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!