1. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Protein synthesis in bacteria Dr Mike Dyall-Smith, lab 3.07, mlds@unimelb.edu.au Aims: Understand the process of translation in Bacteria Know the the major components involved Know the typical genetic signals needed to allow translation of a mRNA Familiar with the process of protein folding References:, Schaecter et al., Microbe, pp149-58 or Any recent molecular biology textbook.

2. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Lecture outline: 1. Overview of translation 1. Overview of translation in bacteria (comparison with eucarya) components, genetic code, process. 2. Features of mRNA and tRNA 4. Ribosome structure and function 4. Ribosome structure and function (compared to eucarya) 5. Initiation and termination factors 5. Initiation and termination factors and the process of translation 6. Briefly touch on protein folding. *export and processing covered in future lecture.

3. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Central dogma Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 DNA RNA PROTEIN Transcription Translation

4. Translation The decoding (translation) of the 3-letter code of nucleotide bases in the mRNA, to direct the synthesis of proteins. A much more complex operation than transcription.

5. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Overall process of translation in bacteria Initiation Elongation Termination Initiation factors mRNA Requires aa-tRNAs, ATP, GTP Speed: ~ 15 aa/sec

6. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Protein synthesis in bacteria How do ribosomes bind to mRNA? Small subunit binds initiation factorsSmall subunit binds initiation factors This complex binds to mRNA, initiator tRNA can then bind.This complex binds to mRNA, initiator tRNA can then bind. The large subunit joins the complexThe large subunit joins the complex Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301

7. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Transfer RNA Acceptor Arm Anticodon Extra Arm D Arm T  Arm

8. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Transfer RNA (tRNA) structure Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Linkage to aa tRNA Arm Anticodon

9. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 tRNA is actually ‘L’ shaped Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Anticodon arm

10. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 UNIVERSAL GENETIC CODE

11. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Ribosomes are large enough to bind 2 tRNAs and cover 40 nt of mRNA

12. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301

13. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301

14. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 70 S Ribosomes

15. Ribosomes contain many proteins and 3 rRNAs 2/3 of the mass of bacterial ribosomes is rRNA

16. Initiation of translation:

18. IF-2 puts the fMet-tRNA f initiator in the P site. IF-2, bound to GTP, associates with the P site of the 30S subunit. fMet-tRNA f. then binds to the IF- 2 on the 30S subunit. fMet-tRNA f. then binds to the IF- 2 on the 30S subunit. IF-2 then transfers the tRNA into the partial P site 50S subunit binds and IF1-3 are released.

19. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Where do ribosomes bind on mRNA ? Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301

20. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Ribosome binding sites bind to the 3’ end of 16S rRNA Shine-Dalgarno sequence RBS or SDS

21. Initiator tRNA in bacteria is tRNA f Initiator tRNA in bacteria is tRNA f MET N-formyl-methionyl tRNA. It is only used for initiating translation. All proteins start with this amino acid. Internal methionines use another tRNA, tRNA m MET

22. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 tRNA and mRNA move in the same direction through the ribosome Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301

23. A site : site where an aminoacyl-tRNA enters to base pair with a codon. P site : site occupied by a peptidyl-tRNA Deacylated tRNA: has no amino acid or polypeptide chain attached Translocation: the movement of the ribosome, one codon at a time, along mRNA after the addition of an amino acid to the polypeptide chain. Elongation: the stage in a macromolecular synthesis reaction (replication, transcription, or translation) when the nucleotide or polypeptide chain is extended by the addition of individual subunits.

24. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Two sites for binding tRNAs on the ribosome P A Peptide bond formed Translocation P and A sites occupied Ribosome movement

25. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Typical polycistronic bacterial mRNA Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301

26. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Reinitiation on polycistronic mRNA Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301

27. Termination at stop codons: UAA, UGA, UAG Termination codons are recognised by ‘release factor’ proteins (RF1/RF2, RF3). RF1 or RF2 bind to stop codons in the A site of ribosomes, and activate the ribosome to hydrolyse the adjacent peptidyl tRNA (in the P site), to release the protein. RF3 releases the RF1 or RF2. Finally, RRF (ribosome recycling factor) dissociates the remaining mRNA, tRNA and ribosome subunits.

28. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 mRNA degradation in bacteria is rapid Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Half-life of 1-3 min

29. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 mRNA degradation in bacteria is rapid Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Q: What happens if the ribosome reaches the end of a broken mRNA without seeing a stop codon ? A: it gets stalled !! TGA

30. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 tmRNA - how to overcome stalled ribosomes Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 tmRNA is part tRNA part mRNA. It completes translation, putting a small peptide sequence at the end of a protein. This is a tag for protein degradation. http://www.indiana.edu/~tmrna/

31. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Overall process of translation in Bacteria Initiation Elongation Termination Initiation factors mRNA Requires aa-tRNAs, ATP, GTP Speed: ~ 15 aa/sec

32. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Lecture outline: 1. Overview of translation 1. Overview of translation in bacteria (and comparison with eucarya) components, genetic code, process. 2. Features of mRNA and tRNA 3. Ribosome structure and function 3. Ribosome structure and function (compared to eucarya) 4. Initiation and termination factors 4. Initiation and termination factors and the process of translation 5. Outline of protein folding Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301

33. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Signal sequence of HGH This is removed (by the pituitary cells) during secretion, to form the mature hormone.

34. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Review of Paper:

35. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 ABSTRACT Goeddel et al., Nature 281:544 (1979)

36. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Structure of pig growth hormone. alpha helix S-S bond N C S.Abdel-Meguid, Monsanto Co. 191 aa

37. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Signal sequence of HGH This is removed (by the pituitary cells) during secretion, to form the mature hormone.

38. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Problems in expressing a eukaryotic protein like HGH in a prokaryote like E.coli Goeddel et al., Nature 281:544 (1979) HGH is normally produced as a pre-protein, with a signal sequence (for secretion) that is cut off on export from the cell. Need to get rid of introns Need to supply E.coli sequences for transcription and translation.

39. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Strategy for cloning and expressing HGH Goeddel et al., Nature 281:544 (1979) Construct a gene that will express the mature form of HGH (ie. no signal sequence) Combine cDNA and synthetic DNA to construct the ORF so that the ATG is perfectly positioned with a lac promoter.

40. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Construction of pHGH107 Goeddel et al., Nature 281:544 (1979) 2x lacUV5 promoters HGH and TetR genes transcribed together

41. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Adjusting the distance between the Shine-Dalgarno sequence and the start codon. Goeddel et al., Nature 281:544 (1979) In the cloning steps, the spacing was 11bp. This was reduced to 7bp, as in the natural lacZ gene.

42. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Expression of HGH in E.coli Goeddel et al., Nature 281:544 (1979) Note the mid-log and stationary phase levels differ. Levels are lower in stationary phase cells.

43. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Expression of HGH in E.coli Goeddel et al., Nature 281:544 (1979) pHGH107-1 has the 7bp spacing between S- D and ATG (as in lacZ) It is worse than the 11bp version!

44. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Expression of HGH in E.coli Goeddel et al., Nature 281:544 (1979) LacI overproducer strain (D1210) shows good control of gene expression (+/- IPTG inducer).

45. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 HGH production in E.coli - analysed by SDS-PAGE Goeddel et al., Nature 281:544 (1979) purification from E.coli pure HGH immuno precipitation Multiple bands ?? pBR322 pure HGH pHGH

46. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Summary: What they achieved. Goeddel et al., Nature 281:544 (1979) “This is the first time that a human polypeptide has been directly expressed in E.coli in a non-precursor form.” Made a gene hybrid (cDNA and synthetic DNA) that coded for the processed form of HGH. Able to express HGH protein in E.coli at high levels (could purify it easily) Expressed HGH was able to react with anti-HGH antibodies

47. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Summary: What they didn’t achieve. Goeddel et al., Nature 281:544 (1979) Did it contain f-Met or not? Was it folded correctly? Did it have the required S-S bridges? Was it active as a growth hormone? Why was there so much proteolytic degradation?

48. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Fig. 1. Construction of plasmid pHGH31, containing the coding sequence (cDNA) for amino acids 24-191 of Human Growth Hormone. Goeddel et al., Nature 281:544 (1979)

49. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Plasmid cloning vector cDNA copy of HGH mRNA Cut in the middle of the Amp R gene Tailed 3’ ends with poly G Tailed cut ends with poly C Goeddel et al., Nature 281:544 (1979)

50. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Fig 1. Cloned HGH gene using polyC-polyG ‘sticky ends’ E.coli HaeIII and PstI sites regenerated Goeddel et al., Nature 281:544 (1979)

51. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Sequence of the Human GH cDNA Goeddel et al., Nature 281:544 (1979)

52. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Construction of the sequence coding for aa 1-24 of mature HGH

53. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Construction of the coding sequence for the first 24aa of mature HGH Goeddel et al., Nature 281:544 (1979) Small synthetic DNA sequences were assembled and ligated together. The terminal oligos contained restriction site ‘sticky ends’.

54. 526-301 Biotechnology Dr Mike Dyall-Smith, 2007 Combining the DNA segments coding for aa 1-24 and aa 24-191 Goeddel et al., Nature 281:544 (1979)