1. Project
Studying Synechococcus elongatus
for biophotovoltaics

2. 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!

3. In bacteria transcription and translation are initially coupled

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

5. 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

6. Bacteria have > 1 protein/mRNA (polycistronic)
euk have 1 protein/mRNA

7. Bacteria have > 1 protein/mRNA (polycistronic)
Mutations can have polar effects: mutations in upstream genes may affect expression of perfectly good downstream genes!

8. Transcription
Prokaryotes have one RNA polymerase
makes all RNA
core polymerase = complex of 5 subunits (a1aIIbb’w)

9. 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

10. Initiating transcription in Prokaryotes
1) Core RNA polymerase is promiscuous

11. Initiating transcription in Prokaryotes
Core RNA polymerase is promiscuous
sigma factors provide specificity

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

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

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

15. Initiating transcription in Prokaryotes
3) Once bound, RNA polymerase
“melts” the DNA
4) rNTPs bind template

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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

23. Structure of Prokaryotic promoters
Other sequences also often influence transcription! Our plasmid contains the nickel promoter.

24. Structure of Prokaryotic promoters
Other sequences also often influence transcription! Our plasmid contains the nickel promoter. ↵

25. Structure of Prokaryotic promoters
Other sequences also often influence transcription! Our plasmid contains the nickel promoter.
nrsBACD encode nickel transporters

26. 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

27. 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

28. 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

29. Termination of transcription in prokaryotes
1) Sometimes go until ribosomes fall too far behind

30. 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”

31. 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

32. Homologous recombination
1) DNA strands must be capable of base-pairing

33. Homologous recombination
DNA strands must be capable of base-pairing
DNA must get cut

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

35. 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)

36. 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!

37. 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

38. 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

39. 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

40. 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

41. 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

42. Finding Orthologs
Go to
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

43. Go to http://www.ncbi.nlm.nih.gov/ Select “structure”
Finding Orthologs
Go to
Select “structure”
Enter name of protein in search window
Select name of a promising sequence
Select “protein”
Select “run BLAST”
Pick out interesting orthologs
Slr1962 protein
TRAP dicarboxylate transporter