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1 Lectures 33-34 GENETIC CODE and PROTEIN SYNTHESIS Mukund Modak, Ph.D.. Adapted from M. Mathews, Ph.D. LecLtures 33 and 34
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2 Proteins are important… ~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. 2
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3 I.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 3
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4 POLYSOMES E.M.
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5 The central dogma states that once “information” has passed into protein it cannot get out again. The transfer of information from nucleic acid to nucleic acid, or from nucleic acid to protein, may be possible, but transfer from protein to protein, or from protein to nucleic acid is impossible. Information means here the precise determination of sequence, either of bases in the nucleic acid or of amino acid residues in the protein. Francis Crick, 1958 DNARNAPROTEIN CENTRAL DOGMA N- or amino- terminus C- or carboxy- terminus 5’5’ 3’ RNA protein
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6 Coupled transcription & translation in bacteria [ 5’ to 3’ ] [ N terminus to C terminus ] Not so in Eukaryotes
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7 U C A G Phe Leu Ser Tyr STOP Cys STOP Trp Leu Pro His HIs Gln Arg Ile Met Thr Asn Lys Ser Arg Val Ala Asp Glu Gly 1 st position (5’ end) 2 nd position3 rd position (3’ end) UCAG UCAGUCAG UCAGUCAG UCAGUCAG UCAGUCAG GENETIC CODE Normal Hb – β Sickle cell Hb – β S CCU Pro GAG Glu GAG Glu CCU Pro GAG Glu GUG Val 56 7 codon #
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8 Degenerate (or redundant) GENETIC CODE: Nearly universal – variations in mitochondria, mycoplasma, ciliates Unpunctuated – although some codons are signals Non-overlapping Co-linear triplet code Mutations - in coding region can cause various ill-effects, such as, change in desired amino acids, early or late stop, insertion, etc.
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9 I.INTRODUCTION II.TRANSLATIONAL MACHINERY Ribosomes: prokaryotic / eukaryotic Messenger RNA Transfer RNA Aminoacyl-tRNA synthetases; Met-tRNA forms (m, f, i) Initiation, elongation and termination enzymes III.MECHANISM OF TRANSLATION AND INHIBITORS OF PROTEIN SYNTHESIS IV. ENERGETICS AND REGULATION OF TRANSLATION 99
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10 1. Ribosomes (large and small subunits) 2. Messenger RNA (mRNA) 3. Transfer RNAs (tRNAs) 4. Amino Acids (aa’s) 5. Enzymes (“factors”) 6. Energy (ATP, GTP) TRANSLATIONAL COMPONENTS
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11 1. Ribosome Structure
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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, yellow, etc.: rRNA Blue: Ribosomal proteins White: Nascent polypeptide 12
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13 2. mRNA Eukaryotic: cap only 1 AAA ~150 5’ UTR 7-MeGpppGXY 3’ UTRpoly A 5’ end 3’ end Monocistronic (spliced) 5’3’ ppp ( >1 coding region ) Polycistronic Prokaryotic: # 1 # 2 # 3 ( 1 coding region ) Cistron = coding region = open reading frame (ORF)
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14 3. tRNA Translational Adaptor
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15 AA + tRNA + ATP Overall free energy change for aminoacylation of tRNA AA ~tRNA + AMP + PP i PP i + H 2 O2 P i G = -6.6 Kcal/mole G ~ -6.6 Kcal/mole G ~0 Kcal/mole (1) (2) tRNAs carry “activated” amino acids: aaRS PPase aaRS = aminoacyl-tRNA synthetase PPase = pyrophosphatase 4. Amino Acids
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16 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”
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17 Genetic Code 61 Codons for AA’s 20 AA’s Translation Machinery 20 ~50 AA – tRNA synthases ( i.e., 1 per AA ) tRNA species (at least 1 per AA, but less than 1 per codon) CODONmRNA 123 1235’ ANTI-CODON tRNA 3’ “WOBBLE” Pairing GAA GAG 2 codons anti-codon stem-loop of tRNA Wobble Position e.g. CUU 1 anti–codon
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18 2 tRNAs for AUG / Methionine: 2 different functions N-formyl in bacteria: F-Met Initiation Codon Internal Met Codon 1 5’ 3’ AUG UAC CCA Met CCA Met 5’ 3’ Met – tRNA F or I Met – tRNA M
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19 Translation Step Charging of tRNA 1. Initiation 2. Elongation 3. Termination Modifications, cleavage, etc. Enzymes ProkaryotesEukaryotes Aminoacyl – tRNA synthetases IF1- IF3 eIF1- eIF5 (multiple) EF1, EF2 eEF1, eEF2 RF1- RF3 eRF1, eRF3 5. Translation Factors
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20 I.INTRODUCTION II.TRANSLATIONAL MACHINERY III.MECHANISM OF TRANSLATION AND INHIBITORS OF PROTEIN SYNTHESIS Initiation Elongation Termination Antibiotics Toxins IV. ENERGETICS AND REGULATION OF TRANSLATION 20
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21 2. Internal ribosome entry AUG.. HOW RIBOSOMES FIND THEIR INITIATION SITES 1. Cap - dependent scanning cap AUG... AUG.. Shine - Dalgarno box 40S 30S S - D eukaryotes prokaryotes ---------------IRES----------- 16S rRNA Next step: large subunit 50S/60S subunit joining
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22 30S ribosomal subunit initiation at S-D sequence
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23 2. Internal ribosome entry AUG... HOW RIBOSOMES FIND THEIR INITIATION SITES 1. Cap - dependent scanning cap AUG.. Shine - Dalgarno box 40S 30S S - D STREPTOMYCIN eukaryotes prokaryotes ---------------IRES----------- Streptomycin, Gentamycin, Tobramycin, Amikacin, etc. are aminoglycosides. They also cause miscoding during elongation 16S rRNA
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24 CLINDAMYCIN Macrolides e.g. ERYTHROMYCIN TETRACYCLINES SPECTINOMYCIN AA – tRNA binding EF 1A, 1B (EF-Tu, Ts) [eEF 1 α, eEF1 βγ ] Peptidyl Transfer Peptidyl transferase (50S / 60S) Translocation EF2 [eEF2] DIPHTHERIA TOXIN PUROMYCIN CHLORAMPHENICOL ELONGATION RICIN -SARCIN GTP P Site E Site A Site
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25 Puromycin Tyrosinyl-tRNA Puromycin imitates AA-tRNA
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26 1) Diphtheria toxin inactivates eEF2 2) Erythromycin inhibits EF2 Inhibition of ribosome translocation 26
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27 stop codons UAG UAA UGA RF 1,2,3 [eRF1,3] Termination & Release TERMINATION
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28 ENERGETICS OF PROTEIN SYNTHESIS 1.Charging 2.Initiation Unwinding and scanning Met-tRNA i binding 3.Elongation AA-tRNA binding Translocation 4.Termination ATP, 2~ ATP (several), 1~ GTP, 1~ GTP, 1~ (see later) GTP, 1~ GTP (number unknown), 1~ TOTAL: 4~ per AA polymerized + initiation + termination > 1200~ for an average protein Compared to 36-38 ATP’s generated by Glucose CO 2
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29 Down-regulation of the supply of initiator Met-tRNAi via eIF2 eIF2B eIF2 GDP eIF2 GTP eIF2 GTP Met-tRNA i PROTEIN SYNTHESIS eIF2 supplies Met- tRNA i to 40S subunit
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30 Control : Down-regulation of the supply of initiator Met-tRNAi via eIF2 kinases eIF2B eIF2 GDP eIF2 GTP eIF2 GTP Met-tRNA i PROTEIN SYNTHESIS eIF2 supplies Met- tRNA i to 40S subunit eIF2 phosphorylation inhibits initiation kinases eIF2 eIF2B eIF2 Trapped eIF2B INITIATION INHIBITED P P eIF2 kinases HRI: reticulocytes minus heme PKR: interferon plus virus- infection (dsRNA) PERK: ER stress GCN2: amino acid starvation
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31 EF-Ts EF-Tu GDP EF-Tu GTP EF-Tu GTP aa-tRNA PROTEIN SYNTHESIS GTP/GDP exchange during elongation by (e)EF1 (aka EF-Tu) Terminology PROK. EUK. Old New Tu 1A 1α Ts 1B 1βγ aa-tRNA complex GEF This factor supplies aa- tRNA to ribosome during elongation.
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32 CYTOPLASM cytosolic protein “free” polysome endoplasmic reticulum lumen secreted protein membrane-bound polysome on “rough” ER nuclear membrane cell membrane
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33 Target 30S 50S Ile-tRNA synthase 50S, 60S 80S eEF2 60S Action (1) Inhibits initiation (2) Causes misreading Inhibits binding of AA-tRNA to A-site Inhibits peptidyl transferase Inhibit translocation Inhibits isoleucine tRNA charging Premature release of nascent polypeptide Inhibits translocation Inhibits translocation¤ Inhibits binding of AA-tRNA to A-site♦ Inhibits binding of AA-tRNA to A-site & translocation# Inhibitor STREPTOMYCIN, Gentamicin, Kanamycin, Neomycin, etc. TETRACYCLINE, doxycycline CHLORAMPHENICOL ERYTHROMYCIN, Clarithromycin, Azithromycin Clindamycin, Lincomycin Mupirocin (pseudomonic acid) PUROMYCIN Cycloheximide DIPHTHERIA TOXIN RICIN (castor beans) -Sarcin (fungus) Aminoglycosides Tetracylines Macrolides Lincosamides Class Inhibitors of Protein Synthesis: Antibiotics and Toxins Catalytic activities of toxins ¤ ADP ribosylation ♦ 28S rRNA depurination (A) # 28S rRNA cleavage CAPITALIZED: most important
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34 Nucleus Transcription & translation mRNA Ribosomes Initiator Site selection PROKARYOTES EUKARYOTES No Coupled Polycistronic 70S (50S, 30S) f Met – tRNA i Shine-Dalgarno mediated internal initiation Yes Separated Monocistronic, Capped & Polyadenylated 80S (60S, 40S) Met – tRNA i 1) Scanning 2) IRES mediated internal entry Initiation factors Order of events 3 1) mRNA binding 2) f Met – tRNA i binding >12 1) Met – tRNA i binding 2) mRNA binding Antibiotics SensitiveResistant ToxinsResistant Sensitive
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35 Protein Modifications 1.Phosphorylation - (Tyr, Ser,Threo) Metabolic Regulation, Signal transduction, etc 2.Hydroxylation - (Proline) in collagen, Endoplasmic Reticulum 3.Glycosylation – (O-linked as with Ser/Threo- OH or N-Linked as in lysine) 4.Other - biotinilation, farnesyl, etc Protein Degradation - Mostly thru specific proteases and ubiquitin-proteosome system
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