7 주차 수업자료

1. 7주차 수업자료

2. Chapter 13. Protein Synthesis

3. One gene  how many protein? • gene vs. genome • protein vs. proteome

4. Amino acid to polypeptide

5. Amino acid Enantiomer (optical isomer) CORN rule (COOH  R  NH2  H) L-form: left-handed, counter-clockwise D-form: right-handed, clockwise L + D: racemic mixture

6. Amino acid Enantiomer (optical isomer) CORN rule (COOH  R  NH2  H) L-form: left-handed, counter-clockwise D-form: right-handed, clockwise L + D: racemic mixture

7. Decoding genetic code by tRNA • codon vs. anticodon • tRNA: acceptor stem, anticodon-loop, TψC and DHU loop (modified bases) • The first base of tRNAanticodon can wobble (for codon/anticodon pairing)

8. Charging tRNA with amino acid • Aminoacyl tRNA synthetase • attach aa to tRNA (uncharged  charged tRNA) • highly specific to amino acid and tRNA

9. Ribosome • New protein synthesis • Bacteria: 70S = 30S (small subunit) + 50S (large subunit) • Eukaryote: 80S = 40S (small subunit) + 60S (large subunit) • Ribosomal subunit = rRNA + ribosomal protein (S1, S2, …, L1, L2, …) • 23S or 28S rRNA: ribozyme (peptidyl transferase) • Open reading frame (ORF)

10. Start of protein synthesis • Initiator tRNA • fMet (bacteria) • Met (eukaryote) • In bacteria, to find out the start AUG codon by ribosome • Shine-Dalgano sequence: ribosome binding site (RBS) • Anti-S-D sequence in 16S rRNA • In eukaryote, 5’-cap of mRNA is recognized

11. Assembly of initiation complex • Binding of 30S small subunit to mRNA • Initiator tRNA with fMet binding to form 30S initiation complex with the help of initiation factors (IF1-3) • Binding of 50S large subunit to form 70S initiation complex • 3 GC pairs in anticodon stem allow initiator tRNA’s entry into P site

12. Elongation of polypeptide • Three ribosomal sites for tRNA: A, P, and E sites • tRNA enters the A site (exception: fMet-initiator tRNA) • Two elongation factors required: EF-Tu/EF-Ts and EF-G • Peptidyl transferase (aminoacyltransferase): for polypeptide growth

13. Termination of protein synthesis • Stop codon is recognized by release factor (RF1 or RF2), not by tRNA • Hydrolysis of the bond between polypeptide chain and tRNA in P-site by peptidyl transferase • RF3 releases RF1 or RF2 from ribosome • Ribosome recycling factor (RRF) dissociates ribosomal subunits

14. Polysome (polyribosome) • Several ribosomes attach to mRNA simultaneously (~100 bp apart) • Linear in bacteria • Circular in eukaryote: by protein-protein interaction (eIF4 and polyA-binding protein)

15. Protein synthesis in bacteria • Polycistronic mRNA • Coupled transcription-translation • Chromosomes and ribosomes in the same cellular compartment • Impossible for eukaryotes: DNA in nucleus and ribosome in cytoplasm

16. Rescue of stalled ribosome in bacteria • Defective mRNA with no stop codon? • tmRNA • functions as tRNA+mRNA • Contains short mRNA for 11 aa ssrA tag (AANDENYALAA) • Contains stop codon • No anticodon loop and D-loop (SmpB required) • ssrA tag is recognized by several proteases (tail-specific protease such as Clp protease) for degradation of defective protein

17. Assembly of the eukaryotic initiation complex • Assembly of 40S small subunit and initiator Met-tRNA with eIF3 (binds to small subunit) and eIF2 (binds to initiator Met-tRNA) • CAP-binding protein (eIF4) binds to 5’-cap of mRNA • mRNA binding to 40S small subunit with the help of eIF4 to form 40S initiation complex and start scanning to find AUG with Kozak consensus sequence (GCCRCCAUGG)

18. Start of protein synthesis in eukaryotes • Binding to 60S large subunit with the help of eIF5 to form to form 80S initiation complex (eIF 2 and eIF3 are released) • Internal ribosome entry site (IRES)

19. Control of protein synthesis • In bacteria, • Ribosome modulation factor (RMF) inactivates ribiosomes under low nutrient/energy condition (stress condition) • In eukaryotes, • eIF2 is phosphorylated and GDP cannot be removed under low nutrient/energy condition (stress condition)

20. Protein export • Signal sequence (SS) or leader sequence: N-terminus, cleaved after export • In bacteria, • Signal recognition protein (SecA) binds SS and brings it to translocase complex in the cell membrane (cytoplasmic membrane) • Cotranslational export: protein export and synthesis happens at the same time • Leader peptidase cuts off SS • SecB (secretory chaperonin) binds polypeptide to prevent premature folding • Folding occurs after all the polypeptide is exported

21. Molecular chaperone for protein folding • Hsp70 type • to prevent premature folding • GroEL/ES type • GroEL (=Hsp60) • GroES (=Hsp10), lid • to refold misfolded or damaged proteins

22. Chaperonin: trigger factor • Folding problem of newly synthesized protein?

23. Mitochondria and chloroplasts • Symbiotic theory • Mitochondria and chloroplasts contain their own ribosomes and make some of their own proteins • Translocase: transport of cytoplasmic protein (with leader sequence) into mitochondria (TOM and TIM) and chloroplasts (TOC and TIC)

24. Post-translational modifications

25. Unusual amino acids By post-translational modification • Selenocysteine insertion sequence (SECIS element): forms a stem-and-loop structure • Sec-tRNA binds to SelB (or eEFsec) and then to stem-and-loop • Different stem-and-loop region between bacteria and mammals (21st aa) (22nd aa)

26. Protein degradation by proteases Lysosomal degradation Proteasomal degradation