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. Next Assignment
Presentation on genome editing
Presentation on expressing a eukaryotic protein in bacteria
Presentation on expressing a eukaryotic protein in another eukaryote

4. Cloning Orthologs
1) Identify sequence from an organism that we can obtain
2) Identify coding sequence:
Most sites identify Start and Stop codons
We need 5’ and 3’ UTR!
At Genbank or others can often obtain additional flanking sequence, eg, by clicking “CDS”
Or, get gene name and check at TAIR, MPSS or GRAMENE
If not, will need to obtain it based on position
3) Design primers to obtain entire CDS + minimal flanking sequence from suitable source!

5. Cloning Orthologs
Design primers to obtain entire CDS + minimal flanking sequence from suitable source!
Design nested primers that start at ATG and end at stop codon and add CACC at 5’ end
Probably will need to do this manually!
Will break all the rules for primer design
May get away with it if only use them on
Amplicon
Limited templates to bind

6. 5’-TACTCGAAAGCAAAAGTCGTAG 62°C 3’-TTAGGCCGGGTAGCCACGC 60°C
Cloning Orthologs
Design primers to obtain entire CDS + minimal flanking sequence from suitable source!
Design nested primers that start at ATG and end at stop codon and add CACC at 5’ end
Probably will need to do this manually!
Will break all the rules for primer design
May get away with it if only use them on
Amplicon
Limited templates to bind
Test at
5’-TACTCGAAAGCAAAAGTCGTAG 62°C
3’-TTAGGCCGGGTAGCCACGC 60°C

7. Cloning Orthologs
Design primers to obtain entire CDS + minimal flanking sequence from suitable source!
Design nested primers that start at ATG and end at stop codon and add CACC at 5’ end
Probably will need to do this manually
Extract high MW genomic DNA from E.coli
& S. elongatus

8. Cloning Orthologs
Design primers to obtain entire CDS + minimal flanking sequence from suitable source!
Design nested primers that start at ATG and end at stop codon and add CACC at 5’ end
Probably will need to do this manually!
Extract high MW genomic DNA from E.coli
& S. elongatus
5) Do the two PCR rxns: if rxn 2 gives band of
expected size clone it into pSyn_1/D-TOP

9. Cloning Orthologs
Design primers to obtain entire CDS + minimal flanking sequence from suitable source!
Design nested primers that start at ATG and end at stop codon and add CACC at 5’ end
Probably will need to do this manually!
Extract high MW genomic DNA from E.coli
& S. elongatus
5) Do the two PCR rxns: if rxn 2 gives band of
expected size clone it into pSyn_1/D-TOP
6) Identify clones by PCR & restriction digests

10. Cloning Orthologs
Design primers to obtain entire CDS + minimal flanking sequence from suitable source!
Design nested primers that start at ATG and end at stop codon and add CACC at 5’ end
Probably will need to do this manually!
Extract high MW genomic DNA from E.coli
& S. elongatus
5) Do the two PCR rxns: if rxn 2 gives band of
expected size clone it into pSyn_1/D-TOP
Identify clones by PCR & restriction digests
Verify by sequencing

11. Regulating gene expression
Goal is
controlling
Proteins
How many?
Where?
How active?
8 levels (two not
shown are mRNA
localization & prot
degradation)

12. Regulating gene expression
1) initiating
transcription
most important
~50% of control

13. Microarrays
Compare chromatin modification in different tissues or treatments by immuno-precipitating chromatin with antibodies specific for a particular histone modification, eg H3K4me3 then labeling precipitated DNA & controls with a different dye = ChIP-chip

14. Selected tissue samples
Sequencing
Compare chromatin modification in different tissues or treatments by immuno-precipitating chromatin with antibodies specific for a particular histone modification, eg H3K4me3 then sequencing precipitated DNA = ChIP-seq
Selected tissue samples
Total RNA
Chromatin
Genomic DNA
small RNA
mRNA
ChIP DNA
McrBC
Solexa sequencing libraries
Solexa sequencing

15. Transcription in Eukaryotes
3 RNA polymerases
all are multi-subunit
complexes
5 in common
3 very similar
variable # unique ones
Now have Pols IV & V in plants
Make siRNA

16. Transcription in Eukaryotes
RNA polymerase I: 13 subunits ( unique)
acts exclusively in nucleolus to make 45S-rRNA precursor

17. Transcription in Eukaryotes
Pol I: acts exclusively in nucleolus to make 45S-rRNA precursor
accounts for 50% of total RNA synthesis

18. Transcription in Eukaryotes
Pol I: acts exclusively in nucleolus to make 45S-rRNA precursor
accounts for 50% of total RNA synthesis
insensitive to -aminitin

19. Transcription in Eukaryotes
Pol I: only makes 45S-rRNA precursor
50 % of total RNA synthesis
insensitive to -aminitin
Mg2+ cofactor
initiation frequency

20. promoter is 5' to "coding sequence" 2 elements
RNA polymerase I
promoter is 5' to "coding sequence"
2 elements
1) essential core includes transcription start site +1
-100
coding sequence
UCE
core

21. promoter is 5' to "coding sequence" 2 elements
RNA polymerase I
promoter is 5' to "coding sequence"
2 elements
1) essential core includes transcription start site
2) UCE (Upstream Control Element) at ~ stimulates transcription x +1
-100
coding sequence
UCE
core

22. Initiation of transcription by Pol I
Order of events was determined by in vitro reconstitution
1) UBF (upstream binding factor) binds UCE and core element
UBF is a transcription factor: DNA-binding proteins which recruit polymerases and tell them where to begin

23. Initiation of transcription by Pol I
1) UBF binds UCE and core element
2) SL1 (selectivity factor 1) binds UBF (not DNA)
SL1 is a coactivator
proteins which
bind transcription
factors and
stimulate
transcription

24. Initiation of transcription by Pol I
1) UBF binds UCE and core element
2) SL1 (selectivity factor 1) binds UBF (not DNA)
SL1 is a complex of
4 proteins including
TBP (TATAA-
binding protein)

25. Initiation of transcription by Pol I
1) UBF binds UCE and core element
2) SL1 (selectivity factor 1) binds UBF (not DNA)
3) complex recruits Pol I

26. Initiation of transcription by Pol I
1) UBF binds UCE and core element
2) SL1 (selectivity factor 1) binds UBF (not DNA)
3) complex recruits Pol I
4) Pol I transcribes until
it hits a termination site

27. Processing rRNA
~ 200 bases are methylated
Box C/D snoRNA picks sites

28. Processing rRNA
~ 200 bases are methylated
Box C/D snoRNA picks sites
One for each!

29. Processing rRNA
~ 200 bases are methylated
Box C/D snoRNA picks sites
One for each!
2) Another ~ 200 are pseudo-uridylated
Box H/ACA snoRNAs pick sites

30. Processing rRNA
~ 400 bases are methylated or pseudo-uridylated
snoRNAs pick sites
One for each!
2) 45S pre-rRNA is cut into 28S, 18S and 5.8S products

31. Processing rRNA
~ 200 bases are methylated
2) 45S pre-rRNA is cut into 28S,
18S and 5.8S products
3) Ribosomes are assembled w/in nucleolus

32. RNA Polymerase III
Reconstituted in vitro
makes ribosomal 5S and tRNA
(+ some snRNA & scRNA)

33. RNA Polymerase III
makes ribosomal 5S and tRNA
(+ some snRNA & scRNA)
>100 different kinds of genes
~10% of all RNA synthesis

34. RNA Polymerase III
makes ribosomal 5S and tRNA
(+ some snRNA & scRNA)
>100 different kinds of genes
~10% of all RNA synthesis
Cofactor = Mn2+ cf Mg2+

35. RNA Polymerase III
makes ribosomal 5S and tRNA
(+ some snRNA & scRNA)
>100 different kinds of genes
~10% of all RNA synthesis
Cofactor = Mn2+ cf Mg2+
sensitive to high [-aminitin]

36. RNA Polymerase III
makes ribosomal 5S and tRNA (also some snRNA and some scRNA)
Has the most subunits

37. RNA Polymerase III
makes ribosomal 5S and tRNA (also some snRNA and some scRNA)
Has the most subunits
initiation frequency

38. Initiation of transcription by Pol III
promoter is often w/in "coding" sequence!

39. Initiation of transcription by Pol III
promoter is w/in "coding" sequence!
5S & tRNA promoters differ
5S has single “C” box

40. Initiation of transcription by Pol III
1) TFIIIA binds C box in 5S
2) recruits TFIIIC

41. Initiation of transcription by Pol III
TFIIIA binds C box
recruits TFIIIC
3) TFIIIB binds
TBP & 2 others

42. Initiation of Pol III transcription
1) TFIIIA binds C box
2) recruits TFIIIC
3) TFIIIB binds
4) Complex recruits Pol III

43. Initiation of Pol III transcription
1) TFIIIA binds C box
2) recruits TFIIIC
3) TFIIIB binds
4) Complex recruits Pol III
5) Pol III goes until
hits > 4 T's

44. Initiation of transcription by Pol III
promoter is w/in coding sequence!
5S & tRNA promoters differ
tRNA genes have “A” & “B” boxes

45. Initiation of transcription by Pol III
tRNA genes have “A” and “B” boxes
1) TFIIIC binds B box

46. Initiation of transcription by Pol III
tRNA genes have “A” and “B” boxes
1) TFIIIC binds B box
2) recruits TFIIIB

47. Initiation of transcription by Pol III
1) TFIIIC binds box B
2) recruits TFIIIB
3) complex recruits Pol III

48. Initiation of transcription by Pol III
1) TFIIIC binds box B
2) recruits TFIIIB
3) complex recruits Pol III
4) Pol III runs until
hits > 4 Ts

49. Processing tRNA
tRNA is trimmed
5’ end by RNAse P
(contains RNA)
3’ end by RNAse Z
Or by exonucleases

50. Processing tRNA
tRNA is trimmed
Transcript is spliced
Some tRNAs are
assembled from 2 transcripts

51. transferase (no template)
Processing tRNA
tRNA is trimmed
Transcript is spliced
CCA is added to 3’ end
By tRNA nucleotidyl
transferase (no template)
tRNA +CTP -> tRNA-C + PPi tRNA-C +CTP--> tRNA-C-C + PPi tRNA-C-C +ATP -> tRNA-C-C-A + PPi

52. Processing tRNA
tRNA is trimmed
Transcript is spliced
CCA is added to 3’ end
Many bases are modified
Significance unclear

53. Processing tRNA
tRNA is trimmed
Transcript is spliced
CCA is added to 3’ end
Many bases are modified
No cap! -> 5’ P
(due to 5’ RNAse P cut)