Translation in Prokaryote & Eukaryotes. Siripan Limsirichaikul
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1 Translation in Prokaryote & Eukaryotes Siripan Limsirichaikul
2 Translation 1. mrna : RBS, start-stop stop codon 2. Ribosome : rrna + protein 3. trna : trna charging 4. Amino acid : genetic code
3 Transcription Unit or RNA coding region or?gene? Regulatory region *Gene*?Gene? DNA 5 3 terminator 3 Promoter RNA of gene A 5 ( _ UTR xx) (+xx) upstream downstream 3 -UTR 5 3 START codon STOP codon (+ sense) (- antisense) Transcription start site (TSS) (+1) Transcription terminator site
4 1. Messenger RNA : mrna Transcription Start site Transcription terminator site DNA mrna Prokaryote 5 UTR Start codon Stop codon AUG NNN NNN NNN NNN UAA 3 UTR mrna Eukaryote Ribosome Binding Site (RBS) in prokaryote : Shine-dalgarno seq in Eukaryotes : 5 CAP (m 7 G) m 7 G 5 UTR AUG NNN NNN NNN NNN UAA 3 UTR AAAAA Mature mrna Open Reading Frame (ORF) UTR = untranslated region
5 Genetic code : triplet code/ codon Antisense DNA 5 TCC CAG CAT 3 3 AGG GUC GUA 5 mrna 5 AUG CUG GGA 3 Met Leu Gly 4 3 = 64 codons for 20 amino acids + 3 Stop codons
6 Genetic code is not universal
7 Open reading frame Begin from start codon & End at stop codon mrna 5 AUG GCU UGU UUA AGA UAU 3 Frame 1 Protein Met Ala Cys Leu Arg Tyr mrna Protein 5 A UGG CUU GUU UAA GAU AU 3 Trp Leu Val Stop Frame 2 mrna 5 AU GGC UUG UUU AAG AUA U 3 Frame 3 Protein Gly Leu Phe Lys Iso
8 Ribosome Binding Site (RBS) Shine-Dalgarno (SD) seq In prokaryotes Bind to ribosome to initiate translation Locate upstream of start codon (AUG) Consensus seq is AGG AGG
9 Ribosome Binding Site (RBS) In Eukaryotes 5 cap of mrna (7-methyl guanosine cap) pola promote recycling of ribosome Kozak seq : upstream of start (-3)( Not a RBS but promote promote translation initiation
10 2. Ribosome : rrna + protein Small subunit mrna binding site Decode mrna Large subunit : peptidyl E site : Exit peptidyl transferase P site : Peptidyl trna binding site A site : acceptor site
11 Prokayotes / Eukaryotes
12 Cycling of Ribosome in cell
13 3. trna (transfer RNA) 3 End : Acceptor arm Anticodon Loop
14 trna Charging: amino acid activation trna + amino acid = aminoacyl trna STEP 1 : Adenyl-lylation of amino acid STEP 2 : Adenyl-lylated amino acid +trna
15 trna charging : step 2 trna synthetase : tryptophan synthetase Specific trna corresponding amino acid ATP
16 translation Initiation Elongation Termination
17 Translation Initiation in Eukaryotes / Prokaryotes 30s ribosome bind to RBS (ribosome binding site) 5 CAP (Eukaryotes) SD (Prokaryotes) Add first amino acid at start codon (ATG) Met (Eukaryotes) f-met (Prokaryotes)
18 Translation initiation in prokaryote Initiation factor & small subunit ( (16S rrna) ) bind to SD seq Aminoacyl trna bind to start codon (AUG) Large subunit joins complex
19 Small subunit of Ribosome (16s) bind to mrna (SD seq) Translation initiation in prokaryote 16s rrna : 5 CUCCU 3 Shine Dalgarno Seq : 5 AGGAG 3
20 Translation initiation in Eukaryote 1. Preinitiation complex : Initation factor + Met-tRNA trna + small subunit (ternary complex) 2. Initiation complex : add cap-binding complex for binding to 5 5 cap 3. Start codon recognition ternary complex bind to AUG 4. Large subunit (60s) joins complex
21 Translation elongation Direction : 5 3 mrna N C terminal end of protein Peptide bond formation ElongationFactor1α-GTPs aminoacyl trna Peptidyl tranferase in large subunit
22 1. Second aminocyl trna bind to A site 2. GTP hydrolysis ribosome changes conformation. 3. Peptide bond formation : peptidyl transferase 4. Ribosome translocation trna at P E and eject trna at A P
23 Translation elongation
24 GTP
25 Translation Termination Releasing factor at A-site A at STOP codon no trna code stop codon Stop codon Ocher : UAA Amber : UAG Opal : UGA
26 Circular Polysomes and Recycling ribosome
27 Post translational modifications Cleavage of initiator methionine f-met in prokaryotes Disulfide bond formation/ folding Chemical modification Glycosylation Acetylation Phosphorylation Note: for Host selection in genetic engineering, and modified for functional protein
28
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