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TRANSLASI

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TRANSLASI

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  1. TRANSLASI Dr. YektiAsihPurwestri, M.Si.

  2. Asam amino danikatanpeptida • Ada 20 asam amino yang dikodeoleh DNA • Semuamempunyagugus amino group (-NH2) danguguskarboksil (-COOH). Terikatpada C sentralnyaadalahrantaisamping R. Gugusasampadaasamminoterikatpadagugus amino padaasam amino berikutnya, membentukikatanpeptida

  3. Dari gen menjadi protein DNA Transcription mRNA Translation Sequence of a.a Primary structure of protein

  4. Translasi merupakanprosespembacaankodondanmenggabungkanasam amino melaluiikatanpeptida • Komponenprosestranslasi • mRNA  tersusunataskodegenetik • Ribosom • tRNAbersamadenganasam amino • Enzim2

  5. Tahapprosestranslasi • Inisiasi • Elongasi • Terminasi Inisiasi Aktivasiasam amino untukbergabungmembentuk protein

  6. Activation of amino acids for incorporation into proteins.

  7. Genetic code  Three nucleotides - codon - code for one amino acid in a protein Codon  sequence of three nucleotides in a mRNA that specifies the incorporation of a specific amino acid into a protein. The relationship between codons and the amino acids they code for is called the genetic code.

  8. Not all codons are used with equal frequency. There is a considerable amount of variation in the patterns of codon usage between different organisms.

  9. KodeGenetik • Masing2 kelompok yang terdiridari 3 nukleotidapada mRNA disebutkodon. Karena 4 basa , kodon yang terbentukadalah 43 = 64, yang harusmengkode 20 asam amino yang berbeda. • Lebihdarisatukodondigunakanuntukbanyakasam amino : kodegenetikbersifat “degenerate”. Hal iniberartibahwatidakmungkinmengambilsekuen protein danmenterjemahkannyakedalamsekuenbasa gen tersebut. • Dalambanyakhal, basaketigadarikodon (the wobble base) dapatdiubahtanpamengubahasamaminonya. • AUG digunakansebagai start kodon. Awaltranslasisemua protein adalahmetionin, meskipunseringdihilangkansetelahtranslasi. Adajuga internal metionin yang dikodeolehkodon AUG yang sama. • Terdapat 3 stop kodon, yang disebut “nonsense” kodon. Protein berakhirdengan stop kodon yang tidakmengkodesuatuasam amino

  10. KodeGenetik • Kebanyakankodegenetikbersifat universal. Digunakanbaikpadaprokariotmaupuneukariot. • Namun, beberapavariandijumpai, terutamapadamitokondria yang hanyamemilikisedikit gen. • Contoh: CUA umumnyamengkode, tetapipadamitokondria yeast mengkodetreonin. AGA umumnyamengkodearginin, tetapipadamitokondriamanusiadan Drosophila merupakan stop kodon.

  11. Wobble Hypothesis

  12. Relationships of DNA to mRNA to polypeptide chain.

  13. Translation is accomplished by the anticodon loop of tRNA forming base pairs with the codon of mRNA in ribosomes

  14. composed of  a nucleic acid and a specific amino acid  provide the link between the nucleic acid sequence of mRNA and the amino acid sequence it codes for. An anticodon a sequence of 3 nucleotides in a tRNA that is complementary to a codon of mRNA Transfer RNA (tRNA) Structure of tRNAs

  15. Transfer RNA • Transfer RNA molecules are short RNAs that fold into a characteristic cloverleaf pattern. Some of the nucleotides are modified to become things like pseudouridine and ribothymidine. • Each tRNA has 3 bases that make up the anticodon. These bases pair with the 3 bases of the codon on mRNA during translation. • Each tRNA has its corresponding amino acid attached to the 3’ end. A set of enzymes, the “aminoacyltRNAsynthetases”, are used to “charge” the tRNA with the proper amino acid. • Some tRNAs can pair with more than one codon. The third base of the anticodon is called the “wobble position”, and it can form base pairs with several different nucleotides.

  16. Only tRNAfMetis accepted to form the initiation complex. • All further charged tRNAs require fully assembled (i.e., 70S) ribosomes • The Shine-Dalgarnosequence  help ribosomes and mRNA aligns correctly for the start of translation. • Ribosome consists of • A site aminoacyl • P site  peptidyl • - E site exit • Two initiation factors (IF1 &IF3) bind to a 70S ribosome. • promote the dissociation of 70S ribosomes into free 30S and 50S subunits. • mRNA and IF2, which carries • GTP • the charged tRNA • bind to a free 30S subunit. •  After these have all bound, the 30S initiation complex is complete.

  17. Peptide bond formation catalyzed by an enzyme complex called peptidyltransferase Peptidyltransferase consists of some ribosomal proteins and the ribosomal RNA  acts as a ribozyme. The process is repeated until a termination signal is reached.

  18. Termination of translationoccurs when one of the stop codons (UAA, UAG, or UGA) appears in the A site of the ribosome. No tRNAs correspond to those sequences, so no tRNA is bound during termination. Proteins called release factors participate in termination

  19. Posttranslational Processing of Proteins • Folding • Amino acid modification (some proteins) • Proteolytic cleavage FOLDING Before a newly translated polypeptide can be active, it must be folded into the proper 3-D structure and it may have to associate with other subunits.

  20. Enzymes/protein involve in folding process 1. Cis-trans isomerase for proline  Proline is the only amino acid in proteins  forms peptide bonds in which the trans isomer is only slightly favored (4 to 1 versus 1000 to 1 for other residues). Thus, during folding, there is a significant chance that the wrong proline isomer will form first. Cells have enzymes to catalyze the cis-trans isomerization necessary to speed correct folding. 2. disulfide bond making enzymes 3. Chaperonins (molecular chaperones)  a protein to help keep it properly folded and non-aggregated.

  21. Insulin is synthesized  single polypeptide preproinsulin has leader sequence (help it be transported through the cell membrane) Specific protease cleaves leader sequence proinsulin. Proinsulin folds into specific 3D structure and disulfide bonds form Another protease cuts molecule insulin 2 polypeptide chains

  22. Chaperones Function to keep a newly synthesized protein from either improperly folding or aggregating After synthesized, protein needs to fold in order to have its function The folding pattern is dictated in the amino acid sequence of the protein. • Some proteins capable to fold into its proper 3-D structure by itself without any help of other molecules • Some proteins need chaperones to fold (example in human hsp 70) • Some proteins need bigger protein  chaperonins to be able to fold correctly. Chaperonins  a polysubunit protein form “a cage” like shape  give micro environment to protein

  23. Protein Targeting Nascent proteins  contain signal sequence  determine their ultimate destination. Bacteria  newly synthesized protein can: stay in the cytosol, send to the plasma membran, outer membrane, periplasmic, extracellular. Eukaryotes  can direct proteins to internal sites  lysosomes, mitochondria etc. Nascent polypeptide E.R and glycosylated  golgi complex and modified  sorted for delivery to lysosomes, secretory vesicle and plasma membrane.

  24. Translocation • The protein to be translocated (called a pro-protein) is complexed in the cytoplasm with a chaperone • The complex keeps the protein from folding prematurely, which would prevent it from passing through the secretory porean ATPase that helps drive the translocation • after the pro-protein is translocated, the leader peptide is cleaved by a membrane-bound protease and the protein can fold into its active 3-d form.

  25. Signal recognition particle (SRP) detects signal sequence and brings ribosome to the ER membrane

  26. Most mitochondrial proteins are synthesized in the cytosol and imported into the organelle

  27. Initiation of Translation • In prokaryotes, ribosomes bind to specific translation initiation sites. There can be several different initiation sites on a messenger RNA: a prokaryotic mRNA can code for several different proteins. Translation begins at an AUG codon, or sometimes a GUG. The modified amino acid N-formyl methionine is always the first amino acid of the new polypeptide. • In eukaryotes, ribosomes bind to the 5’ cap, then move down the mRNA until they reach the first AUG, the codon for methionine. Translation starts from this point. Eukaryotic mRNAs code for only a single gene. (Although there are a few exceptions, mainly among the eukaryotic viruses). • Note that translation does not start at the first base of the mRNA. There is an untranslated region at the beginning of the mRNA, the 5’ untranslated region (5’ UTR).

  28. More Initiation • The initiation process involves first joining the mRNA, the initiator methionine-tRNA, and the small ribosomal subunit. Several “initiation factors”--additional proteins--are also involved. The large ribosomal subunit then joins the complex.

  29. Elongation • The ribosome has 2 sites for tRNAs, called P and A. The initial tRNA with attached amino acid is in the P site. A new tRNA, corresponding to the next codon on the mRNA, binds to the A site. The ribosome catalyzes a transfer of the amino acid from the P site onto the amino acid at the A site, forming a new peptide bond. • The ribosome then moves down one codon. The now-empty tRNA at the P site is displaced off the ribosome, and the tRNA that has the growing peptide chain on it is moved from the A site to the P site. • The process is then repeated: • the tRNA at the P site holds the peptide chain, and a new tRNA binds to the A site. • the peptide chain is transferred onto the amino acid attached to the A site tRNA. • the ribosome moves down one codon, displacing the empty P site tRNA and moving the tRNA with the peptide chain from the A site to the P site.

  30. Elongation

  31. Termination • Three codons are called “stop codons”. They code for no amino acid, and all protein-coding regions end in a stop codon. • When the ribosome reaches a stop codon, there is no tRNA that binds to it. Instead, proteins called “release factors” bind, and cause the ribosome, the mRNA, and the new polypeptide to separate. The new polypeptide is completed. • Note that the mRNA continues on past the stop codon. The remaining portion is not translated: it is the 3’ untranslated region (3’ UTR).

  32. Post-Translational Modification • New polypeptides usually fold themselves spontaneously into their active conformation. However, some proteins are helped and guided in the folding process by chaperone proteins • Many proteins have sugars, phosphate groups, fatty acids, and other molecules covalently attached to certain amino acids. Most of this is done in the endoplasmic reticulum. • Many proteins are targeted to specific organelles within the cell. Targeting is accomplished through “signal sequences” on the polypeptide. In the case of proteins that go into the endoplasmic reticulum, the signal seqeunce is a group of amino acids at the N terminal of the polypeptide, which are removed from the final protein after translation.