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How to bioengineer a novel system? Obtain a sequence by PCR, then clone it into a suitable plasmid

How to bioengineer a novel system? Obtain a sequence by PCR, then clone it into a suitable plasmid We ’ re adding DNA, but want E. coli to make a protein!. In bacteria transcription and translation are initially coupled . In Bacteria transcription and translation are initially coupled

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How to bioengineer a novel system? Obtain a sequence by PCR, then clone it into a suitable plasmid

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  1. How to bioengineer a novel system? • Obtain a sequence by PCR, then clone it into a suitable plasmid • We’re adding DNA, but want E. coli to make a protein!

  2. In bacteria transcription and translation are initially coupled

  3. In Bacteria transcription and translation are initially coupled • RNA polymerase quits if ribosomes lag too much

  4. In Bacteria transcription and translation are initially coupled • RNA polymerase quits if ribosomes lag too much • Recent studies show that ribosomes continue translating once mRNA is complete; i.e after transcription is done

  5. Bacteria have > 1 protein/mRNA (polycistronic) • http://bmb-it-services.bmb.psu.edu/bryant/lab/Project/Hydrogen/index.html#section1 • euk have 1 protein/mRNA

  6. Bacteria have > 1 protein/mRNA (polycistronic) • Mutations can have polar effects: mutations in upstream genes may affect expression of perfectly good downstream genes!

  7. Regulating transcription Telling RNA pol to copy a DNA sequence

  8. Regulating transcription Telling RNA pol to copy a DNA sequence Transcription factors bind promoters & control initiationof transcription

  9. Regulating transcription • Telling RNA pol to copy a DNA sequence • Transcription factors bind promoters & control initiationof transcription • 1/signal gene senses

  10. Regulating transcription • Telling RNA pol to copy a DNA sequence • Transcription factors bind promoters & control initiationof transcription • 1/signal gene senses • 1 binding site/signal gene senses

  11. Transcription factors Bind surface -> base-pairs form unique patterns in major & minor grooves

  12. Transcription factors Bind surface -> base-pairs form unique patterns in major & minor grooves Scan DNA for correct pattern

  13. Transcription factors Bind surface -> base-pairs form unique patterns in major & minor grooves Scan DNA for correct pattern need 15 - 20 H-bonds = 5-8 base-pairs

  14. Transcription Prokaryotes have one RNA polymerase makes all RNA core polymerase = complex of 5 subunits (a1aIIbb’w)

  15. Transcription Prokaryotes have one RNA polymerase makes all RNA core polymerase = complex of 5 subunits (a1aIIbb’w) w not absolutely needed, but cells lacking w are very sick

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

  17. Initiating transcription in Prokaryotes Core RNA polymerase is promiscuous sigma factors provide specificity

  18. Initiating transcription in Prokaryotes • Core RNA polymerase is promiscuous • sigma factors provide specificity • Bind promoters

  19. Initiating transcription in Prokaryotes • Core RNA polymerase is promiscuous • sigma factors provide specificity • Bind promoters • Different sigmas bind different promoters

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

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

  23. 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

  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 s 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 s factor • 2)” -35 region”: 5’-TTGACA-3’ : bound by s 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 s factor • 2)” -35 region”: 5’-TTGACA-3’ : bound by s factor • 3) UP element : -57: bound by a 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 s factor • 2)” -35 region”: 5’-TTGACA-3’ : bound by s factor • 3) UP element : -57: bound by a factor

  28. 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 s factor • 2)” -35 region”: 5’-TTGACA-3’ : bound by s factor • 3) UP element : -57: bound by a factor • Other sequences also often influence transcription! Eg Trp operator

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

  30. Prok gene regulation Copy genes whenno trp Repressor stops operon if [trp]

  31. Prok gene regulation • Repressor stops operon if [trp] • trp allostericallyregulates repressor • can't bind operator until 2 trp bind

  32. 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

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

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

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

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

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

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

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

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

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

  42. 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 lactoseis present and glucoseis not

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

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