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Proteins are important Bcos They are…
~44% of the dry wt. of the human body. ~5% of human caloric intake goes for protein synthesis.
catalyze most of the reactions in living organisms.
serve many roles (enzymatic, structural, transport, regulation, ...)
…in sickness and in health
protein synthesis is tightly regulated by environmental stimuli as well as intrinsic processes (e.g., hormonal, developmental). dysregulation can cause disease. many antibiotics act at the level of protein synthesis.
INTRODUCTION Central Dogma Ribosomes and polysomes Genetic Code Mutations with effects at the translation level
II. TRANSLATIONAL MACHINERY III. MECHANISM OF TRANSLATION AND INHIBITORS OF PROTEIN SYNTHESIS IV. ENERGETICS AND REGULATION OF TRANSLATION
POLYSOMES E. 4 .M.
Francis Crick. or from nucleic acid to protein.or aminoterminus RNA 3’ C. or from protein to nucleic acid is impossible. 1958 5’ N. Information means here the precise determination of sequence.or carboxyterminus 5 protein . but transfer from protein to protein. The transfer of information from nucleic acid to nucleic acid.CENTRAL DOGMA DNA RNA PROTEIN The central dogma states that once “information” has passed into protein it cannot get out again. may be possible. either of bases in the nucleic acid or of amino acid residues in the protein.
Coupled transcription & translation in bacteria [ N terminus to [ 5’ to 3’ ] C terminus ] Not so in Eukaryotes 6 .
1st position (5’ end) 2nd position U Phe Phe Leu Leu Leu Leu Leu Leu Ile Ile Ile Met Val Val Val Val C Ser Ser Ser Ser Pro Pro Pro Pro Thr Thr Thr Thr Ala Ala Ala Ala A Tyr Tyr STOP STOP His HIs Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu G Cys Cys STOP Trp Arg Arg Arg Arg Ser Ser Arg Arg Gly Gly Gly Gly 3rd position (3’ end) GENETIC CODE U C A G U C A G U C A G U C A G U C A G codon # 5 6 7 CCU GAG GAG Pro Glu Glu Normal Hb – β Sickle cell Hb – βS 7 CCU GUG GAG Pro Val Glu .
mycoplasma. change in desired amino acids. etc.GENETIC CODE: Co-linear triplet code Nearly universal – variations in mitochondria. such as. ciliates Degenerate (or redundant) Non-overlapping Unpunctuated – although some codons are signals Mutations . early or late stop. insertion. 8 .in coding region can cause various ill-effects.
elongation and termination enzymes III. Met-tRNA forms (m.I. ENERGETICS AND REGULATION OF TRANSLATION 9 9 . f. MECHANISM OF TRANSLATION AND INHIBITORS OF PROTEIN SYNTHESIS IV. TRANSLATIONAL MACHINERY Ribosomes: prokaryotic / eukaryotic Messenger RNA Transfer RNA Aminoacyl-tRNA synthetases. INTRODUCTION II. i) Initiation.
Energy (ATP. Amino Acids (aa’s) 5.TRANSLATIONAL COMPONENTS 1. Transfer RNAs (tRNAs) 4. GTP) 10 . Enzymes (“factors”) 6. Ribosomes (large and small subunits) 2. Messenger RNA (mRNA) 3.
1. Ribosome Structure 11 .
etc. yellow.: rRNA Blue: Ribosomal proteins White: Nascent polypeptide 12 12 .Section through 50S ribosomal subunit Peptidyl transferase is RNA Polypeptide exit tunnel is 40~50 aa long C: Central protuberance PT: Peptidyl tranferase center Red.
2. mRNA Eukaryotic: Monocistronic (spliced) 5’ end cap 7-MeGpppGXY ( 1 coding region ) 5’ UTR only 1 3’ UTR 3’ end poly A AAA ~150 Cistron = coding region = open reading frame (ORF) Prokaryotic: Polycistronic 5’ ppp #1 ( >1 coding region ) #2 #3 13 3’ .
3. tRNA Translational Adaptor 14 .
6 Kcal/mole aaRS = aminoacyl-tRNA synthetase PPase = pyrophosphatase 15 .4. Amino Acids tRNAs carry “activated” amino acids: aaRS (1) AA + tRNA + ATP PPase AA ~tRNA + AMP + PPi G ~0 Kcal/mole 2 Pi G = -6.6 Kcal/mole (2) PPi + H2O Overall free energy change for aminoacylation of tRNA G ~ -6.
Formation of aminoacyl-tRNA The amino acid is first activated by reacting with ATP The activated amino acid is transferred from aminoacyl-AMP to tRNA These enzymes are vital for the fidelity of protein synthesis: 2 steps allow “proofreading” 16 .
Genetic Code 20 AA’s 61 Codons for AA’s Translation Machinery 20 AA – tRNA synthases ( i. 1 per AA ) ~50 tRNA species (at least 1 per AA.g. but less than 1 per codon) “WOBBLE” Pairing anti-codon stem-loop of tRNA Wobble Position e..e. CUU 1 anti–codon tRNA GAA GAG 2 codons 3’ 5’ ANTI-CODON 3 2 1 5’ mRNA 1 2 3 CODON 17 .
2 tRNAs for AUG / Methionine: 2 different functions N-formyl in bacteria: F-Met Met Met CCA Met – tRNA M CCA Met – tRNA F or I 3’ 5’ UAC AUG 1 5’ 3’ UAC AUG 5’ 3’ Initiation Codon Internal Met Codon 18 .
etc. Elongation EF1.IF3 eIF1.RF3 eRF1.eIF5 (multiple) 2. Termination RF1. Initiation IF1. Translation Factors Translation Step Enzymes Prokaryotes Eukaryotes Charging of tRNA Aminoacyl – tRNA synthetases 1. eEF2 3. eRF3 Modifications. 19 .5. cleavage. EF2 eEF1.
INTRODUCTION II.I. TRANSLATIONAL MACHINERY III. MECHANISM OF TRANSLATION AND INHIBITORS OF PROTEIN SYNTHESIS Initiation Elongation Termination Antibiotics Toxins IV. ENERGETICS AND REGULATION OF TRANSLATION 20 20 .
dependent scanning 40S 16S rRNA prokaryotes 30S cap AUG. Internal ribosome entry 40S AUG. S-D AUG. ---------------IRES----------- Next step: large subunit 50S/60S subunit joining 21 .HOW RIBOSOMES FIND THEIR INITIATION SITES eukaryotes 1...Dalgarno box 2... Shine . Cap .
30S ribosomal subunit initiation at S-D sequence 22 .
Cap . are aminoglycosides. They also cause miscoding during elongation AUG. etc. Gentamycin.. Shine . Amikacin.. Internal ribosome entry 40S Streptomycin..dependent scanning 40S 16S rRNA prokaryotes STREPTOMYCIN 30S cap AUG.Dalgarno box 2. S-D AUG. ---------------IRES----------- 23 .HOW RIBOSOMES FIND THEIR INITIATION SITES eukaryotes 1. Tobramycin..
1B (EF-Tu.g. eEF1βγ ] Peptidyl Transfer CLINDAMYCIN Macrolides e. ERYTHROMYCIN Translocation RICIN -SARCIN GTP PUROMYCIN CHLORAMPHENICOL Peptidyl transferase (50S / 60S) EF2 [eEF2] DIPHTHERIA TOXIN 24 .ELONGATION P Site A Site AA – tRNA binding E Site TETRACYCLINES SPECTINOMYCIN EF 1A. Ts) [eEF 1α.
Puromycin imitates AA-tRNA Puromycin Tyrosinyl-tRNA 25 .
Inhibition of ribosome translocation 1) Diphtheria toxin inactivates eEF2 2) Erythromycin inhibits EF2 26 26 .
3 [eRF1.TERMINATION stop codons UAG UAA UGA Termination & Release RF 1.3] 27 .2.
Charging Initiation Unwinding and scanning Met-tRNAi binding Elongation AA-tRNA binding Translocation ATP. Termination GTP (number unknown). 1~ 4. 1~ (see later) GTP. 1~ TOTAL: 4~ per AA polymerized + initiation + termination > 1200~ for an average protein Compared to 36-38 ATP’s generated by Glucose CO2 28 . 1~ GTP.ENERGETICS OF PROTEIN SYNTHESIS 1. 1~ 3. 2. GTP. 2~ ATP (several).
Down-regulation of the supply of initiator Met-tRNAi via eIF2 eIF2 • GDP eIF2B eIF2 • GTP eIF2 • GTP • Met-tRNAi PROTEIN SYNTHESIS eIF2 supplies Met.tRNAi to 40S subunit 29 .
Control : Down-regulation of the supply of initiator Met-tRNAi via eIF2 kinases kinases eIF2 eIF2 kinases P HRI: reticulocytes minus heme PKR: interferon plus virusinfection (dsRNA) PERK: ER stress GCN2: amino acid starvation eIF2 • GDP eIF2B eIF2 • GTP eIF2B eIF2 P Trapped eIF2B eIF2 • GTP • Met-tRNAi INITIATION INHIBITED PROTEIN SYNTHESIS eIF2 supplies Met.tRNAi to 40S subunit eIF2 phosphorylation inhibits initiation 30 .
tRNA to ribosome during elongation. 31 . 1α 1βγ EF-Tu • GTP • aa-tRNA PROTEIN SYNTHESIS This factor supplies aa. Old New aa-tRNA complex Tu 1A GEF Ts 1B EUK.GTP/GDP exchange during elongation by (e)EF1 (aka EF-Tu) Terminology EF-Tu • GDP EF-Ts EF-Tu • GTP PROK.
nuclear membrane membrane-bound polysome on “rough” ER endoplasmic reticulum lumen secreted protein “free” polysome CYTOPLASM cytosolic protein cell membrane 32 .
60S Inhibit translocation Inhibits isoleucine tRNA charging Premature release of nascent polypeptide Cycloheximide DIPHTHERIA TOXIN RICIN (castor beans) -Sarcin (fungus) 80S eEF2 60S 60S Inhibits translocation Inhibits translocation¤ Inhibits binding of AA-tRNA to A-site♦ Inhibits binding of AA-tRNA to A-site & translocation# CAPITALIZED: most important Catalytic activities of toxins ¤ ADP ribosylation ♦ 28S rRNA depurination (A) # 28S rRNA cleavage 33 . TETRACYCLINE. doxycycline CHLORAMPHENICOL ERYTHROMYCIN.Inhibitors of Protein Synthesis: Antibiotics and Toxins Inhibitor STREPTOMYCIN. Azithromycin Clindamycin. Neomycin. Clarithromycin. Kanamycin. etc. Gentamicin. Lincomycin Mupirocin (pseudomonic acid) PUROMYCIN Class Aminoglycosides Tetracylines Macrolides Target 30S 30S 50S 50S Action (1) Inhibits initiation (2) Causes misreading Inhibits binding of AA-tRNA to A-site Inhibits peptidyl transferase Inhibit translocation Lincosamides 50S Ile-tRNA synthase 50S.
30S) f Met – tRNAi Shine-Dalgarno mediated internal initiation EUKARYOTES Yes Separated Monocistronic.PROKARYOTES Nucleus No Transcription & translation Coupled mRNA Polycistronic 70S (50S. Capped & Polyadenylated 80S (60S. 40S) Met – tRNAi 1) Scanning 2) IRES mediated internal entry >12 1) Met – tRNAi binding 2) mRNA binding Resistant Sensitive 34 Ribosomes Initiator Site selection Initiation factors Order of events 3 1) mRNA binding 2) f Met – tRNAi binding Sensitive Resistant Antibiotics Toxins .
biotinilation. Ser. Phosphorylation .(Proline) in collagen.Protein Modifications 1. farnesyl. Hydroxylation .Threo) Metabolic Regulation.(Tyr. Glycosylation – (O-linked as with Ser/Threo.Mostly thru specific proteases and ubiquitin-proteosome system 35 . etc 2.OH or N-Linked as in lysine) 4. Other . Signal transduction. etc Protein Degradation . Endoplasmic Reticulum 3.
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