elements
EE
: Enhancer Elements
Distinction between UPE & EE is not clear cut
BLE
: Constitutive enhancer
CRE & MRE : Regulated enhancer
Kingsman & Kingsman 282
(Modified)

Family of cysteine-rich protein :

Nearly 30% amino acid residue

Metallothionein
Low molecular
weight protein (3.5 14 kDa)

Production dependent on availability of the:

dietary minerals (Zn, Co, Se)

amino acids (histidin, cystein)

Have the capacity to bind

(through thiol group)

Physiological metal (Zn, Cu, Se)

Xenobiotic (Cd, Hg, Ag)

TRANSCRIPTION CONTROL IN EUKARYOTES

REPRESSORS & SILENCERS
A : Activator
R : Repressor
CA : Co activator
BTC : Basal Transcription Complex
AS : Activating Sequences
(UPE, EE)
RS : Repressing Sequences
(Silencers)

Transcription

METILASI DNA

Perangkat signaling epigenetik gen pada

posisi off
Berperan a.l. pada :

Embryonic development
Cell differentiation
Repression of transcription

Percobaan :

Mice tanpa DNMT metilasi tdk terjadi

mati pada masa perkembangan

METILASI DNA

C 5 Methylcytosine oleh

DNA metil transferase

McGhee & Ginder (1979)

Beta-globin locus :

Tak termetilasi pada sel yang

mengekspresikan
Termetilasi pada sel yang tak
mengekspresikan

TF

RNA pol
Active
transcription

Unmethylated CpG
island
TF
CH3

CH3

RNA pol
CH3

Methylated CpG island

Repressed
transcription

GENE EXPRESSION REGULATION BY METHYLATION (1)

CELL A
Gene X Expressed
C.P. : Core Promoter

CELL B
Gene X not Expressed

GENE EXPRESSION REGULATION BY METHYLATION (2)

Replication
Old Strand
(Methylated)
New Strand
(Un Methylated)
DNA Methylase

Both Strand
Methylated

GENE EXPRESSION REGULATION BY METHYLATION (2)

GENE EXPRESSION MODULATION

BY
DNA METHYLATION - DEMETHYLATION

 CpG

islands

----- are genomic regions that contain a high

frequency of CG dinucleotides.
----- CpG islands particularly occur at or near
the transcription start site of
housekeeping genes.

 Housekeeping gene : -:
A gene involved in basic functions is required
for the sustenance of the cell.
Housekeeping genes are constitutively
expressed
Luxury gene :are those coding for specialized functions
synthesized (usually) in large amounts in
particular cell types.