From Gene to Phenotype- part 3. Lecture Outline 11/9/05. The genetic code. Translation: overview

Transcription

1 DN mrn From ene to Phenotype- part 3 TRNSRIPTION DN 1 RN is transcribed from a DN template. 5 RN RN transcript polymerase RN PROESSIN Exon 2 In eukaryotes, the RN transcript RN transcript (premrn) is spliced and (pre-mrn) Intron modified to produce mrn, which moves minoacyl-trn from the nucleus to the synthetase cytoplasm. NLES mino FORMTION OF acid INITITION OMPLEX MINO ID TIVTION YTOPLSM trn 3 fter leaving the 4 Each amino acid nucleus, mrn attaches attaches to its proper trn to the ribosome. with the help of a specific enzyme and TP. mrn rowing polypeptide ctivated amino acid Ribosomal subunits Poly- ap Poly- Poly- Lecture Outline 11/9/05 Review translation: Initiation, elongation, termination EP model Post-translational modification of polypeptides Signal sequences Mutations (again) polypeptide 5 ap E odon TRNSLTION 5 succession of trns add their amino acids to the polypeptide chain nticodon as the mrn is moved through the ribosome one codon at a time. (When completed, the polypeptide is released from the ribosome.) Exam 3 is next Monday. It will cover mitosis and meiosis, DN synthesis, transcription, translation, genetics of viruses. (chapters 12, 13, 16, 17, part of 18 (to page 345)) Translation: overview TRNSRIPTION TRNSLTION The ribosome is the machine that builds the polypeptide DN mrn mrn Trp Phe mino acids trn with amino acid attached ly trn nticodon 5 odons trn serves as an adaptor that brings the correct amino acid to each codon. The genetic code First mrn base (5 end) Second mrn base Phe Tyr ys Leu Trp His Leu Pro rg ln sn lle Thr Met or Lys rg start sp Val la ly lu Third mrn base ( end) 5 Hydrogen bonds 5 - odon in the mrn 1

2 n aminoacyl-trn synthetase joins a specific amino acid to a trn minoacyl-trn synthetase (enzyme) mino acid P P P TP denosine Pyrophosphate 3 ppropriate trn bonds to amino cid, displacing MP. P i P i P P i trn P P denosine MP denosine 1 ctive site binds the amino acid and TP. Each trn has a slightly different shape How does the ribosome find? Prokaryotes have a special binding sequence upstream of the start codon. In Eukaryotes,the ribosome binds to the 5 cap and scans the message for an. 4 ctivated amino acid is released by the enzyme. See the nimation Inhibition of protein synthesis Toxin Mode of action Target forms peptidyl-puromycin, prevents Puromycin Procaryotes translocation blocks the -site, prevents binding of aminoacyl Tetracycline Procaryotes trns hloramphenicol blocks peptidyl transfer Procaryotes ycloheximide blocks peptidyl transferase Eucaryotes Streptomycin inhibits initiation at high concentrations Procaryotes Diphtheria toxin catalyzes DP-ribosylation of residue in eef2 Eucaryotes Erythromycin binds to 50S subunit, inhibits translocation Procaryotes Ricin inactivates 60S subunit, depurinates an adenosine in 23S rrn Eucaryotes NOTE: Prokaryotes (this generally includes protein synthesis in mitochondria and chloroplasts) 2

3 Only the anticodon of trn determines which amino acid is added by a ribosome. Experimental evidence: onvert cystein to alanine chemically, after it is attached to trn (remove SH group) lanine shows up in ystein sites The amino acid carried by a trn is independent of the anticodon sequence Determined by the amino-acyl trn synthetase enzyme trn with mutations in the anticodon still have their normal amino acid at the 3 end. Experiment:. mutate anticodon of trn thr (-->) Now binds to proline codon instead (). Those trn still carry threonine, but now bind to proline sites. Threonine inserted into polypeptide where proline normally goes. lananine trn synthetase minoacyl trn synthetase enzyme is specific to a particular amino acid and a particular trn lycine doesn t fit.. Quality control Both cap and tail bind to initiation factors to start translation Ensures that mrn is intact Small subunit can detect mis-paired trn and remove them Needs a short delay before peptide bond is formed (to give time for proofreading) Error rate: about

4 ost of protein biosynthesis Synthesis of aminoacyl trns 2 TPs Formation of 1 peptide bond 2 TPs 1 for codon recognition; 1 for translocation Proofreading 1 TP/error onstruction of a specific amino acid sequence is much more costly than formation of a random peptide bond! Transcription and translation can occur simultaneously RN polymerase RN polymerase DN Polyribosome Direction of transcription mrn 0.25 µm DN mrn (5 end) Post translational modifications and sorting Translation begins in the cytosol The signal mechanism for targeting proteins to the ER SRP binds to the signal peptide, ttaches to translocation pore in ER membrane synthesized into the ER Signal peptide removed Folds to final shape Signalrecognition particle (SRP) YTOSOL mrn Signal peptide SRP receptor protein Signal peptide removed ER membrane Protein lycosylation Signal directs protein to the right compartment Translocation complex 4

5 Signal peptide determines where it goes Destined for ER Imported during translation Stays within the membrane system Destined for cytosol or other organelles Imported after translation Brooker Figure haperones help fold proteins Hsp 70 covers exposed hydrophobic patches until the protein can fold Mis-folded proteins are marked for destruction with ubiquitin biquitin tail Hsp60 is like an isolation chamber Proteosome acts as garbage disposal 5

6 The molecular basis of sickle-cell disease: a point mutation Mutations (again) Wild-type hemoglobin DN Mutant hemoglobin DN 5 5 mrn T T T mrn 5 5 In the DN, the mutant template strand has an where the wild-type template has a T. The mutant mrn has a instead of an in one codon. Normal hemoglobin lu Sickle-cell hemoglobin Val The mutant (sickle-cell) hemoglobin has a valine (Val) instead of a glutamic acid (lu). Base-pair substitution Wild type mrn 5 Protein Met Lys Phe ly mino end arboxyl end Base-pair substitution No effect on amino acid sequence instead of Missense Met Lys Phe ly Met Lys Phe Nonsense instead of Met instead of Base-pair insertion or deletion Wild type mrn 5 Protein Met Lys Phe ly mino end arboxyl end Base-pair insertion or deletion Frameshift causing immediate nonsense Extra Met Frameshift causing extensive missense Missing Met Lys Leu la Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid Missing Met Phe ly 6

7 Mutations in the 3rd position of a codon are often silent Second mrn base For amino acids that have only two codons, the 3rd base will either both be purines or both be pyrimidines First mrn base (5 end) Phe Tyr ys Leu Trp Leu His Pro ln rg sn lle Thr Met or Lys start rg sp Val la ly lu Third mrn base ( end) Wobble in 3rd position 7