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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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Transcript of 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!
1) In bacteria transcription and translation are initially coupled
1) In Bacteria transcription and translation are initially coupled• RNA polymerase quits if ribosomes lag too much
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
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
Bacteria have > 1 protein/mRNA (polycistronic)•Mutations can have polar effects: mutations in upstream genes may affect expression of perfectly good downstream genes!
Regulating transcription
Telling RNA pol to copy a DNA sequence
Regulating transcription
Telling RNA pol to copy a DNA sequence
Transcription factors bind promoters & control initiation of transcription
Regulating transcription
Telling RNA pol to copy a DNA sequence
Transcription factors bind promoters & control initiation of transcription
1/signal gene senses
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
Transcription factorsBind surface -> base-pairs form unique patterns in major & minor grooves
Transcription factorsBind surface -> base-pairs form unique patterns in major & minor groovesScan DNA for correct pattern
Transcription factorsBind surface -> base-pairs form unique patterns in major & minor groovesScan DNA for correct patternneed 15 - 20 H-bonds = 5-8 base-pairs
Transcription
Prokaryotes have one RNA polymerase
makes all RNA
core polymerase = complex of 5 subunits (’)
Transcription
Prokaryotes have one RNA polymerase
makes all RNA
core polymerase = complex of 5 subunits (’)
not absolutely needed, but cells lacking are very sick
Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous
Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity
Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity• Bind promoters
Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity• Bind promoters• Different sigmas bind different promoters
Initiating transcription in Prokaryotes1) Core RNA polymerase is promiscuous2) sigma factors provide specificity• Bind promoters3) Once bound, RNA polymerase “melts” the DNA
Initiating transcription in Prokaryotes3) Once bound, RNA polymerase “melts” the DNA4) rNTPs bind template
Initiating transcription in Prokaryotes3) Once bound, RNA polymerase “melts” the DNA4) rNTPs bind template5) RNA polymerase catalyzes phosphodiester
bonds, melts and unwinds template
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
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
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
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
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
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
Prok gene regulation5 genes (trp operon) encode trp enzymes
Prok gene regulationCopy genes when no trpRepressor stops operon if [trp]
Prok gene regulationRepressor stops operon if [trp] trp allosterically regulates repressor can't bind operator until 2 trp bind
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
lac operon
Make these enzymes only if:
1) - glucose
lac operon
Make these enzymes only if:
1) - glucose
2) + lactose
lac operon
Regulated by 2 proteins
1) CAP protein : senses [glucose]
lac operonRegulated by 2 proteins1) CAP protein : senses [glucose]2) lac repressor: senses [lactose]
lac operonRegulated by 2 proteins1) CAP protein : senses [glucose]2) lac repressor: senses [lactose]
encoded by lac i geneAlways on
lac operon2 proteins = 2 binding sites1) CAP site: promoter isn’t active until CAP binds
lac operon2 proteins = 2 binding sites1) CAP site: promoter isn’t active until CAP binds2) Operator: repressor blocks transcription
lac operonRegulated by 2 proteins1) CAP only binds if no glucose -> no activation
lac operonRegulated by 2 proteins1) CAP only binds if no glucose -> no activation2) Repressor blocks transcription if no lactose
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
Result[-galactosidase]rapidly rises if noglucose & lactoseis presentW/in 10 minutes is 6% of total protein!