Download 12042016 Ekspresi Gen dan Rekombinasi DNA(1)

Priyambodo, M.Sc.
Dogma Sentral Biologi
KODON (Kode Genetik)
• Kodon
Suatu aturan yang menetapkan bahwa pada
sintesis protein suatu triplet nukleotida akan
mengkode asam amino tertentu.
• Degenerasi Kode Genetik
Suatu keadaan yang memperlihatkan bahwa
satu asam amino dikode oleh lebih dari satu
kodon disebut degenerasi.
Kodon START dan STOP
• Adapun kodon AUG berfungsi sebagai inisiator,
sehingga disebut kodon inisiator atau START
CODON.
• Tiga macam kodon yaitu UAA, UAG, dan UGA
disebut kodon terminator atau STOP CODON,
karena berfungsi untuk mengakhiri suatu proses
sintesa protein. Ketiga kodon tersebut juga
disebut kodon nonsense, karena tidak
mengkode satu asam amino pun.
Degenerasi Kode Genetik
Outline Ekspresi Gen
5`
3`
5`
3`
coding DNA strand
template DNA strand
Transcription
5`
3`
mRNA
Translation
N
C
Peptide chain
•
•
Molekul RNA merupakan antiparalel terhadap template DNA strand atau
paralel terhadap coding DNA strand.
Pada molekul RNA, T diganti oleh U.
Ekspresi Gen
Ekspresi Gen pada Eukariotik
dan Prokariotik
Ekspresi Gen pada Eukariotik
DNA
TRANSCRIPTION
1 RNA is transcribed
from a DNA template.
3
RNA
transcript
5
RNA
polymerase
RNA PROCESSING
Exon
2 In eukaryotes, the
RNA transcript (premRNA) is spliced and
modified to produce
mRNA, which moves
from the nucleus to the
cytoplasm.
RNA transcript
(pre-mRNA)
Intron
Aminoacyl-tRNA
synthetase
NUCLEUS
Amino
acid
tRNA
FORMATION OF
INITIATION COMPLEX
CYTOPLASM 3 After leaving the
nucleus, mRNA attaches
to the ribosome.
mRNA
AMINO ACID ACTIVATION
4
Each amino acid
attaches to its proper tRNA
with the help of a specific
enzyme and ATP.
Growing
polypeptide
Activated
amino acid
Ribosomal
subunits
5
TRANSLATION
5
E
A
AAA
UG GUU UA U G
Codon
Ribosome
A succession of tRNAs
add their amino acids to
Anticodon the polypeptide chain
as the mRNA is moved
through the ribosome
one codon at a time.
(When completed, the
polypeptide is released
from the ribosome.)
Rantai DNA Sense
Transkripsi
Promoter
Transcription unit
5
3
3
5
Start point
RNA polymerase
DNA
Initiation. After RNA polymerase binds to
the promoter, the DNA strands unwind, and
the polymerase initiates RNA synthesis at the
start point on the template strand.
1
5
3
Unwound
DNA
3
5
Template strand of
DNA
transcript
2 Elongation. The polymerase moves downstream, unwinding the
DNA and elongating the RNA transcript 5  3 . In the wake of
transcription, the DNA strands re-form a double helix.
Rewound
RNA
RNA
5
3
3
5
3
5
RNA
transcript
3 Termination. Eventually, the RNA
transcript is released, and the
polymerase detaches from the DNA.
5
3
3
5
5
Completed RNA
transcript
3
Fase Inisiasi Transkripsi
1 Eukaryotic promoters
TRANSCRIPTION
DNA
RNA PROCESSING
Pre-mRNA
mRNA
Ribosome
TRANSLATION
Polypeptide
Promoter
5
3
3
5
T A T A A AA
AT AT T T
T
TATA box
Start point
Template
DNA strand
Several transcription
factors
2
Transcription
factors
5
3
3
5
3 Additional transcription
factors
RNA polymerase II
Transcription factors
5
3
3
5
5
RNA transcript
Figure 17.8
Transcription initiation complex
Mekanisme post-transkripsi
A modified guanine nucleotide
added to the 5 end
TRANSCRIPTION
RNA PROCESSING
50 to 250 adenine nucleotides
added to the 3 end
DNA
Pre-mRNA
5
mRNA
Protein-coding segment
Polyadenylation signal
3
G P P P
AAUAAA
AAA…AAA
Ribosome
TRANSLATION
5 Cap
Polypeptide
5 UTR
Start codon Stop codon
3 UTR
Poly-A tail
Mekanisme post-transkripsi
TRANSCRIPTION
RNA PROCESSING
DNA
Pre-mRNA
5 Exon Intron
Pre-mRNA 5 Cap
30
31
1
Coding
segment
mRNA
Ribosome
Intron
Exon
Exon
3
Poly-A tail
104
105
146
Introns cut out and
exons spliced together
TRANSLATION
Polypeptide
mRNA 5 Cap
1
3 UTR
Poly-A tail
146
3 UTR
Mekanisme post-transkripsi
RNA transcript (pre-mRNA)
5
Intron
Exon 1
Exon 2
Protein
1
Other proteins
snRNA
snRNPs
Spliceosome
2
5
Spliceosome
components
3
5
mRNA
Exon 1
Exon 2
Cut-out
intron
RNA splicing Is carried out
by spliceosomes in some
cases
Macam RNA
Translasi
Free
amino
acids
Growing
Protein Chain
AA:tRNA
free
tRNA
mRNA
Ribosome
Codon
Codon for
specific AA
Anti-codon
Translasi
TRANSCRIPTION
DNA
mRNA
Ribosome
TRANSLATION
Polypeptide
Amino
acids
Polypeptide
tRNA with
amino acid
Ribosome attached
Gly
tRNA
Anticodon
A A A
U G G U U U G G C
Codons
5
mRNA
3
RNA Transfer
• A tRNA molecule
– Consists of a single RNA strand that is only about
80 nucleotides long
3
A
Amino acid
– Is roughly L-shaped
C
attachment site
C
A 5
C G
G C
C G
U G
U A
A U
A U
U C
UA
A G *
C A C AG
*
G
* CUC
*
G U G U *
G
C
C G A G
A G G
* *
U C
*
G
A
*
G C
Hydrogen
G C
U A
bonds
G
*
A
*
A
C
Two-dimensional structure. The four base-paired regions and three
U
*
A
G
loops are characteristic of all tRNAs, as is the base sequence of the
A
amino acid attachment site at the 3 end. The anticodon triplet is
Anticodon
unique
to each tRNA type. (The asterisks mark bases that have been
Figure
17.14a
chemically modified, a characteristic of tRNA.)
Ribosome Association and Initiation of
Translation
• The initiation stage of translation
– Brings together mRNA, tRNA bearing the first amino
acid of the polypeptide, and two subunits of a
ribosome
P site
3 U A C 5
5 A U G 3
Initiator tRNA
Large
ribosomal
subunit
GTP
GDP
E
A
mRNA
5
Start codon
mRNA binding site
1
Figure 17.17
3
Small
ribosomal
subunit
A small ribosomal subunit binds to a molecule of
mRNA. In a prokaryotic cell, the mRNA binding site
on this subunit recognizes a specific nucleotide
sequence on the mRNA just upstream of the start
codon. An initiator tRNA, with the anticodon UAC,
base-pairs with the start codon, AUG. This tRNA
carries the amino acid methionine (Met).
5
3
Translation initiation complex
2 The arrival of a large ribosomal subunit completes
the initiation complex. Proteins called initiation
factors (not shown) are required to bring all the
translation components together. GTP provides
the energy for the assembly. The initiator tRNA is
in the P site; the A site is available to the tRNA
bearing the next amino acid.
Elongation of the Polypeptide Chain
• In the elongation stage of translation
– Amino acids are added one by one to the preceding
amino acid
TRANSCRIPTION
Amino end
of polypeptide
DNA
mRNA
Ribosome
TRANSLATION
Polypeptide
mRNA
Ribosome ready for
next aminoacyl tRNA
E
3
P A
site site
5
1 Codon recognition. The anticodon
of an incoming aminoacyl tRNA
base-pairs with the complementary
mRNA codon in the A site. Hydrolysis
of GTP increases the accuracy and
efficiency of this step.
2 GTP
2 GDP
E
E
P
Figure 17.18
3 Translocation. The ribosome
translocates the tRNA in the A
site to the P site. The empty tRNA
in the P site is moved to the E site,
where it is released. The mRNA
moves along with its bound tRNAs,
bringing the next codon to be
translated into the A site.
P
A
GDP
GTP
E
P
A
A
2 Peptide bond formation. An
rRNA molecule of the large
subunit catalyzes the formation
of a peptide bond between the
new amino acid in the A site and
the carboxyl end of the growing
polypeptide in the P site. This step
attaches the polypeptide to the
tRNA in the A site.
Termination of Translation
• The final stage of translation is termination
– When the ribosome reaches a stop codon in the
mRNA
Release
factor
Free
polypeptide
5
3
3
5
5
3
Stop codon
(UAG, UAA, or UGA)
1 When a ribosome reaches a stop 2 The release factor hydrolyzes 3 The two ribosomal subunits
codon on mRNA, the A site of the
the bond between the tRNA in and the other components of
ribosome accepts a protein called
the P site and the last amino
the assembly dissociate.
a release factor instead of tRNA.
acid of the polypeptide chain.
The polypeptide is thus freed
from the ribosome.
Figure 17.19
REKOMBINASI DNA
Kunang-kunang
Luciferase
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KLONING GEN MELALUI REKOMBINAN DNA
Rekombinan DNA merupakan gabungan materi DNA dari sumber materi
genetik yang berbeda.
Teknik rekombinan DNA merupakan dasar dari teknologi rekayasa genetik
atau manipulasi genetik.
Teknik rekombinan DNA juga menjadi dasar untuk analisa genetika
molekular,
antara lain untuk mengkarakterisasi gen, promoter.
Prinsip dari teknik rekombinan DNA yakni adanya DNA yang berperan
sebagai
pembawa atau vektor (misal:plasmid, fagmid, yeast artificial
chromosome/YAC)
dan fragmen DNA yang akan disisipkan.
Perkembangan teknologi rekombinan DNA sudah sangat pesat, sudah
banyak
produk yang dihasilkan maupun pemahaman tentang proses biologi
molekular
yang menggunakan bantuan teknik rekombinan DNA.
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staff.unila.ac.id/priyambodo
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Prinsip dasar dari
teknik rekombinan
DNA
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Pemotongan dan penyambungan DNA pada teknik rekombinan DNA
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Salah satu teknik membuat konstruksi DNA rekombinan yakni menggunakan plasmid
alami dengan menggantikan gen X (kecuali bagian promoternya/arsir kuning) dengan
gen Cat (chloramphenicol acetyltransferase). Ekspresi dari gen Cat dapat
diuji dari aktivitas Cat yang menghasilkan produk berupa CAM (chloramphenicol).
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Enzim Endonuklease Restriksi
(Enzim Restriksi)
•
Endonuklease restriksi mengkatalisis pemotongan DNA pada
daerah tertentu yang mempunyai urutan nukleotida yang
spesifik.
• Urutan nukleotida pada DNA yang dapat dibaca sama dari kiri
ke kanan atau sebaliknya disebut urutan palindromik,
misalnya urutan 5’ AAGC 3’ akan terbaca sama dengan 3’
CGAA 5’.
• Enzim restriksi akan mengenali daerah palindromik dan
memutus ikatan fosfodiester dari rantai DNA.
• Endonuklease restriksi memiliki banyak jenis dan masingmasingmemilikiurutan palindromik yang khas.
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Konstruksi plasmid
vektor pBR322.
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