how to bioengineer a novel system? obtain a sequence by pcr, then clone it into a suitable plasmid
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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 - PowerPoint PPT PresentationTRANSCRIPT
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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!
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1) In bacteria transcription and translation are initially coupled
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1) In Bacteria transcription and translation are initially coupled• RNA polymerase quits if ribosomes lag too much
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1) 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
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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
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Bacteria have > 1 protein/mRNA (polycistronic)•Mutations can have polar effects: mutations in upstream genes may affect expression of perfectly good downstream genes!
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Regulating transcription
Telling RNA pol to copy a DNA sequence
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Regulating transcription
Telling RNA pol to copy a DNA sequence
Transcription factors bind promoters & control initiation of transcription
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Regulating transcription
Telling RNA pol to copy a DNA sequence
Transcription factors bind promoters & control initiation of transcription
1/signal gene senses
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Regulating transcriptionTelling RNA pol to copy a DNA sequenceTranscription factors bind promoters & control initiation of transcription
1/signal gene senses1 binding site/signal gene senses
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Transcription factorsBind surface -> base-pairs form unique patterns in major & minor grooves
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Transcription factorsBind surface -> base-pairs form unique patterns in major & minor groovesScan DNA for correct pattern
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Transcription factorsBind surface -> base-pairs form unique patterns in major & minor groovesScan DNA for correct patternneed 15 - 20 H-bonds = 5-8 base-pairs
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Transcription
Prokaryotes have one RNA polymerase
makes all RNA
core polymerase = complex of 5 subunits (’)
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Transcription
Prokaryotes have one RNA polymerase
makes all RNA
core polymerase = complex of 5 subunits (’)
not absolutely needed, but cells lacking are very sick
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Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous
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Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity
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Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity• Bind promoters
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Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity• Bind promoters• Different sigmas bind different promoters
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Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity• Bind promoters3) Once bound, RNA polymerase “melts” the DNA
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Initiating transcription in Prokaryotes3) Once bound, RNA polymerase “melts” the DNA4) rNTPs bind template
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Initiating transcription in Prokaryotes3) Once bound, RNA polymerase “melts” the DNA4) rNTPs bind template5) RNA polymerase catalyzes phosphodiester
bonds, melts and unwinds template
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Initiating transcription in Prokaryotes3) Once bound, RNA polymerase “melts” the DNA4) rNTPs bind template5) RNA polymerase catalyzes phosphodiester
bonds, melts and unwinds template6) sigma falls off after ~10 bases are added
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Structure of Prokaryotic promotersThree 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
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Structure of Prokaryotic promotersThree 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
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Structure of Prokaryotic promotersThree 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 factor3) UP element : -57: bound by factor
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Structure of Prokaryotic promotersThree 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 factor3) UP element : -57: bound by factor
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Structure of Prokaryotic promotersThree 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 factor3) UP element : -57: bound by factorOther sequences also often influence transcription! Eg Trp operator
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Prok gene regulation5 genes (trp operon) encode trp enzymes
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Prok gene regulationCopy genes when no trpRepressor stops operon if [trp]
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Prok gene regulationRepressor stops operon if [trp] trp allosterically regulates repressor can't bind operator until 2 trp bind
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lac operonSome operons use combined “on” & “off” switches E.g. E. coli lac operon Encodes enzymes to use lactose
lac Z = -galactosidaselac Y= lactose permeaselac A = transacetylase
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lac operon
Make these enzymes only if:
1) - glucose
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lac operon
Make these enzymes only if:
1) - glucose
2) + lactose
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lac operon
Regulated by 2 proteins
1) CAP protein : senses [glucose]
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lac operonRegulated by 2 proteins1) CAP protein : senses [glucose]2) lac repressor: senses [lactose]
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lac operonRegulated by 2 proteins1) CAP protein : senses [glucose]2) lac repressor: senses [lactose]
encoded by lac i geneAlways on
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lac operon2 proteins = 2 binding sites1) CAP site: promoter isn’t active until CAP binds
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lac operon2 proteins = 2 binding sites1) CAP site: promoter isn’t active until CAP binds2) Operator: repressor blocks transcription
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lac operonRegulated by 2 proteins1) CAP only binds if no glucose -> no activation
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lac operonRegulated by 2 proteins1) CAP only binds if no glucose -> no activation2) Repressor blocks transcription if no lactose
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lac operonRegulated by 2 proteins1) CAP only binds if no glucose 2) Repressor blocks transcription if no lactose3) Result: only make enzymes for using lactose if lactose is present and glucose is not
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Result[-galactosidase]rapidly rises if noglucose & lactoseis presentW/in 10 minutes is 6% of total protein!