From Gene to Phenotype- part 3

1. DNA TRANSCRIPTION RNA is transcribed from a DNA template. 2 3 4 5 1 3 Poly-A RNA transcript RNA polymerase 5 Exon RNA PROCESSING In eukaryotes, the RNA transcript (pre- mRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cytoplasm. RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase Cap NUCLEUS Amino acid FORMATION OF INITIATION COMPLEX AMINO ACID ACTIVATION tRNA CYTOPLASM After leaving the nucleus, mRNA attaches to the ribosome. Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Growing polypeptide mRNA Activated amino acid Poly-A Poly-A Ribosomal subunits Cap 5 TRANSLATION C A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome one codon at a time. (When completed, the polypeptide is released from the ribosome.) C A U A E A C Anticodon A A A U G G U G U U U A Codon Ribosome From Gene to Phenotype- part 3 DNA mRNA polypeptide

2. Lecture Outline 11/9/05 • Review translation: • Initiation, elongation, termination • EPA model • Post-translational modification of polypeptides • Signal sequences • Mutations (again) Exam 3 is next Monday. It will cover mitosis and meiosis, DNA synthesis, transcription, translation, genetics of viruses. (chapters 12, 13, 16, 17, part of 18 (to page 345))

3. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly tRNA C C C G G Anticodon A A A A G G G U G U U U C Codons 5 3 mRNA Translation: overview tRNA serves as an “adaptor” that brings the correct amino acid to each codon. The ribosome is the machine that builds the polypeptide

4. Second mRNA base U C A G U C A G U C A G U UAU UUU UCU UGU Tyr Cys Phe UAC UUC UCC UGC C U U Ser UCA UUA UAA Stop Stop UGA A Leu UAG UCG Stop UUG UGG Trp G U C A G CCU U CUU CAU CGU His C CUC CCC CAC CGC C C Arg Pro Leu CUA CCA CAA CGA A Gln Third mRNA base (3 end) CCG CUG CAG CGG G First mRNA base (5 end) U C A G U AUU ACU AAU AGU Asn A Ser C lle AUC ACC AAC AGC A Thr A AUA ACA AAA AGA Lys Arg Met or start G AUG ACG AAG AGG U C A G U GUU GCU GAU GGU G Asp C GCC GAC GGC GUC G Val Ala Gly GUA GCA GAA GGA A Glu GUG GCG GAG GGG G 3 A C C 5 A C G The genetic code C G C G U G U A A U U A U C G * G U A C A C A * A U C C * G * U G U G G * G A C C G * C * A G U G * * G A G C Hydrogen bonds G C U A G * A * A C * U A G A 5’-AUGCAAUUCGGAAAC Codon in the mRNA

5. Active site binds the amino acid and ATP. 4 1 3 An aminoacyl-tRNA synthetase joins a specific amino acid to a tRNA Amino acid Aminoacyl-tRNA synthetase (enzyme) P P P Adenosine ATP Each tRNA has a slightly different shape Adenosine P Pyrophosphate P Pi Pi Pi tRNA Appropriate tRNA bonds to amino Acid, displacing AMP. Adenosine P AMP Activated amino acid is released by the enzyme.

6. How does the ribosome find AUG? • 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 AUG.

7. See the Animation • www.dnai.org

8. Inhibition of protein synthesis NOTE: Prokaryotes (this generally includes protein synthesis in mitochondria and chloroplasts)

9. Only the anticodon of tRNA determines which amino acid is added by a ribosome. • Experimental evidence: • Convert cystein to alanine chemically, after it is attached to tRNA (remove SH group) • Alanine shows up in Cystein sites

10. The amino acid carried by a tRNA is independent of the anticodon sequence • Determined by the amino-acyl tRNA synthetase enzyme • tRNA with mutations in the anticodon still have their normal amino acid at the 3’ end. • Experiment:. mutate anticodon of tRNAthr(AGU-->AGG) • Now binds to proline codon instead (CCU). • Those tRNA still carry threonine, but now bind to proline sites. • Threonine inserted into polypeptide where proline normally goes.

11. Aminoacyl tRNA synthetase enzyme is specific to a particular amino acid and a particular tRNA Alananine tRNA synthetase Glycine doesn’t fit . .

12. Quality control • Both cap and tail bind to initiation factors to start translation • Ensures that mRNA is intact • Small subunit can detect mis-paired tRNA and remove them • Needs a short delay before peptide bond is formed (to give time for proofreading) • Error rate: about 10-4

13. Cost of protein biosynthesis • Synthesis of aminoacyl tRNAs 2 ATPs • Formation of 1 peptide bond 2 GTPs • 1 for codon recognition; 1 for translocation • Proofreading 1 ATP/error • Construction of a specific amino acid sequence is much more costly than formation of a random peptide bond!

14. Transcription and translation can occur simultaneously RNA polymerase DNA mRNA Polyribosome Direction of transcription 0.25 m RNA polymerase DNA Polypeptide Ribosome mRNA (5 end)

15. Post translational modifications and sorting Glycosylation Signal directs protein to the right compartment

16. The signal mechanism for targeting proteins to the ER Signal peptide removed Translation begins in the cytosol Polypeptide synthesized into the ER Attaches to translocation pore in ER membrane Folds to final shape SRP binds to the signal peptide, 2 3 1 4 5 6 Ribosome mRNA Signal peptide ER membrane Signal peptide removed Signal- recognition particle (SRP) Protein SRP receptor protein CYTOSOL Translocation complex

17. Signal peptide determines where it goes Destined for cytosol or other organelles Destined for ER Imported during translation Imported after translation Stays within the membrane system Brooker Figure 13.22

19. Chaperones help fold proteins Hsp 70 covers exposed hydrophobic patches until the protein can fold Hsp60 is like an isolation chamber

20. Mis-folded proteins are marked for destruction with ubiquitin Ubiquitin tail Proteosome acts as garbage disposal

21. Mutations (again)

22. Wild-type hemoglobin DNA Mutant hemoglobin DNA In the DNA, the mutant template strand has an A where the wild-type template has a T. 3 5 3 5 T T C A T C mRNA mRNA The mutant mRNA has a U instead of an A in one codon. G A A U A G 5 3 5 3 Normal hemoglobin Sickle-cell hemoglobin The mutant (sickle-cell) hemoglobin has a valine (Val) instead of a glutamic acid (Glu). Val Glu The molecular basis of sickle-cell disease: a point mutation

23. Wild type A U G A A G U U U G G C U A A mRNA 5 3 Protein Lys Met Phe Gly Stop Amino end Carboxyl end Base-pair substitution No effect on amino acid sequence U instead of C A U G A A G U U U G G U U A A Lys Met Phe Gly Stop Missense A instead of G A A A U G A A G U U U A G U U Lys Met Phe Ser Stop Nonsense U instead of A G A U U G G A G U A U U U C A Met Stop Base-pair substitution

24. Wild type G A U A A G U U U G G C U A A mRNA 3 5 Gly Met Lys Phe Protein Stop Amino end Carboxyl end Base-pair insertion or deletion Frameshift causing immediate nonsense Extra U A G U U A A G U U U G G C U A Met Stop Frameshift causing extensive missense Missing U A G U A A G U U G G C U A A Met Lys Ala Leu Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid Missing A A G A G U U U U G G C U A A Met Phe Gly Stop Base-pair insertion or deletion

25. Mutations in the 3rd position of a codon are often silent Second mRNA base U C A G U UAU UUU UCU UGU For amino acids that have only two codons, the 3rd base will either both be purines or both be pyrimidines Tyr Cys Phe UAC UUC UCC UGC C U Ser UCA UUA UAA Stop Stop UGA A Leu UAG UCG Stop UUG UGG Trp G CCU U CUU CAU CGU His CUC CCC CAC CGC C C Arg Pro Leu CUA CCA CAA CGA A Gln CCG CUG CAG CGG G Third mRNA base (3 end) First mRNA base (5 end) U AUU ACU AAU AGU Asn Ser C lle AUC ACC AAC AGC A Thr A AUA ACA AAA AGA Lys Arg Met or start G AUG ACG AAG AGG U GUU GCU GAU GGU Asp C GCC GAC GGC GUC G Val Ala Gly GUA GCA GAA GGA A Glu GUG GCG GAG GGG G

26. Wobble in 3rd position