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Project
Studying Synechococcus elongatus
for biophotovoltaics
How to bioengineer a novel bio-photovoltaic system?
Obtain a sequence by PCR, then clone it into a suitable plasmid
•We’re adding DNA, but want Synechococcus 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-itservices.bmb.psu.edu/bryant/lab/Project/Hydrogen/index.html#secti
on1
•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!
Transcription
Prokaryotes have one RNA polymerase
makes all RNA
core polymerase = complex of 5 subunits (a1aIIbb’w)
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
Initiating transcription in Prokaryotes
1) Core RNA polymerase is promiscuous
Initiating transcription in Prokaryotes
1) Core RNA polymerase is promiscuous
2) sigma factors provide specificity
Initiating transcription in Prokaryotes
1) Core RNA polymerase is promiscuous
2) sigma factors provide specificity
• Bind promoters
Initiating transcription in Prokaryotes
1) Core RNA polymerase is promiscuous
2) sigma factors provide specificity
• Bind promoters
• Different sigmas bind different promoters
Initiating transcription in Prokaryotes
1) Core RNA polymerase is promiscuous
2) sigma factors provide specificity
• Bind promoters
3) Once bound, RNA polymerase
“melts” the DNA
Initiating transcription in Prokaryotes
3) Once bound, RNA polymerase
“melts” the DNA
4) rNTPs bind template
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
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
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
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
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
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
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
Structure of Prokaryotic promoters
Other sequences also often influence transcription! Our plasmid
contains the nickel promoter.
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
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
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
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
Termination of transcription in prokaryotes
1) Sometimes go until ribosomes fall too far behind
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”
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
Homologous recombination
1) DNA strands must be capable of base-pairing
Homologous recombination
1)DNA strands must be capable of base-pairing
2)DNA must get cut
Homologous recombination
1) DNA strands must be capable of base-pairing
2) DNA must get cut
3) Ends are processed to form single-strand overhangs
1)
2)
3)
4)
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)
1)
2)
3)
4)
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!
1)
2)
3)
4)
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
1)
2)
3)
4)
5)
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
Homologous recombination
1)DNA strands must be capable of base-pairing
2)DNA must get cut
3)Ends are processed to form single-strand overhangs
4)Single strand invades homolog (with help of RecA protein): forms
Holliday junction
5)Branch migrates
6)Holliday jn is
cut & DNA is ligated
Homologous recombination
1)DNA strands must be capable of base-pairing
2)DNA must get cut
3)Ends are processed to form single-strand overhangs
4)Single strand invades homolog (with help of RecA protein): forms
Holliday junction
5)Branch migrates
6)Holliday jn is
cut & DNA is ligated
7) Use mismatch repair
to fix mismatches
Homologous recombination
1)DNA strands must be capable of base-pairing
2)DNA must get cut
3)Ends are processed to form single-strand overhangs
4)Single strand invades homolog (with help of RecA protein): forms
Holliday junction
5)Branch migrates
6)Holliday jn is
cut & DNA is ligated
7) Use mismatch repair
to fix mismatches
•Why add selectable
marker to new sequence
Finding Orthologs
1)Go to http://www.ncbi.nlm.nih.gov/
2)Enter name of gene in search window
3)Select “nucleotide”
4)Select name of a promising sequence
5)Select “run BLAST”: optimize for somewhat similar sequences
(blastn)
6)Pick out interesting orthologs
Finding Orthologs
1)Go to http://www.ncbi.nlm.nih.gov/
2)Select “structure”
3)Enter name of protein in search window
4)Select name of a promising sequence
5)Select “protein”
6)Select “run BLAST”
7)Pick out interesting orthologs