1. Translation Chapter 27

2. Central Dogma Genetic code contained w/in 4 deoxy- nucleotide bases of DNA 1 gene  1 polypeptide DNA is template for codes, but not direct template –DNA transcr’d  mRNA –mRNA is direct template for polypeptides Translation = cell uses mRNA to construct polypeptides

3. The Genetic Code Discovered in 1960’s (27-7) Same code for almost all prokaryotes, eukaryotes Codon = 3 nucleotide bases of mRNA that code for 1 aa –REMEMBER: this info was originally “held” as deoxynucleotides w/in gene of DNA Most aa’s have >1 codon –Only aa’s w/ single 3-base codon = met, trp –“Wobble” @ 3 rd nucleotide

4. Fig.27-7

5. The Genetic Code – cont’d AUG = initiation codon –Codes for met UAA, UAG, UGA = stop codons –Code for termination of polypeptide synth No spacing between codons –Reading frame = 3 nucleotides –If insertion or deletion  frame shift (27-3) May  improper aa’s inc’d into polypeptide

6. Ribonucleic Acids Messenger RNA (mRNA) – described in transcr’n (Chpt. 26) –Size varies according to gene size So polypeptide size –Contains genetic code w/ 3 nucleotides/aa Ribosomal RNA (rRNA) – RNA that helps make up the ribosome Transfer RNA (tRNA) – carries aa to proper site on ribosome

7. The Ribosome Complex of enz’s, rRNA’s, aa’s to accomplish translation Made up of subunits Different sizes rRNA, prot’s, subunits (27-11) –Bacterial 70S = 50S + 30S subunits –Eukaryotic 80S = 60S + 40S subunits S = Svedberg unit = size unit based on centrifugation properties

8. Fig.27-11

10. The Ribosome – cont’d rRNA’s have complicated 2 o structures (27-12) –Needed to position mRNA/tRNA –May act like enzymes – assist in catalyzing form’n of peptide bonds

11. Transfer RNA (27-15) Interacts w/ both m and rRNA’s as well as aa’s At least 1 tRNA for each aa 1 region of tRNA becomes covalently linked to its aa –“Amino acid arm”

12. Transfer RNA – cont’d Covalent binding of aa to tRNA by tRNA synthetase –1 synthetase enz for each diff tRNA –Forms high-energy intermediate between tRNA + aa Anhydride link w/ AMP (27-16) “Activated” This intermediate links to aa arm –Forms high energy ester linkage –Energy held in these high potential energy bonds is used in peptide bond form’n

13. Fig.27-16

15. Transfer RNA – cont’d tRNA synthetase has proofreading ability –Where proper aa “knows” to be att’d to correct tRNA 1 region of tRNA contains “anticodon” –“Anticodon arm” –Opposite Amino acid arm –Has seq of 3 nucleotide bases which interact (base-pair) w/ codon of mRNA (27-14)

16. Fig.27-14

17. Transfer RNA – cont’d Other arms differ slightly in shape, nucleotides –Help synthetases distinguish proper tRNAs (27-18) Also has sites for attachment to 70 or 80S rRNA and mRNA

18. “Ingredients” for Translation mRNA –Contains genetic code for proper aa sequence to synthesize polypeptide tRNA attached to aa (through high energy bond)(27-17) –This complexes w/ mRNA @ codon for aa –First base of mRNA codon base-pairs w/ 3 rd base of anticodon on tRNA (27-8)

19. Fig.27-17

20. Fig.27-8

21. “Ingredients” for Translation – cont’d rRNA assoc’d w/ proteins  ribosome –Ribosome draws all structures together properly to facilitate translation Note: translation has initiation, elongation, termination steps. Book uses E. coli system as model

22. Initiation of Translation Begins @ amino terminus of new polypeptide New polypeptide always begins w/ met –BUT altered met: N- formylmethionine (p.1044) –fMet has particular codon that specifies it AUG Called initiation codon

23. Initiation – cont’d 30S subunit of ribosome binds IF-3 and IF-1 (27-22) –IF-3 (or 1) = Initiation Factor 3 (or 1) (proteins) (Table 27-9) –IF-3 prevents early binding of ribosomal subunits –IF-1 prevents improper binds of tRNA to wrong site mRNA binds 30S-IF3-IF1 complex –Binding such that initiation codon of mRNA is at specific site on subunit Called P site (for “Peptidyl” site) Lies next to A site (for “Aminoacyl” site)

24. Fig.27-22

25. Initiation – cont’d –Shine-Dalgarno sequence helps w/ proper placement of mRNA into P site (27-23) Specific seq along mRNA @ partic site relative to intitiation codon Recognized by rRNA of 30S ribosome Helps “line up” mRNA initiation codon @ P site on 30S subunit

26. Fig.27-23

27. Initiation – cont’d tRNA enters the structure –Must be tRNA that carries fMet –tRNA-fMet complex must be associated w/ IF-2 –IF-2 must be bound to GTP Get 30S-IF3-IF1 complexed with mRNA (w/ initiation codon @ P site), with anticodon region of tRNA-fMet-IF2-GTP base-paired to initiation codon

28. Fig.27-22

29. Initiation – cont’d 50S subunit enters  completed initiation complex –GTP cleaved  GDP + Pi –IF-1, -2, -3 disassociate –Now have 70S ribosome –Now P and A sites have completed conform’ns –Now 3 rd site (E or exit site) is formed next to P site

30. Fig.27-22

31. Initiation – cont’d 3 recognition sites hold the ribosome together –Shine-Dalgarno seq holds ribosome to mRNA –Codon-anticodon holds mRNA to tRNA –P site conform’n holds ribosome to tRNA

32. Elongation – cont’d A site on ribosome is empty –mRNA w/ codon for next aa is @ A site tRNA bound to aa, w/ anticodon that matches codon for next aa is prepared (27-25) –Binds EF-Tu (“Elongation Factor Tu”, a protein), which is bound to GTP –  Complex of tRNA-aa-EF Tu-GTP Complex approaches empty A site on 70S ribosome –“Placed” by base-pairing of codon/anticodon

33. Fig.25-25

34. Elongation – cont’d EF Tu-GTP cleaved from tRNA and GTP hydrolyzed  EF Tu-GDP + Pi –Allows time for proofreading of base pairing of codon/anticodon –GDP cleaved –EF Tu-GTP regenerated  1 st aa (fMet) and aa2 in P and A sites, respectively –Now in proper position to peptide bond

35. Elongation – cont’d  -NH 2 of aa2 attacks carbonyl of fMet (27-26) –  aa2 tRNA now has dipeptide attached –  1 st tRNA now empty (uncharged w/ aa) –REMEMBER: aa had been activated by ester bond form’n w/ tRNA –rRNA of 50S subunit may participate in peptide bonding by catalyzing rxn

36. Fig.27-26

37. Elongation – cont’d Ribosome moves 5’  3’ along mRNA by distance of 1 codon (27-27) –Called “translocation” –Due to conform’l change of ribosome –Requires EF-G att’d to GTP EF-G = translocase GTP cleaved from translocase and Also, GTP hydrolyzed  GDP + Pi

38. Fig.27-27

39. Elongation – cont’d  Empty tRNA @ E site, tRNA-aa2-fMet @ P site, A site empty –mRNA w/ codon for next aa is @ A site –Empty tRNA is expelled from ribosome –Next tRNA can now position next aa for form’n of another peptide bond Note: polypeptide is always attached to tRNA of last aa added @ ribosome

40. Termination of Translation Signaled by 1 of 3 stop codons (UAA, UAG, UGA) on mRNA (27-28) –When stop codon is @ A site –No tRNA recognizes these codons –These codons ARE recognized by Release Factors (RF1, RF2, or RF3) – proteins One of these binds mRNA @ stop codon –RF3 may stimulate release of polypeptide

41. Termination – cont’d tRNA w/ polypeptide chain att’d now “stalled” @ P site –Ester bond between tRNA and peptide hydrolyzed –Also interactions between tRNA and ribosome weaken –Also interactions between 30S, 50S subunits weaken –Ribosome dissociates

42. Fig.27-28

43. Polysomes >1 Ribosome at a time translates a single mRNA (27-29) –Efficient use of single mRNA strand In bacteria, mRNA transcript is translated before transcr’n complete (27-30) –REMEMBER: no nucleus in prokaryotes, so repl’n, transcr’n, transl’n all occur in cytoplasm Not true of eukaryotes

44. Fig.27-29

45. Fig.27-30

46. New Proteins are Processed Must be folded, altered molecularly  biologically active REMEMBER: folding, non-covalent interactions among 1 o structure aa’s/funct’l grps  proper 2 o, 3 o, 4 o structures  functional protein Post-translational modifications –Terminal aa’s modified fMet cleaved or its formyl grp cleaved from amino terminus –Terminal aa’s used for signaling cleaved

47. Post-translational modifications –Individual aa’s covalently modified Phosph’n

48. Post-translational modifications –Individual aa’s covalently modified Carboxyl grps added Methyl grps added

49. Post-Translational Modifications –Carbohydrates, isoprenes added  glycoproteins, lipoproteins –Prosthetic grps added Ex: heme added to hemoglobin, cytochromes –Proteolytic processing Polypeptide chains cleaved  funct’l proteins

50. Eukaryotic Translation REMEMBER: DNA more complicated, mRNA processed One processing step = 5’ cap, polyA tail –Play a role in complexing of mRNA to ribosome (27-24) More (9) initiation factors (Table 27-9) –Various roles, some impt to processed mRNA

51. Fig.27-24