Dogma Sentral Biologi

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

staff.unila.ac.id/priyambodo


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. staff.unila.ac.id/priyambodo


29 staff.unila.ac.id/priyambodo

Prinsip dasar dari teknik rekombinan DNA


Pemotongan dan penyambungan DNA pada teknik rekombinan DNA


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).



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.



Konstruksi plasmid vektor pBR322.


Dogma Sentral Biologi

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