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KODON (Kode Genetik)
• KodonSuatu aturan yang menetapkan bahwa padasintesis protein suatu triplet nukleotida akanmengkode asam amino tertentu.
• Degenerasi Kode GenetikSuatu keadaan yang memperlihatkan bahwasatu asam amino dikode oleh lebih dari satukodon disebut degenerasi.
Kodon START dan STOP• Adapun kodon AUG berfungsi sebagai inisiator,
sehingga disebut kodon inisiator atau STARTCODON.
• Tiga macam kodon yaitu UAA, UAG, dan UGAdisebut kodon terminator atau STOP CODON,karena berfungsi untuk mengakhiri suatu prosessintesa protein. Ketiga kodon tersebut jugadisebut kodon nonsense, karena tidakmengkode satu asam amino pun.
Outline Ekspresi Gen
• Molekul RNA merupakan antiparalel terhadap template DNA strand atauparalel terhadap coding DNA strand.
• Pada molekul RNA, T diganti oleh U.
5`
5` 3`
5`
3`
N
3`
C
Translation
Transcription
mRNA
template DNA strand
Peptide chain
coding DNA strand
Ekspresi Gen pada EukariotikTRANSCRIPTIONRNA is transcribed
from a DNA template.
DNA
RNApolymerase
RNAtranscript
RNA PROCESSING
In eukaryotes, theRNA transcript (pre-mRNA) is spliced andmodified to producemRNA, which movesfrom the nucleus to thecytoplasm.
Exon
RNA transcript(pre-mRNA)
Intron
NUCLEUS
FORMATION OFINITIATION COMPLEX
After leaving thenucleus, mRNA attachesto the ribosome.
CYTOPLASM
mRNA Growingpolypeptide
Ribosomalsubunits
Aminoacyl-tRNAsynthetase
AminoacidtRNA
AMINO ACID ACTIVATION
Each amino acidattaches to its proper tRNAwith the help of a specificenzyme and ATP.
Activatedamino acid
TRANSLATION
A succession of tRNAsadd their amino acids tothe polypeptide chainas the mRNA is movedthrough the ribosomeone codon at a time.(When completed, thepolypeptide is releasedfrom the ribosome.)
AnticodonA A AU G G U U U A U G
E A
Ribosome
1
5
5
3
Codon
2
3 4
5
TranskripsiPromoter Transcription unit
RNA polymeraseStart point
53
35
35
53
53
35
53
35
5
5
Rewound
RNA
RNA
transcript
3
3Completed RNAtranscript
Unwound
DNA
RNA
transcript
Template strand ofDNA
DNA
1 Initiation. After RNA polymerase binds tothe promoter, the DNA strands unwind, andthe polymerase initiates RNA synthesis at thestart point on the template strand.
2 Elongation. The polymerase moves downstream, unwinding theDNA and elongating the RNA transcript 5 3 . In the wake oftranscription, the DNA strands re-form a double helix.
3 Termination. Eventually, the RNAtranscript is released, and thepolymerase detaches from the DNA.
Fase Inisiasi Transkripsi
Figure 17.8
TRANSCRIPTION
RNA PROCESSING
TRANSLATION
DNA
Pre-mRNA
mRNA
Ribosome
Polypeptide
T A T A AA AA T A T T T T
TATA box Start point TemplateDNA strand
53
35
Transcriptionfactors
53
35
Promoter
53
355
RNA polymerase IITranscription factors
RNA transcript
Transcription initiation complex
Eukaryotic promoters1
Several transcriptionfactors
2
Additional transcriptionfactors
3
Mekanisme post-transkripsi
A modified guanine nucleotideadded to the 5 end
50 to 250 adenine nucleotidesadded to the 3 end
Protein-coding segment Polyadenylation signal
Poly-A tail3 UTRStop codonStart codon5 Cap 5 UTR
AAUAAA AAA…AAA
TRANSCRIPTION
RNA PROCESSING
DNA
Pre-mRNA
mRNA
TRANSLATIONRibosome
Polypeptide
G P P P5 3
Mekanisme post-transkripsi
TRANSCRIPTION
RNA PROCESSING
DNA
Pre-mRNA
mRNA
TRANSLATIONRibosome
Polypeptide
5 CapExon Intron
1
5
30 31
Exon Intron
104 105 146
Exon 3Poly-A tail
Poly-A tail
Introns cut out andexons spliced together
Codingsegment
5 Cap1 146
3 UTR3 UTR
Pre-mRNA
mRNA
Mekanisme post-transkripsiRNA transcript (pre-mRNA)
Exon 1 Intron Exon 2
Other proteinsProteinsnRNA
snRNPs
Spliceosome
Spliceosomecomponents
Cut-outintronmRNA
Exon 1 Exon 2
5
5
5
1
2
3
RNA splicing Is carried outby spliceosomes in somecases
Translasi
mRNARibosome Codon for
specific AA
Codon
Freeaminoacids
AA:tRNA
GrowingProtein Chain
freetRNA
Anti-codon
Translasi
TRANSCRIPTION
TRANSLATION
DNA
mRNARibosome
Polypeptide
PolypeptideAminoacids
tRNA withamino acidattachedRibosome
tRNA
Anticodon
mRNA
Gly
A A AU G G U U U G G C
Codons5 3
RNA Transfer• A tRNA molecule
– Consists of a single RNA strand that is only about80 nucleotides long
– Is roughly L-shaped
Figure 17.14a
Two-dimensional structure. The four base-paired regions and threeloops are characteristic of all tRNAs, as is the base sequence of theamino acid attachment site at the 3 end. The anticodon triplet isunique to each tRNA type. (The asterisks mark bases that have beenchemically modified, a characteristic of tRNA.)
3CCACGCUUAAGACACCU*
GC * *
G U G U *CU* G AG
GU**A
*A A GUC
AGACC*
C G A G A G GG*
*GACUC*AUUUAGGCG5
Amino acidattachment site
Hydrogenbonds
Anticodon
A
• The initiation stage of translation– Brings together mRNA, tRNA bearing the first amino
acid of the polypeptide, and two subunits of aribosome Large
ribosomalsubunit
The arrival of a large ribosomal subunit completesthe initiation complex. Proteins called initiationfactors (not shown) are required to bring all thetranslation components together. GTP providesthe energy for the assembly. The initiator tRNA isin the P site; the A site is available to the tRNAbearing the next amino acid.
2
Initiator tRNA
mRNA
mRNA binding site Smallribosomalsubunit
Translation initiation complex
P site
GDPGTP
Start codon
A small ribosomal subunit binds to a molecule ofmRNA. In a prokaryotic cell, the mRNA binding siteon this subunit recognizes a specific nucleotidesequence on the mRNA just upstream of the startcodon. An initiator tRNA, with the anticodon UAC,base-pairs with the start codon, AUG. This tRNA
carries the amino acid methionine (Met).
1
U A CA U G
E A
35
53
35 35
Figure 17.17
Ribosome Association and Initiation ofTranslation
• In the elongation stage of translation– Amino acids are added one by one to the preceding
amino acid
Figure 17.18
Amino endof polypeptide
mRNA
Ribosome ready fornext aminoacyl tRNA
E
P A
E
P A
E
P A
E
P A
GDPGTP
GTP
GDP2
2
site site5
3
TRANSCRIPTION
TRANSLATION
DNA
mRNARibosome
Polypeptide
Codon recognition. The anticodonof an incoming aminoacyl tRNAbase-pairs with the complementarymRNA codon in the A site. Hydrolysisof GTP increases the accuracy andefficiency of this step.
1
Peptide bond formation. AnrRNA molecule of the largesubunit catalyzes the formationof a peptide bond between thenew amino acid in the A site andthe carboxyl end of the growingpolypeptide in the P site. This stepattaches the polypeptide to thetRNA in the A site.
2
Translocation. The ribosometranslocates the tRNA in the Asite to the P site. The empty tRNAin the P site is moved to the E site,where it is released. The mRNAmoves along with its bound tRNAs,bringing the next codon to betranslated into the A site.
3
Elongation of the Polypeptide Chain
• The final stage of translation is termination– When the ribosome reaches a stop codon in the
mRNA
Figure 17.19
Releasefactor
Freepolypeptide
Stop codon(UAG, UAA, or UGA)
5
3 35
35
When a ribosome reaches a stopcodon on mRNA, the A site of theribosome accepts a protein calleda release factor instead of tRNA.
1 The release factor hydrolyzesthe bond between the tRNA inthe P site and the last aminoacid of the polypeptide chain.The polypeptide is thus freedfrom the ribosome.
2 3 The two ribosomal subunitsand the other components ofthe assembly dissociate.
Termination of Translation
KLONING GEN MELALUI REKOMBINAN DNA
Rekombinan DNA merupakan gabungan materi DNA dari sumber materigenetik yang berbeda.
Teknik rekombinan DNA merupakan dasar dari teknologi rekayasa genetikatau manipulasi genetik.
Teknik rekombinan DNA juga menjadi dasar untuk analisa genetikamolekular,antara lain untuk mengkarakterisasi gen, promoter.
Prinsip dari teknik rekombinan DNA yakni adanya DNA yang berperansebagaipembawa atau vektor (misal:plasmid, fagmid, yeast artificialchromosome/YAC)dan fragmen DNA yang akan disisipkan.
Perkembangan teknologi rekombinan DNA sudah sangat pesat, sudahbanyakproduk yang dihasilkan maupun pemahaman tentang proses biologimolekularyang menggunakan bantuan teknik rekombinan DNA.
staff.unila.ac.id/priyambodo
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Salah satu teknik membuat konstruksi DNA rekombinan yakni menggunakan plasmidalami dengan menggantikan gen X (kecuali bagian promoternya/arsir kuning) dengangen Cat (chloramphenicol acetyltransferase). Ekspresi dari gen Cat dapatdiuji dari aktivitas Cat yang menghasilkan produk berupa CAM (chloramphenicol).
staff.unila.ac.id/priyambodo
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Enzim Endonuklease Restriksi(Enzim Restriksi)• Endonuklease restriksi mengkatalisis pemotongan DNA pada
daerah tertentu yang mempunyai urutan nukleotida yangspesifik.
• Urutan nukleotida pada DNA yang dapat dibaca sama dari kirike kanan atau sebaliknya disebut urutan palindromik,misalnya urutan 5’ AAGC 3’ akan terbaca sama dengan 3’CGAA 5’.
• Enzim restriksi akan mengenali daerah palindromik danmemutus ikatan fosfodiester dari rantai DNA.
• Endonuklease restriksi memiliki banyak jenis dan masing-masingmemilikiurutan palindromik yang khas.
staff.unila.ac.id/priyambodo
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