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SINTESIS PROTEIN Sri Puji Astuti Wahyuningsih FST UNAIR BIODAS II Dept. Biologi FST UNAIR ARUS INFORMASI GENETIK Informasi DNA adalah dalam bentuk urutan tertentu dari nukleotida di sepanjang untai DNA DNA diwariskan oleh organisme ◦ Menghasilkan ciri-ciri tertentu melalui sintesis protein Proses di mana DNA mensintesis protein dinamakan ekspresi gen ◦ Ada dua tahapan, yaitu transkripsi dan translasi BIODAS II Dept. Biologi FST UNAIR RIBOSOM ◦ Adalah organel sel yang menfasilitasi terjadinya translasi (sintesis polipeptida) Figure 17.1 BIODAS II Dept. Biologi FST UNAIR Konsep 1: GEN MENENTUKAN PROTEIN MELALUI TRANSKRIPSI DAN TRANSLASI 1909, Dokter Inggris Archibald Garrod ◦ Adalah orang pertama yang menunjukkan bahwa gen menentukan fenotip melalui enzim yang mengkatalisis reaksi kimia tertentu dalam sel 1920, Beadle dan Edward Tatum: Neurospora crassa (kapang roti) yang dimutasi dengan sinar-X ◦ Menciptakan mutan yang tidak bisa bertahan hidup pada media minimal ◦ Mengembangkan hipotesis "satu gen-satu enzim“ ◦ Fungsi gen adalah untuk menentukan produk enzim tertentu BIODAS II Dept. Biologi FST UNAIR PRINSIP DASAR TRANSKRIPS DAN TRANSLASI Transkripsi ◦ Adalah sintesis RNA di bawah arahan DNA ◦ Menghasilkan messenger RNA (mRNA) atau RNA duta Translasi ◦ Adalah sintesis polipeptida yang terjadi di bawah arahan mRNA ◦ Terjadi dalam ribosom BIODAS II Dept. Biologi FST UNAIR Pada Prokariota ◦ Transkripsi dan translasi terjadi bersamaan (translasi mRNA dimulai saat transkripsi masih berlangsung) TRANSCRIPTION DNA mRNA Ribosome TRANSLATION Polypeptide Figure 17.3a (a) Prokaryotic cell. In a cell lacking a nucleus, mRNA produced by transcription is immediately translated without additional processing. BIODAS II Dept. Biologi FST UNAIR Pada Figure 17.3b Eukariota ◦ Transkrip RNA atau transkrip primer atau pre-mRNA dimodifikasi sebelum menjadi mRNA Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION Polypeptide (b) Eukaryotic cell. The nucleus provides a separate compartment for transcription. The original RNA transcript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA. BIODAS II Dept. Biologi FST UNAIR Aliran informasi genetik adalah ◦ DNA RNA protein Aliran Informasi Genetik ◦ Dikodekan sebagai urutan triplet basa yang tidak tumpang tindih yang disebut kodon BIODAS II Dept. Biologi FST UNAIR Selama transkripsi ◦ Gen menentukan urutan basa di sepanjang molekul mRNA Gene 2 DNA molecule Gene 1 Gene 3 DNA strand (template) 3 A C C A A A C C G A G T U G G U U U G G C U C A 5 TRANSCRIPTION mRNA 5 3 Codon TRANSLATION Protein Figure 17.4 Trp Phe Gly Ser Amino acid BIODAS II Dept. Biologi FST UNAIR KODE GENETIK Kodon dalam mRNA Figure 17.5 Second mRNA base U C A G UAU UUU UCU UGU Tyr Cys Phe UAC UUC UCC UGC U UUA UCA Ser UAA Stop UGA Stop Leu UAG Stop UGG Trp UUG UCG CUU CUC C CUA CUG AUU AUC A AUA AUG GUU G GUC GUA GUG Leu lle Met or start Val CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG U C A G U CGU CGC C Arg CGA A CGG G U AGU Ser C AGC A AGA Arg G AGG Pro CAU His CAC CAA Gln CAG Thr AAU Asn AAC AAA Lys AAG Ala U GAU GGU Asp C GAC GGC Gly GAA GGA A Glu GAG GGG G Third mRNA base (3 end) First mRNA base (5 end) ◦ Diterjemahkan menjadi asam amino atau berfungsi sebagai sinyal mulai (start) dan berhenti (stop) dari translasi BIODAS II Dept. Biologi FST UNAIR Konsep 2: TRANSKRIPSI ADALAH SINTESIS RNA YANG DIARAHKAN DNA Komponen Molekular Transkripsi Sintesis RNA dikatalisis oleh RNA polimerase. Berfungsi untuk memisahkan untai DNA dan menghasilkan nukleotida RNA dari arah 5’ 3’ Prokariota hanya mempunyai 1 jenis RNA polimerase. Eukatiota memiliki 3 jenis RNA polimerase, yang digunakan untuk sintesis mRNA adalah RNA polimerase II BIODAS II Dept. Biologi FST UNAIR Sintesis Transkrip RNA Tahapan transkripsi ◦ Inisiasi ◦ Elongasi ◦ Terminasi 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 Figure 17.7 Completed RNA transcript 3 BIODAS II Dept. Biologi FST UNAIR Pengikatan RNA Polimerase dan Inisiasi Transkripsi TRANSCRIPTION RNA PROCESSING 1 Eukaryotic promoters DNA Pre-mRNA mRNA Promotor : sekuen DNA tempat melekatnya RNA polimerase dan tempat inisiasi untuk sintesis RNA Ribosome TRANSLATION Polypeptide Promoter 5 3 3 5 T A T A A A A A T A T T T T TATA box Start point Template DNA strand Several transcription factors 2 Transcription factors Faktor Transkripsi 5 3 3 5 3 Additional transcription factors ◦ Membantu RNA polimerase untuk mengenali sekuen promoter pada Eukariota RNA polymerase II Transcription factors 5 3 3 5 5 RNA transcript FigureFigure 17.8 17.8 Transcription initiation complex BIODAS II Dept. Biologi FST UNAIR Non-template strand of DNA Elongation RNA nucleotides RNA polymerase A T C C A A 3 3 end U 5 A E G C A T A G G T T Direction of transcription (“downstream”) 5 Template strand of DNA Newly made RNA BIODAS II Dept. Biologi FST UNAIR Elongasi Transkripsi RNA polimerase bergerak sepanjang DNA ◦ Enzim menguraikan DNA double helix, sekitar 1020 basa DNA dibuka dan dibaca untuk menghasilkan pasangannya, yaitu nukleotida RNA Terminasi Transkripsi RNA polimerase telah selesai membaca DNA. Telah dihasilkan seluruh untaian nukleotida RNA BIODAS II Dept. Biologi FST UNAIR Konsep 3: SEL EUKARIOTIK MEMODIFIKASI RNA SETELAH TRANSKRIPSI Enzim di inti sel pada eukariotik ◦ Modifikasi pre-mRNA dengan cara tertentu sebelum pesan genetik dikirim ke sitoplasma BIODAS II Dept. Biologi FST UNAIR Pengubahan ujung mRNA Setiap ujung molekul pre-mRNA dimodifikasi dengan cara tertentu ◦ Ujung 5 dimodifikasi dengan penambahan nukeotida tudung (cap) ◦ Ujung 3 dengan penambahan ekor poli-A 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 G Protein-coding segment Polyadenylation signal 3 P P P AAUAAA AAA…AAA Ribosome TRANSLATION 5 Cap 5 UTR Start codon Stop codon 3 UTR Poly-A tail Polypeptide Figure 17.9 BIODAS II Dept. Biologi FST UNAIR Split Genes dan RNA Splicing Penyambungan RNA / RNA splicing ◦ Memindahkan intron dan menggabungkan exon TRANSCRIPTION RNA PROCESSING DNA 5 Exon Intron Pre-mRNA 5 Cap 30 31 1 Intron Exon Exon 3 Poly-A tail 104 105 146 Pre-mRNA Coding segment mRNA Ribosome Introns cut out and exons spliced together TRANSLATION Polypeptide mRNA 5 Cap 1 3 UTR Poly-A tail 146 3 UTR Figure 17.10 BIODAS II Dept. Biologi FST UNAIR Dilakukan oleh spliceosome, yaitu enzim yang berfungsi untuk memindahkan intron dan menggabungkan exon RNA transcript (pre-mRNA) 5 Intron Exon 1 Exon 2 Protein 1 Other proteins snRNA snRNPs Spliceosome 2 5 Spliceosome components Figure 17.11 3 5 mRNA Exon 1 Cut-out intron Exon 2 BIODAS II Dept. Biologi FST UNAIR Fungsi dan Pentingnya Intron Adanya intron ◦ Memungkinkan satu gen dapat mengkode lebih dari satu jenis polipeptida BIODAS II Dept. Biologi FST UNAIR Protein sering memiliki arsitektur modular ◦ Terdiri dari daerah struktural dan fungsional yang disebut domain ◦ Kode exon akan berbeda untuk domain yang berbeda pada protein Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Figure 17.12 Polypeptide BIODAS II Dept. Biologi FST UNAIR Konsep 4: TRANSLASI ADALAH SINTESIS POLIPEPTIDA YANG DIARAHKAN OLEH RNA Komponen Molekular Translasi Sel mentranslasi pesan mRNA menjadi protein dengan bantuan RNA transfer (tRNA) BIODAS II Dept. Biologi FST UNAIR Konsep Dasar Translasi TRANSCRIPTION DNA mRNA Ribosome TRANSLATION Polypeptide Amino acids Polypeptide Ribosome tRNA with amino acid attached Gly tRNA Anticodon A A A U G G U U U G G C Codons 5 Figure 17.13 3 mRNA BIODAS II Dept. Biologi FST UNAIR Struktur dan Funsi RNA Transfer Molekul tRNA A ◦ Tersusun dari untai RNA tunggal yang panjangnya hanya C C sekitar 80 nukleotida 3 A Amino acid ◦ Berbentuk L C attachment site (a) Two-dimensional structure. The four base-paired regions and three loops are characteristic of all tRNAs, as is the base sequence of the amino acid attachment site at the 3 end. The anticodon triplet is unique to each tRNA type. (The asterisks mark bases that have been chemically modified, a characteristic of tRNA.) Figure 17.14a C A C G C U U A A U C C A C AG * G G U G U * C * * U C * G AG G U * * A * A 5 G C G G A U U U A * C U C C G A G * C C A G A A G * * G A G G Hydrogen bonds C U G A Anticodon BIODAS II Dept. Biologi FST UNAIR Amino acid attachment site 5 3 Hydrogen bonds A A G 3 Anticodon (b) Three-dimensional structure 5 Anticodon (c) Symbol used in this book Figure 17.14b BIODAS II Dept. Biologi FST UNAIR Enzim aminoacyl-tRNA synthetase ◦ Menggabungkan masing-masing asam amino ke tRNA yang benar Amino acid P P Aminoacyl-tRNA synthetase (enzyme) 1 Active site binds the amino acid and ATP. P Adenosine ATP 2 ATP loses two P groups and joins amino acid as AMP. P Pyrophosphate Pi Phosphates P Adenosine Pi Pi tRNA 3 Appropriate tRNA covalently Bonds to amino Acid, displacing AMP. P Adenosine AMP 4 Activated amino acid is released by the enzyme. Figure 17.15 Aminoacyl tRNA (an “activated amino acid”) BIODAS II Dept. Biologi FST UNAIR Ribosom ◦ Menfasilitasi ikatan antikodon tRNA dengan kodon mRNA selama sintesis protein DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Subunit ribosomal ◦ Disusun oleh protein dan molekul RNA yang dinamakan RNA ribosomal atau rRNA Polypeptide Exit tunnel Growing polypeptide tRNA molecules Large subunit E P A Small subunit 5 mRNA (a) 3 Computer model of functioning ribosome. This is a model of a bacterial ribosome, showing its overall shape. The eukaryotic ribosome is roughly similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules and proteins. Figure 17.16a BIODAS II Dept. Biologi FST UNAIR Ribosom mempunyai 3 sisi pengikatan (binding sites) tRNA ◦ Sisi P ◦ Sisi A ◦ Sisi E P site (Peptidyl-tRNA binding site) A site (AminoacyltRNA binding site) E site (Exit site) Large subunit E mRNA binding site P A Small subunit (b) Schematic model showing binding sites. A ribosome has an mRNA binding site and three tRNA binding sites, known as the A, P, and E sites. This schematic ribosome will appear in later diagrams. Figure 17.16b BIODAS II Dept. Biologi FST UNAIR Amino end Growing polypeptide Next amino acid to be added to polypeptide chain tRNA 3 mRNA 5 Codons (c) Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site. Figure 17.16c BIODAS II Dept. Biologi FST UNAIR Pembentukan Polipeptida Kita dapat membagi translasi menjadi 3 tahapan ◦ Inisiasi ◦ Elongasi ◦ Terminasi BIODAS II Dept. Biologi FST UNAIR Asosiasi Ribosom dan Inisiasi Translasi Tahap inisiasi translasi ◦ Menyatukan mRNA, tRNA yang membawa asam amino pertama dari polipeptida, dan dua subunit ribosom Large ribosomal subunit P site 3 U A C 5 5 A U G 3 Initiator tRNA GTP GDP E A mRNA 5 Start codon mRNA binding site 1 Figure 17.17 5 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). 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. BIODAS II Dept. Biologi FST UNAIR Elongasi Translasi ◦ Asam amino ditambahkan satu per satu ke asam amino sebelumnya 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 P A GDP 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. 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. BIODAS II Dept. Biologi FST UNAIR Terminasi Translasi Tahap akhir translasi adalah ketika ribosom sampai kodon stop pada 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 BIODAS II Dept. Biologi FST UNAIR Poliribosom Sejumlah ribosom dapat mentranslasi satu molekul mRNA tunggal secara simultan. Hanya pada sel prokariota Completed polypeptide Growing polypeptides Incoming ribosomal subunits Start of mRNA (5 end) End of mRNA (3 end) (a) An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. Ribosomes mRNA 0.1 µm Figure 17.20a, b (b) This micrograph shows a large polyribosome in a prokaryotic cell (TEM). BIODAS II Dept. Biologi FST UNAIR Protein Fungsional Rantai polipeptida /protein menjalani modifikasi setelah proses translasi Protein dimodifikasi - untuk membentuk molekul tiga dimensi / folding (pelipatan) - Untuk dapat ditranfer ke lokasi yang membutuhkan BIODAS II Dept. Biologi FST UNAIR Protein dibutuhkan dalam sistem endomembran (contoh: enzim lisosim yang ada di organel lisosom) atau disekresikan (contoh: enzim pencernaan seperti amilase). Protein harus ditransport ke retikulum endoplasma (ER) Signal peptida pada ribosom binding pada signal-recognition particle (SRP). Kompleks keduanya binding pada protein reseptor SRP di ER BIODAS II Dept. Biologi FST UNAIR Mekanisme signal untuk protein target pada ER 1 Polypeptide synthesis begins on a free ribosome in the cytosol. 2 An SRP binds to the signal peptide, halting synthesis momentarily. 3 The SRP binds to a receptor protein in the ER membrane. This receptor is part of a protein complex (a translocation complex) that has a membrane pore and a signal-cleaving enzyme. 4 The SRP leaves, and the polypeptide resumes growing, meanwhile translocating across the membrane. (The signal peptide stays attached to the membrane.) 5 The signalcleaving enzyme cuts off the signal peptide. 6 The rest of the completed polypeptide leaves the ribosome and folds into its final conformation. Ribosome mRNA Signal peptide Signalrecognition particle (SRP) SRP receptor CYTOSOL protein ERLUMEN Figure 17.21 Translocation complex Signal peptide removed ER membrane Protein Ringkasan transkripsi dan translasi pada sel eukariotik DNA TRANSCRIPTION 1 RNA is transcribed from a DNA template. 3 5 RNA transcript RNA polymerase Exon RNA PROCESSING 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 FORMATION OF INITIATION COMPLEX CYTOPLASM AMINO ACID ACTIVATION tRNA 3 After leaving the 4 Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. nucleus, mRNA attaches to the ribosome. mRNA Growing polypeptide Activated amino acid Ribosomal subunits 5 TRANSLATION 5 E A AAA UGGUU UA U G Codon Figure 17.26 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.) BIODAS II Dept. Biologi FST UNAIR 39