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Priyambodo, M.Sc.

Dogma Sentral Biologi

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.

Degenerasi Kode Genetik

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

Ekspresi Gen pada Eukariotikdan Prokariotik

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

Rantai DNA Sense

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

Macam RNA

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

REKOMBINASI DNA

Luciferase

Kunang-kunang

staff.unila.ac.id/priyambodo

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.


29staff.unila.ac.id/priyambodo

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Prinsip dasar dariteknik rekombinanDNA


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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 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).


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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.


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Konstruksi plasmidvektor pBR322.


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