1. Engineering magnetosomes to express novel proteins Which ones? Must be suitable for expressing in Magnetospyrillum! Can’t rely on glycosylation, disulphide bonds, lipidation, selective proteolysis, etc for function! Best bets are bacterial proteins Alternatives are eukaryotic proteins that don’t need any of the above Short peptides Tweaking p18 Linker Deleting or replacing GFP TRZN Oxalate decarboxylases Lactate dehydrogenase or other oxalate metab enzyme

2. DNA replication Proteins replicating both strands are in replisome Attached to membrane & feed DNA through it lagging strand loops out so make both strands in same direction

3. Magnetospirillum gryphiswaldense Borg optimised rbs, promoter & codon usage

4. Magnetospirillum gryphiswaldense Borg optimised rbs, promoter & codon usage Developed inducible system based on tetracycline

5. Magnetospirillum gryphiswaldense Borg optimised rbs, promoter & codon usage Developed inducible system based on tetracycline Fuse protein to C-terminus of mamC

6. Magnetospirillum gryphiswaldense Borg optimised rbs, promoter & codon usage Developed inducible system based on tetracycline Fuse protein to mamC C-terminus: exposed at surface

7. Magnetospirillum gryphiswaldense Borg optimised rbs, promoter & codon usage Developed inducible system based on tetracycline Fuse protein to mamC C-terminus: exposed at surface Purify with magnets

8. Assignment Design a mamC C-terminal protein fusion Design DNA sequence encoding a useful protein

9. Assignment Design a mamC C-terminal protein fusion Design DNA sequence encoding a useful protein Replace eGFP of pJH3 with your protein Best to use MluI and NheI sites

10. Assignment Best to use MluI and NheI sites Design oligos that add MluI in frame at 5’ end and NheI at 3’end

11. Assignment Best to use MluI and NheI sites  Design oligos that add MluI in frame at 5’ and NheI at 3’end  Digest vector & clone with MluI and NheI then ligate

12. Assignment Best to use MluI and NheI sites  Design oligos that add MluI in frame at 5’ and NheI at 3’end  Digest vector & clone with MluI and NheI then ligate  Find & analyze clones

13. Transcription Prokaryotes have one RNA polymerase makes all RNA core polymerase = complex of 5 subunits (      ’  )

14. Transcription Prokaryotes have one RNA polymerase makes all RNA core polymerase = complex of 5 subunits (      ’  )  not absolutely needed, but cells lacking  are very sick

15. Initiating transcription in Prokaryotes 1) Core RNA polymerase is promiscuous

16. Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity

17. Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity Bind promoters

18. Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity Bind promoters Different sigmas bind different promoters

19. Initiating transcription in Prokaryotes 1)Core RNA polymerase is promiscuous 2)sigma factors provide specificity Bind promoters 3) Once bound, RNA polymerase “melts” the DNA

20. Initiating transcription in Prokaryotes 3) Once bound, RNA polymerase “melts” the DNA 4) rNTPs bind template

21. Initiating transcription in Prokaryotes 3) Once bound, RNA polymerase “melts” the DNA 4) rNTPs bind template 5) RNA polymerase catalyzes phosphodiester bonds, melts and unwinds template

22. Initiating transcription in Prokaryotes 3) Once bound, RNA polymerase “melts” the DNA 4) rNTPs bind template 5) RNA polymerase catalyzes phosphodiester bonds, melts and unwinds template 6) sigma falls off after ~10 bases are added

23. Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5’ to transcription start) 5’-TATAAT-3’ determines exact start site: bound by  factor

24. Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5 ’ to transcription start) 5 ’ -TATAAT-3 ’ determines exact start site: bound by  factor 2) ” -35 region ” : 5 ’ -TTGACA-3 ’ : bound by  factor

25. Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5 ’ to transcription start) 5 ’ -TATAAT-3 ’ determines exact start site: bound by  factor 2) ” -35 region ” : 5 ’ -TTGACA-3 ’ : bound by  factor 3) UP element : -57: bound by  factor

26. Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5 ’ to transcription start) 5 ’ -TATAAT-3 ’ determines exact start site: bound by  factor 2) ” -35 region ” : 5 ’ -TTGACA-3 ’ : bound by  factor 3) UP element : -57: bound by  factor

27. Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5 ’ to transcription start) 5 ’ -TATAAT-3 ’ determines exact start site: bound by  factor 2) ” -35 region ” : 5 ’ -TTGACA-3 ’ : bound by  factor 3) UP element : -57: bound by  factor Other sequences also often influence transcription! Eg Trp operator

28. Prok gene regulation 5 genes (trp operon) encode trp enzymes

29. Prok gene regulation Copy genes when no trp Repressor stops operon if [trp]

30. Prok gene regulation Repressor stops operon if [trp] trp allosterically regulates repressor can't bind operator until 2 trp bind

31. lac operon Some operons use combined “on” & “off” switches E.g. E. coli lac operon Encodes enzymes to use lactose lac Z =  -galactosidase lac Y= lactose permease lac A = transacetylase

32. lac operon Make these enzymes only if: 1) - glucose

33. lac operon Make these enzymes only if: 1) - glucose 2) + lactose

34. lac operon Regulated by 2 proteins 1) CAP protein : senses [glucose]

35. lac operon Regulated by 2 proteins 1)CAP protein : senses [glucose] 2)lac repressor: senses [lactose]

36. lac operon Regulated by 2 proteins 1)CAP protein : senses [glucose] 2)lac repressor: senses [lactose] encoded by lac i gene Always on

37. lac operon 2 proteins = 2 binding sites 1) CAP site: promoter isn’t active until CAP binds

38. lac operon 2 proteins = 2 binding sites 1)CAP site: promoter isn’t active until CAP binds 2)Operator: repressor blocks transcription

39. lac operon Regulated by 2 proteins 1) CAP only binds if no glucose -> no activation

40. lac operon Regulated by 2 proteins 1) CAP only binds if no glucose -> no activation 2) Repressor blocks transcription if no lactose

41. lac operon Regulated by 2 proteins 1) CAP only binds if no glucose 2) Repressor blocks transcription if no lactose 3) Result: only make enzymes for using lactose if lactose is present and glucose is not

43. Result [  -galactosidase] rapidly rises if no glucose & lactose is present W/in 10 minutes is 6% of total protein!

44. Structure of Prokaryotic promoters Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter.

45. Structure of Prokaryotic promoters Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter. ↵

46. Structure of Prokaryotic promoters Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter. nrsBACD encode nickel transporters

47. Structure of Prokaryotic promoters Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter. nrsBACD encode nickel transporters nrsRS encode “two component” signal transducers nrsS encodes a his kinase nrsR encodes a response regulator

48. Structure of Prokaryotic promoters nrsRS encode “two component” signal transducers nrsS encodes a his kinase nrsR encodes a response regulator When nrsS binds Ni it kinases nrsR

49. Structure of Prokaryotic promoters nrsRS encode “two component” signal transducers nrsS encodes a his kinase nrsR encodes a response regulator When nrsS binds Ni it kinases nrsR nrsR binds Ni promoter and activates transcription of both operons

50. Termination of transcription in prokaryotes 1) Sometimes go until ribosomes fall too far behind

51. Termination of transcription in prokaryotes 1) Sometimes go until ribosomes fall too far behind 2) ~50% of E.coli genes require a termination factor called “rho”

52. Termination of transcription in prokaryotes 1) Sometimes go until ribosomes fall too far behind 2) ~50% of E.coli genes require a termination factor called “rho” 3) rrnB first forms an RNA hairpin, followed by an 8 base sequence TATCTGTT that halts transcription

53. Transcription in Eukaryotes 3 RNA polymerases all are multi-subunit complexes 5 in common 3 very similar variable # unique ones Now have Pols IV & V in plants Make siRNA

54. Transcription in Eukaryotes RNA polymerase I: 13 subunits (5 + 3 + 5 unique) acts exclusively in nucleolus to make 45S-rRNA precursor

55. Transcription in Eukaryotes Pol I: acts exclusively in nucleolus to make 45S-rRNA precursor accounts for 50% of total RNA synthesis

56. Transcription in Eukaryotes Pol I: acts exclusively in nucleolus to make 45S-rRNA precursor accounts for 50% of total RNA synthesis insensitive to  -aminitin

57. Transcription in Eukaryotes Pol I: only makes 45S-rRNA precursor 50 % of total RNA synthesis insensitive to  -aminitin Mg 2+ cofactor Regulated @ initiation frequency

58. RNA polymerase I promoter is 5' to "coding sequence" 2 elements 1) essential core includes transcription start site UCE -100 core +1 coding sequence

59. RNA polymerase I promoter is 5' to "coding sequence" 2 elements 1) essential core includes transcription start site 2) UCE (Upstream Control Element) at ~ -100 stimulates transcription 10-100x UCE -100 core +1 coding sequence

60. Initiation of transcription by Pol I Order of events was determined by in vitro reconstitution 1) UBF (upstream binding factor) binds UCE and core element UBF is a transcription factor: DNA-binding proteins which recruit polymerases and tell them where to begin

61. I nitiation of transcription by Pol I 1) UBF binds UCE and core element 2) SL1 (selectivity factor 1) binds UBF (not DNA) SL1 is a coactivator proteins which bind transcription factors and stimulate transcription