Dogma Sentral Biologi

Transkripsi

1 Priyambodo, M.Sc.

2

3 Dogma Sentral Biologi

4 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.

5 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.

6 Degenerasi Kode Genetik

7 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.

8 Ekspresi Gen

9 Ekspresi Gen pada Eukariotik dan Prokariotik

10 Ekspresi Gen pada Eukariotik DNA TRANSCRIPTION 1 RNA is transcribed from a DNA template. RNA transcript 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 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.)

11 Rantai DNA Sense

12 Transkripsi Promoter Transcription unit 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 Unwound DNA Template strand of DNA transcript 2 Elongation. The polymerase moves downstream, unwinding the DNA and elongating the RNA transcript 3. In the wake of transcription, the DNA strands re-form a double helix. Rewound RNA RNA RNA transcript 3 Termination. Eventually, the RNA transcript is released, and the polymerase detaches from the DNA. Completed RNA transcript

13 Fase Inisiasi Transkripsi 1 Eukaryotic promoters TRANSCRIPTION DNA RNA PROCESSING Pre-mRNA mrna Ribosome TRANSLATION Polypeptide Promoter T A T A A AA AT AT T T T TATA box Start point Template DNA strand Several transcription factors 2 Transcription factors 3 Additional transcription factors RNA polymerase II Transcription factors RNA transcript Figure 17.8 Transcription initiation complex

14 Mekanisme post-transkripsi A modified guanine nucleotide added to the end TRANSCRIPTION RNA PROCESSING 50 to 250 adenine nucleotides added to the end DNA Pre-mRNA mrna Protein-coding segment Polyadenylation signal G P P P AAUAAA AAA AAA Ribosome TRANSLATION Cap Polypeptide UTR Start codon Stop codon UTR Poly-A tail

15 Mekanisme post-transkripsi TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA Exon Intron Pre-mRNA Cap Coding segment mrna Ribosome Intron Exon Exon Poly-A tail Introns cut out and exons spliced together TRANSLATION Polypeptide mrna Cap 1 UTR Poly-A tail 146 UTR

16 Mekanisme post-transkripsi RNA transcript (pre-mrna) Intron Exon 1 Exon 2 Protein 1 Other proteins snrna snrnps Spliceosome 2 Spliceosome components 3 mrna Exon 1 Exon 2 Cut-out intron RNA splicing Is carried out by spliceosomes in some cases

17 Macam RNA

18 Translasi Free amino acids Growing Protein Chain AA:tRNA free trna mrna Ribosome Codon Codon for specific AA Anti-codon

19 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 mrna

20 RNA Transfer A trna molecule Consists of a single RNA strand that is only about 80 nucleotides long A Amino acid Is roughly L-shaped C attachment site C A 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 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.)

21 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 U A C A U G Initiator trna Large ribosomal subunit GTP GDP E A mrna Start codon mrna binding site 1 Figure 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). 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.

22 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 P A site site 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 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.

23 Termination of Translation The final stage of translation is termination When the ribosome reaches a stop codon in the mrna Release factor Free polypeptide 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

24 REKOMBINASI DNA

25 Kunang-kunang Luciferase staff.unila.ac.id/priyambodo

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27 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

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30 Prinsip dasar dari teknik rekombinan DNA 30 staff.unila.ac.id/priyambodo

31 Pemotongan dan penyambungan DNA pada teknik rekombinan DNA 31 staff.unila.ac.id/priyambodo

32 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). 32 staff.unila.ac.id/priyambodo

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34 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. 34 staff.unila.ac.id/priyambodo

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36 Konstruksi plasmid vektor pbr staff.unila.ac.id/priyambodo