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 Something that may turn into thesis project!

2. Magnetospirillum gryphiswaldense Can propagate plasmids (but pBAM requires pir gene) Or can insert into chromosome via tnpA (Tn5)-based transposition

3. Magnetospirillum gryphiswaldense Can propagate plasmids (but pBAM requires pir gene) Can insert into genome by transposition no variation in expression due to copy # or growth stage

4. DNA replication Replication begins at origins of replication DNA polymerases are dumb! other proteins tell where to start

5. DNA replication Replication begins at origins of replication DNA polymerases are dumb! other proteins tell where to start bind origins & position DNA polymerase at start

6. DNA replication bind origins & position DNA polymerase at start Prokaryotes have one origin/DNA molecule 10-20 copies of DnaA bind E. coli chromosomal ori C (cyan boxes). This unwinds adjacent DNA, allows DnaC to load helicase (DnaB) on yellow boxes to start assembling replisome

7. DNA replication 10-20 copies of DnaA bind E. coli chromosomal ori C (cyan boxes). This unwinds adjacent DNA, allows DnaC to load helicase (DnaB) on 13 bp repeats

8. Prokaryotes have one origin/DNA molecule 1 on chromosome and one on each plasmid Plasmid numbers vary depending upon origin sequence i.e. factors that bind to them

9. Prokaryotes have one origin/DNA molecule 1 on chromosome and one on each plasmid Plasmid numbers vary depending upon origin i.e. factors that bind to them i.e. sequences and structure vary

10. Plasmid numbers vary depending upon origin i.e. factors that bind to them Plasmids in same “incompatibility group” use same factors, so should not be cotransformed

11. Plasmid numbers vary depending upon origin Plasmids in same “incompatibility group” use same factors, so should not be cotransformed Some, eg colE1 are controlled by antisense RNA

12. Others (eg R6K) are controlled by iterons: binding sites for regulatory proteins (eg Pi or RepA) DNA pol III needs primer RNA & DnaA to replicate

13. Many plasmids (pBS, pR6K) replicate by” theta mode”

14. SS-phage and some plasmids replicate by “rolling circle”

15. Other replicate by ”strand displacement”

16. Initiating DNA replication Prokaryotes have one origin/DNA molecule Pulse-chase experiments show eukaryotes have multiple origins/chromosome

17. Initiating DNA replication In Euk ORC (Origin Recognition Complex) binds ARS A is invariant, but B1, B2 and B3 vary A B1B2B3

18. Initiating DNA replication In Euk ORC binds ARS licensing factors ensure each ARS is only replicated once/S

19. Initiating DNA replication In Euk ORC binds ARS licensing factors ensure each ARS is only replicated once/S Activation factors initiate DNA replication

20. Initiating DNA replication licensing factors ensure each ARS is only replicated once/S Activation factors initiate DNA replication Licensing & activation factors fall off once replication starts, don't reattach until after mitosis

21. DNA replication must melt DNA @ physiological T Helicase melts DNA

22. DNA replication must melt DNA @ physiological T Helicase melts DNA Forms “replication bubble”

23. DNA replication Helicase melts DNA SSB proteins separate strands until they are copied

24. DNA replication helicase melts DNA unwinding DNA increases supercoiling elsewhere

25. DNA replication helicase melts DNA unwinding DNA increases supercoiling elsewhere DNA gyrase relieves supercoiling

26. Topoisomerases : enzymes that untie knots in DNA Type I nick backbone & unwind once as strand rotates Type II cut both strands: relieve two supercoils/rxn

27. DNA replication 1) where to begin? 2) “melting” 3) “priming” DNA polymerase can only add

28. DNA replication “priming” DNA polymerase can only add primase makes short RNA primers

29. DNA replication DNA polymerase can only add primase makes short RNA primers DNA polymerase adds to primer

30. DNA replication DNA polymerase can only add primase makes short RNA primers DNA polymerase adds to primer later replace primers with DNA

31. DNA replication 1) where to begin? 2) “melting” 3) “priming” 4) DNA replication

32. DNA replication add bases bonding 5’ P to 3’ OH @ growing end

33. DNA replication add bases bonding 5’ P to 3’ OH @ growing end Template holds next base until make bond

34. DNA replication add bases bonding 5’ P to 3’ OH @ growing end Template holds next base until make bond - only correct base fits

35. DNA replication add bases bonding 5’ P to 3’ OH @ growing end Template holds next base until make bond - only correct base fits - energy comes from 2 PO 4

36. DNA replication energy comes from 2 PO 4 "Sliding clamp" keeps polymerase from falling off

37. DNA replication energy comes from 2 PO 4 "Sliding clamp" keeps polymerase from falling off Proof-reading: only correct DNA can exit

38. DNA replication Proof-reading: only correct DNA can exit Remove bad bases & try again

39. DNA replication Only make DNA 5’ -> 3’

40. Leading and Lagging Strands Only make DNA 5’ -> 3’ strands go both ways!

41. Leading and Lagging Strands Only make DNA 5’ -> 3’ strands go both ways! Make leading strand continuously

42. Leading and Lagging Strands Make leading strand continuously Make lagging strand opposite way

43. Leading and Lagging Strands Make leading strand continuously Make lagging strand opposite way wait for DNA to melt, then make Okazaki fragments

44. Leading and Lagging Strands Make lagging strand opposite way wait for DNA to melt, then make Okazaki fragments each Okazaki fragment has its own primer: made discontinuously

45. Leading and Lagging Strands each Okazaki fragment has its own primer made discontinuously DNA replication is semidiscontinuous

46. Leading and Lagging Strands each Okazaki fragment has its own primer made discontinuously DNA replication is semidiscontinuous Okazaki fragments grow until hit one in front

47. RNAse H removes primer & gap is filled

48. Okazaki fragments grow until hit one in front RNAse H removes primer & gap is filled DNA ligase joins fragments

49. Okazaki fragments grow until hit one in front RNAse H removes primer & gap is filled DNA ligase joins fragments Energy comes from ATP-> AMP

50. DNA replication Real process is far more complicated! Proteins replicating both strands are in replisome

51. DNA replication Real process is far more complicated! Proteins replicating both strands are in replisome Attached to membrane & feed DNA through it

52. 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

53. DNA pol detaches when hits previous primer, reattaches at next primer

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

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

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

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

58. 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

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

60. 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

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

62. 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

63. 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

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

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

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

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

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

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

70. 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

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

72. 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

73. 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

74. 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

75. 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

76. 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

77. 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

78. 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

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

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

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

82. 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

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

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

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

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

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

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

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

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

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

92. 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

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

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

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

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

98. 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

99. 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

100. 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

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

102. 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”

103. 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

104. 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

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

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

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

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

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

110. 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

111. 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

112. 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