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Lectures 33-34 GENETIC CODE and PROTEIN SYNTHESIS

LecLtures 33 and 34. Lectures 33-34 GENETIC CODE and PROTEIN SYNTHESIS. Mukund Modak, Ph.D. Adapted from M. Mathews, Ph.D. ~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.

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Lectures 33-34 GENETIC CODE and PROTEIN SYNTHESIS

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  1. LecLtures 33 and 34 Lectures 33-34GENETIC CODE and PROTEIN SYNTHESIS Mukund Modak, Ph.D. Adapted from M. Mathews, Ph.D. .

  2. ~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, ...) 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. Proteins are important… …in sickness and in health 2

  3. INTRODUCTION • Central Dogma • Ribosomes and polysomes • Genetic Code • Mutations with effects at the translation level • TRANSLATIONAL MACHINERY • MECHANISM OF TRANSLATION AND INHIBITORS OF PROTEIN SYNTHESIS • IV. ENERGETICS AND REGULATION OF TRANSLATION 3

  4. POLYSOMES E.M.

  5. CENTRAL DOGMA 5’ RNA 3’ N- or amino- terminus C- or carboxy- terminus protein DNA RNA PROTEIN 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

  6. Coupled transcription & translation in bacteria [ N terminus to C terminus ] [ 5’ to 3’ ] Not so in Eukaryotes

  7. codon # 5 6 7 CCU Pro GAG Glu GAG Glu CCU Pro GUG Val GAG Glu 1st position (5’ end) 2nd position 3rd position (3’ end) U C A G Ser Ser Ser Ser Tyr Tyr STOP STOP Phe Phe Leu Leu Cys Cys STOP Trp U C A G GENETIC CODE U Leu Leu Leu Leu Pro Pro Pro Pro His HIs Gln Gln Arg Arg Arg Arg U C A G C U C A G Ile Ile Ile Met Thr Thr Thr Thr Asn Asn Lys Lys Ser Ser Arg Arg A Val Val Val Val Ala Ala Ala Ala Asp Asp Glu Glu Gly Gly Gly Gly U C A G G Normal Hb – β Sickle cell Hb – βS

  8. Nearly universal – variations in mitochondria, mycoplasma, ciliates Degenerate (or redundant) Non-overlapping Unpunctuated – although some codons are signals GENETIC CODE: 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.

  9. INTRODUCTION • TRANSLATIONAL MACHINERY • Ribosomes: prokaryotic / eukaryotic • Messenger RNA • Transfer RNA • Aminoacyl-tRNA synthetases; Met-tRNA forms (m, f, i) • Initiation, elongation and termination enzymes • MECHANISM OF TRANSLATION AND INHIBITORS OF PROTEIN SYNTHESIS • IV. ENERGETICS AND REGULATION OF TRANSLATION 9 9

  10. TRANSLATIONAL COMPONENTS 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)

  11. 1. Ribosome Structure

  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

  13. 2. mRNA Monocistronic (spliced) Eukaryotic: 3’ end ( 1 coding region ) 5’ end 3’ UTR poly A 5’ UTR AAA ~150 only 1 cap 7-MeGpppGXY Cistron = coding region = open reading frame (ORF) Polycistronic Prokaryotic: ( >1 coding region ) 5’ 3’ # 2 ppp # 1 # 3

  14. 3. tRNA Translational Adaptor

  15. (1) AA ~tRNA + AMP + PPi G ~0 Kcal/mole (2) PPi + H2O 2 Pi G = -6.6 Kcal/mole G ~ -6.6 Kcal/mole 4. Amino Acids tRNAs carry “activated” amino acids: AA + tRNA + ATP aaRS PPase Overall free energy change for aminoacylation of tRNA aaRS = aminoacyl-tRNA synthetase PPase = pyrophosphatase

  16. Formation of aminoacyl-tRNA The amino acid is firstactivated by reacting with ATP The activated amino acid istransferred from aminoacyl-AMP to tRNA These enzymes are vital for the fidelityof protein synthesis: 2 steps allow “proofreading”

  17. Genetic Code Translation Machinery 20 AA – tRNA synthases ( i.e., 1 per AA ) ~50 tRNA species (at least 1 per AA, but less than 1 per codon) GAA GAG 2 codons 1 2 3 20 AA’s 61 Codons for AA’s “WOBBLE” Pairing Wobble Position e.g.CUU1 anti–codon anti-codon stem-loop of tRNA tRNA 3’ 5’ ANTI-CODON 3 2 1 5’ mRNA CODON

  18. 2 tRNAs for AUG / Methionine: 2 different functions N-formyl in bacteria: F-Met Met Met CCA CCA Met – tRNA F or I Met – tRNA M 3’ 5’ 3’ 5’ UAC UAC 5’ 3’ AUG AUG 1 Initiation Codon Internal Met Codon

  19. Enzymes ProkaryotesEukaryotes 5. Translation Factors Translation Step Charging of tRNA 1. Initiation 2. Elongation 3. Termination Modifications, cleavage, etc. Aminoacyl – tRNA synthetases IF1- IF3 eIF1- eIF5 (multiple) EF1, EF2eEF1, eEF2 RF1- RF3eRF1, eRF3

  20. INTRODUCTION • TRANSLATIONAL MACHINERY • MECHANISM OF TRANSLATION AND INHIBITORS OF PROTEIN SYNTHESIS • Initiation • Elongation • Termination • Antibiotics • Toxins • IV. ENERGETICS AND REGULATION OF TRANSLATION 20

  21. HOW RIBOSOMES FIND THEIR INITIATION SITES eukaryotes prokaryotes 1. Cap - dependent scanning 40S 30S 16S rRNA S - D cap AUG.. AUG... Shine - Dalgarno box 2. Internal ribosome entry 40S AUG.. Next step:large subunit 50S/60S subunit joining ---------------IRES-----------

  22. 30S ribosomal subunit initiation at S-D sequence

  23. STREPTOMYCIN AUG.. AUG.. HOW RIBOSOMES FIND THEIR INITIATION SITES eukaryotes prokaryotes 1. Cap - dependent scanning 40S 30S 16S rRNA S - D cap Shine - Dalgarno box 2. Internal ribosome entry Streptomycin, Gentamycin, Tobramycin, Amikacin, etc. areaminoglycosides. They also cause miscoding during elongation 40S AUG... ---------------IRES-----------

  24. TETRACYCLINES SPECTINOMYCIN AA – tRNA binding EF 1A, 1B (EF-Tu, Ts) [eEF 1α, eEF1βγ ] PUROMYCIN CHLORAMPHENICOL Peptidyl Transfer Peptidyl transferase (50S / 60S) CLINDAMYCIN Macrolides e.g. ERYTHROMYCIN EF2 [eEF2] Translocation DIPHTHERIA TOXIN RICIN -SARCIN ELONGATION A Site P Site E Site GTP

  25. Puromycin imitates AA-tRNA Puromycin Tyrosinyl-tRNA

  26. Inhibition of ribosome translocation 1) Diphtheria toxin inactivates eEF2 2) Erythromycin inhibits EF2 26

  27. stop codons UAG UAA UGA TERMINATION Termination & Release RF 1,2,3 [eRF1,3]

  28. TOTAL: 4~ per AA polymerized + initiation + termination > 1200~ for an average protein ENERGETICS OF PROTEIN SYNTHESIS • 1. Charging • 2. Initiation • Unwinding and scanning • Met-tRNAi 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~ Compared to 36-38 ATP’s generated by Glucose CO2

  29. eIF2B Down-regulation of the supply of initiator Met-tRNAi via eIF2 eIF2 • GDP eIF2 • GTP eIF2 • GTP • Met-tRNAi PROTEIN SYNTHESIS eIF2 supplies Met- tRNAi to 40S subunit

  30. kinases P eIF2 eIF2B P eIF2 eIF2B Trapped eIF2B INITIATION INHIBITED eIF2 phosphorylation inhibits initiation Control :Down-regulation of the supply of initiator Met-tRNAi via eIF2 kinases eIF2 kinases HRI: reticulocytes minus heme PKR: interferon plus virus- infection (dsRNA) PERK: ER stress GCN2: amino acid starvation eIF2 • GDP eIF2 • GTP eIF2 • GTP • Met-tRNAi PROTEIN SYNTHESIS eIF2 supplies Met- tRNAi to 40S subunit

  31. EF-Ts GTP/GDP exchange during elongation by (e)EF1 (aka EF-Tu) Terminology PROK.EUK. OldNew Tu 1A1α Ts 1B1βγ EF-Tu • GDP aa-tRNAcomplex GEF EF-Tu • GTP EF-Tu • GTP • aa-tRNA PROTEIN SYNTHESIS This factor supplies aa- tRNA to ribosome during elongation.

  32. membrane-bound polysome on “rough” ER nuclear membrane endoplasmic reticulum lumen secreted protein “free” polysome cytosolic protein CYTOPLASM cell membrane

  33. Class Inhibitor Aminoglycosides Tetracylines Macrolides Lincosamides 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) Inhibitors of Protein Synthesis: Antibiotics and Toxins Target 30S 30S 50S 50S 50S Ile-tRNA synthase 50S, 60S 80S eEF2 60S 60S Action (1) Inhibits initiation (2) Causes misreading Inhibits binding of AA-tRNA to A-site Inhibits peptidyl transferase Inhibit translocation 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# Catalytic activities of toxins ¤ ADP ribosylation ♦ 28S rRNA depurination (A) # 28S rRNA cleavage CAPITALIZED: most important

  34. Initiation factors Order of events 3 1) mRNA binding 2) f Met – tRNAi binding >12 1) Met – tRNAi binding 2) mRNA binding Antibiotics Sensitive Resistant EUKARYOTES PROKARYOTES Yes Separated Monocistronic, Capped & Polyadenylated 80S (60S, 40S) Met – tRNAi 1) Scanning 2) IRES mediated internal entry Nucleus Transcription & translation mRNA No Coupled Polycistronic Ribosomes Initiator Site selection 70S (50S, 30S) f Met – tRNAi Shine-Dalgarno mediated internal initiation Sensitive Toxins Resistant

  35. Protein Modifications Phosphorylation - (Tyr, Ser,Threo) Metabolic Regulation, Signal transduction, etc Hydroxylation - (Proline) in collagen, Endoplasmic Reticulum Glycosylation – (O-linked as with Ser/Threo- OH or N-Linked as in lysine) Other - biotinilation, farnesyl, etc Protein Degradation - Mostly thru specific proteases and ubiquitin-proteosome system

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