Project

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Project Studying Synechococcus elongatus for
biophotovoltaics
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• How to bioengineer a novel bio-photovoltaic
  system?
• Obtain a sequence by PCR, then clone it into a
  suitable plasmid
• Were adding DNA, but want Synechococcus to make
  a protein!

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1. In bacteria transcription and translation are
   initially coupled

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

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• 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 gt 1 protein/mRNA (polycistronic)
• http//bmb-it-services.bmb.psu.edu/bryant/lab/Proj
  ect/Hydrogen/index.htmlsection1
• euk have 1 protein/mRNA

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• Bacteria have gt 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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Transcription Prokaryotes have one RNA
polymerase makes all RNA core polymerase
complex of 5 subunits (a1aIIbbw)
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Transcription Prokaryotes have one RNA
polymerase makes all RNA core polymerase
complex of 5 subunits (a1aIIbbw) w not
absolutely needed, but cells lacking w are very
sick
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Initiating transcription in Prokaryotes 1) Core
RNA polymerase is promiscuous
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• Initiating transcription in Prokaryotes
• Core RNA polymerase is promiscuous
• sigma factors provide specificity

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

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

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

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• Initiating transcription in Prokaryotes
• 3) Once bound, RNA polymerase
• melts the DNA
• 4) rNTPs bind template

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• 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

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• 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

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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
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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
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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
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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
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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 CAP site in lac promoter
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Structure of Prokaryotic promoters Other
sequences also often influence transcription! Our
plasmid contains the nickel promoter.
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Structure of Prokaryotic promoters Other
sequences also often influence transcription! Our
plasmid contains the nickel promoter.
?
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Structure of Prokaryotic promoters Other
sequences also often influence transcription! Our
plasmid contains the nickel promoter. nrsBACD
encode nickel transporters
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• Structure of Prokaryotic promoters
• Other sequences also often influence
  transcription! Our 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

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• 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

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• 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

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Termination of transcription in prokaryotes 1)
Sometimes go until ribosomes fall too far behind
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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
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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) Our terminator
(rrnB) first forms an RNA hairpin, followed by an
8 base sequence TATCTGTT that halts transcription
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Homologous recombination 1) DNA strands must be
capable of base-pairing
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• Homologous recombination
• DNA strands must be capable of base-pairing
• DNA must get cut

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• Homologous recombination
• DNA strands must be capable of base-pairing
• DNA must get cut
• Ends are processed to form single-strand overhangs

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• Homologous recombination
• DNA strands must be capable of base-pairing
• DNA must get cut
• Ends are processed to form single-strand
  overhangs
• Single strand invades homolog (with help of RecA
  protein)

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• Homologous recombination
• DNA strands must be capable of base-pairing
• DNA must get cut
• Ends are processed to form single-strand
  overhangs
• Single strand invades homolog (with help of RecA
  protein)
• Must be capable of forming hybrid molecule!

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• Homologous recombination
• DNA strands must be capable of base-pairing
• DNA must get cut
• Ends are processed to form single-strand
  overhangs
• Single strand invades homolog (with help of RecA
  protein)
• Must be capable of forming hybrid molecule!
• DNA polymerase adds on to end of invading molecule

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• Homologous recombination
• DNA strands must be capable of base-pairing
• DNA must get cut
• Ends are processed to form single-strand
  overhangs
• Single strand invades homolog (with help of RecA
  protein) forms Holliday junction
• Branch migrates

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• Homologous recombination
• DNA strands must be capable of base-pairing
• DNA must get cut
• Ends are processed to form single-strand
  overhangs
• Single strand invades homolog (with help of RecA
  protein) forms Holliday junction
• Branch migrates
• Holliday jn is
• cut DNA is ligated

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• Homologous recombination
• DNA strands must be capable of base-pairing
• DNA must get cut
• Ends are processed to form single-strand
  overhangs
• Single strand invades homolog (with help of RecA
  protein) forms Holliday junction
• Branch migrates
• Holliday jn is
• cut DNA is ligated
• 7) Use mismatch repair
• to fix mismatches

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• Homologous recombination
• DNA strands must be capable of base-pairing
• DNA must get cut
• Ends are processed to form single-strand
  overhangs
• Single strand invades homolog (with help of RecA
  protein) forms Holliday junction
• Branch migrates
• Holliday jn is
• cut DNA is ligated
• 7) Use mismatch repair
• to fix mismatches
• Why add selectable
• marker to new sequence

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• Finding Orthologs
• Go to http//www.ncbi.nlm.nih.gov/
• Enter name of gene in search window
• Select nucleotide
• Select name of a promising sequence
• Select run BLAST optimize for somewhat similar
  sequences (blastn)
• Pick out interesting orthologs

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• Finding Orthologs
• Go to http//www.ncbi.nlm.nih.gov/
• Select structure
• Enter name of protein in search window
• Select name of a promising sequence
• Select protein
• Select run BLAST
• Pick out interesting orthologs