CHAPTER 30: PROTEIN SYNTHESIS

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1 CHAPTER 30: PROTEIN SYNTHESIS (Translation) Translation: mrna protein LECTURE TOPICS Complexity, stages, rate, accuracy Amino acid activation [trna charging] trnas and translating the Genetic Code - Amino acid activation [trna charging] - codon-anticodon recognition - Wobble base pairing and code degeneracy Ribosome Structure-mRNA/tRNA/rRNA base pairings important in translation! TRANSLATION PROCESS - Prokaryotic - Eukaryotic - Inhibitors [specific steps in process]

2 Process Rates for Flow of Genetic Information (prokaryotic) PROCESS "MACHINERY" RATES DNA REPLICATION DNA Polymerase I DNA Polymerase III 10 bases/sec 1000 bases/sec TRANSCRIPTION RNA Polymerase 50 bases/sec synthesis of mrna for 200 a.a. protein takes (200 x 3)/50 = 12 sec TRANSLATION Ribosomes 20 a.a.'s/sec synthesis of a 200 a.a. protein takes 200/20 =10 sec

3 Complexity of Translation: Cast of Characters [amounts in one E.coli cell] Translation machinery is 35% of dry weight of cell! 20,000 ribosomes 200,000 trnas 100,000 proteins and cofactors

4 Complexity of Translation: Stages and Cast of Characters

5

6 trna is a compact A-helix structure! Only a few bases are NOT paired! trna structure/animations: (Excellent!)

7 Threonyl trna Thr synthetase Proofreading (Error correcting) Mistakes (Val, Ser): Actually leaves Serine (1/100 a.a s) Hydrolyzes wrong amino acid (Ser) a.a-amp-enzyme

8 Codon-anticodon recognition and wobble base pairing 3 5 UH2 UH2 Sometimes inosine is at 5`-end of anticodon mrna codon 5 I G C C C G -5 Wobble I:C base pair

9 Wobble Hypothesis: * Don t need 61 trnas! * The anticodon of some trnas can read 2 or 3 codons (5`) ONLY ONLY (3`) Wobble basepairing

10 Ribosome Structure 30S 50S 70S

11 Transcription and translation are coupled in E.coli They happen simultaneously!

12 Translation process is 100% Processive: 1. Make peptide bonds 2. Relative movements (mrna + ribosome) 3. Supply of reagents,etc. Elongation 30S Initiation complex Termination

13 Protein (polypeptide) Synthesis is NH 2 COOH Amino terminus COOH terminus

14 Ribosome has two essential functions in protein synthesis

15 How does a ribosome work? trna-mrna-rrna base-pairing interactions determine accuracy of protein synthesis. 50S ribosome subunit 30S ribosome subunit Three trnas mrna

16 Purine-rich Start mrnas Initiation: Prokaryotic mrna-16s rrna base-pairing Purine-rich Start

17 rrna-mrna(codon)-trna(anticodon) base-pairing: another accuracy factor

18 Three trna binding sites on 30S subunit CCA ends Exit Peptidyl Amino-acyl Codon-Anticodon base pairing

19 [E,P,A] three trna binding sites on 30S subunit NH 2 [Large subunit] 3 trnas [small subunit]

20 Protein Translation Factors * EF-Tu 23S rrna ribozyme E P A E P A GTP GDP + Pi EF-G Termination by Release Factors (Stop codon recognition) E P A E P A

21 Elongation : Peptide Bond Synthesis [peptide bond formed and growing peptide moves from P-site to A-site].. * Initiator trna Catalyzed by 23S rrna ribozyme

22 50S Peptidyl transferase : A ribozyme activity A site (Green) P site(red) Adenine 2451 P site A site 23S RNA-catalyzed peptide bond formation A site

23 Protein Synthesis Animation: 3-D model (Nierhaus, 2006) Protein Synthesis Animation

24 Stop Dec 4, 2008

25 Eukaryotic cytoplasmic ribosome (80S)

26 Eukaryotic Initiation complex EIF-2(GTP) for start AUG only 1) Ribosome binds to cap 2) Moves to 1st AUG EIF2-GDP + Pi 3) Large + small subunits associate Elongation

27 Eukaryotic mrna: Protecting the ends! protein-rna and protein-protein interactions circularize mrna eif4e binds to cap eif4e binds to eif4g eif4g binds to PABI

28 Ricinus communis (castor bean) is the source of ricin, a deadly poison that stops eukaryotic protein synthesis (500μg is lethal human dose). A and B chains disulfide linked B chain binds to cell and induces uptake by endocytosis A chain released in cytoplasm is a N-glycosidase that removes A 4324 in 28S rrna and prevents elongation factor binding

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