1. Central Dogma Cytoplasm of eukaryote Cytoplasm of prokaryote DNAmRNA Protein transcription translation replication Translation converts sequence of bases in mRNA to sequence of amino acids in polypeptide

2. Lecture 12 - Translation *Translation Overview Genetic Code tRNA Charging reactions Ribosome Protein Synthesis Initiation - Prokaryotes vs Eukaryotes Elongation Termination

3. Overview: Players in Translation Messenger RNA (mRNA) Ribosome Proteins Ribosomal RNA (rRNA) Transfer RNA (tRNA) Other molecules (proteins, GTP etc.)

4. CGAT -- linear sequence of 4 bases DNARNA CGAU -- linear sequence of 4 bases PROTEIN KRHSTNQAVILMFYWCGPDE linear sequence of 20 amino acids convert mRNA sequence to amino acid sequence Genetic Code How many bases must be read at one time in order to have a unique code for each amino acid?

5. codons Triplet Code Frameshift mutations There are 3 possible frames to read a mRNA sequence

6. Universal (almost) Genetic Code

7. 80 nucleotides Acceptor Stem tRNA ECB 7-23

8. Codon - anticodon base pairing mRNA codon anticodon antiparallel 5’3’ Genetic code is degenerate (redundant) Wobble in 3rd position of codon

9. Aminoacyl-tRNA Synthetase enzymes One tRNA synthetase for each amino acid Synthetase binds tRNA - specificity conferred by the anticodon loop and the acceptor stem. How does the correct aa become attached to the corresponding tRNA?

10. “charged tRNA” Charging reaction and base pairing Energetics - ATP to AMP; equivalent to 2 ATPs to charge tRNA ECB 7-26

11. Amino acid is bonded to 3’ OH of tRNA

12. Genetic Code Translates linear sequence of 4 bases (RNA) to linear sequence of 20 amino acids. Codon 3-base sequence on mRNA that specifies an amino acid Reading Frame Grouping of nucleotide sequence into codons (3 reading frames possible, only one is used) Terminology Anticodon 3-base sequence on tRNA that specifies an amino acid Charging Reaction Adds amino acid to tRNA

13. Eukaryotic ribosomes Prokaryotic ribosomes See ECB 7-28

14. Ribosome has 1 binding site for mRNA and 3 for tRNA mRNA binds small subunit tRNAs bind both subunits (at interface) ECB 7-29

15. Translation Overview Genetic Code tRNA Charging reactions Ribosome *Protein Synthesis Initiation - Prokaryotes vs Eukaryotes Elongation Termination Lecture 12 - Translation

16. Shine-Delgarno sequence is 5’ (upstream) of initiation codon (AUG) on mRNA (in 5’ UTR) ---GGAGGA--- ---GGAGGA--- mRNA -5’ Shine-Delgarno sequence ---ACCUCCUUUA--- rRNA -3’ Initiation in Prokaryotes mRNA binds to small ribosomal subunit by base pairing to 16S rRNA

17. GDP + P i Initiation in Prokaryotes 30S Initiation factors 30S initiation complex 50S 70S initiation complex 30S fmet tRNA GTP IF2 Initiationcodon Initiation codon S-D AUG determines reading frame

18. Translation can be initiated at several sites on prokaryotic mRNA Prokaryotes - In polycistronic mRNA coded by an operon, each coding region must have Shine-Delgarno sequence and AUG ECB7-29 ECB 7-33

19. Initiation in eukaryotes ECB 7-32

20. Stepwise addition of amino acids Elongation factors (EFs) are required 3 Key steps:1. Entry of aminoacyl-tRNA 2. Formation of a peptide bond 3. Translocation - movement of ribosome with respect to the mRNA 3 tRNA binding sites: A, P, E A site = Aminoacyl site, accepts new tRNA P site = Peptidyl site, tRNA with growing polypeptide chain E site = Exit site, release of uncharged tRNA Translation Elongation (eukaryotic and prokaryotic)

21. Start with tRNA + peptide chain in P site (only a singe aa if chain just initiated) E P A Three steps in elongation ECB 7-31

22. N- to C-terminus synthesis Peptidyltranserase reaction- Peptide Bond Formation Proks and euks Does not require input of energy

23. Termination 3 stop codons; UAG, UGA, UAA ECB 7-34

24. Protein synthesis is energetically expensive… Charging aa-tRNA: 2 ATP (ATP -> AMP+2P i )… Binding of aa-tRNA/proofreading: 1 GTP… Translocation of ribosome 1 codon towards 3’ end of mRNA: 1 GTP… Total of at least 4 high energy bonds/aa added… As much as 80% of cells energy devoted to protein synthesis!

25. Peptidyl-tRNA in P site…A site is empty… Adapted from ECB figure 7-31 Polypeptide elongation

27. Step 1: Complex of aa-tRNA and EF1-GTP binds in A-site… Polypeptide elongation

30. proofreading Requirement for GTP hydrolysis and release of EF1 before peptide bond formation imposes a time delay…allowing wrong aa- tRNAs to dissociate from ribosome = proofreading Polypeptide elongation

31. Step 3a: Large subunit shifts relative to small subunit and mRNA… Step 2: Peptide bond formed (energy of 2 ATP from charging of aa-tRNA). Polypeptide elongation

32. Step 3b: Small subunit moves 1 codon (3 nucl.) towards 3’ end. Empty tRNA is ejected. GTP GDP + P i Polypeptide elongation

33. Prokaryotes: ~20 aa/sec… Eukaryotes: ~ 2 aa/sec… Polypeptide elongation 07.6-translation_II.mov

34. Polyribosomes Multiple ribosomes translating one mRNA 5’ to 3’ ECB 7-35

35. Antibiotics that block prokaryotic protein synthesis