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. 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

4. 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

5. Cloning Orthologs
Identify sequence from an organism that we can obtain
For eubacteria DNA will do: no introns

6. Cloning Orthologs
Identify sequence from an organism that we can obtain
For eubacteria DNA will do
For plants, animals, fungi or archaebacteria need mRNA

7. Cloning Orthologs
Identify sequence from an
organism that we can obtain
For eubacteria DNA will do
For plants, animals, fungi or archaebacteria need mRNA: DNA & 1˚ RNA contain introns

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

9. mRNA PROCESSING
Primary transcript is hnRNA
undergoes 3 processing reactions before export to cytosol
All three are coordinated with transcription & affect gene expression: enzymes piggy-back on POLII

10. mRNA PROCESSING
Primary transcript is hnRNA
undergoes 3 processing reactions before export to cytosol
1) Capping addition of 7-methyl G to 5’ end

11. mRNA PROCESSING
Primary transcript is hnRNA
undergoes 3 processing reactions before export to cytosol
1) Capping addition of 7-methyl G to 5’ end
identifies it as mRNA: needed for export & translation

12. mRNA PROCESSING
Primary transcript is hnRNA
undergoes 3 processing reactions before export to cytosol
1) Capping addition of 7-methyl G to 5’ end
identifies it as mRNA: needed for export & translation
Catalyzed by CEC attached to POLII

13. mRNA PROCESSING
1) Capping
2) Splicing: removal of introns
Evidence:
electron microscopy
sequence alignment

14. Splicing: the spliceosome cycle
1) U1 snRNP (RNA/protein complex) binds 5’ splice site

15. Splicing:The spliceosome cycle
1) U1 snRNP binds 5’ splice site
2) U2 snRNP binds “branchpoint”
-> displaces A at branchpoint

16. Splicing:The spliceosome cycle
1) U1 snRNP binds 5’ splice site
2) U2 snRNP binds “branchpoint”
-> displaces A at branchpoint
3) U4/U5/U6 complex
binds intron
displace U1
spliceosome
has now assembled

17. Splicing:
RNA is cut at 5’ splice site
cut end is trans-esterified to branchpoint A

18. Splicing: 5) RNA is cut at 3’ splice site
6) 5’ end of exon 2 is ligated to 3’ end of exon 1
7) everything disassembles -> “lariat intron” is degraded

19. Splicing:The spliceosome cycle

20. Splicing:
Some RNAs can self-splice!
role of snRNPs is to increase rate!
Why splice?

21. Splicing:
Why splice?
1) Generate diversity
exons often encode protein domains

22. Splicing:
Why splice?
1) Generate diversity
exons often encode protein domains
Introns = larger target for insertions, recombination

23. Why splice?
1) Generate diversity
>94% of human genes show alternate splicing

24. Why splice?
1) Generate diversity
>94% of human genes show alternate splicing
same gene encodes
different protein
in different tissues

25. Why splice?
1) Generate diversity
>94% of human genes show alternate splicing
same gene encodes
different protein
in different tissues
Stressed plants use
AS to make variant
stress-response
proteins

26. Why splice?
1) Generate diversity
>94% of human genes show alternate splicing
same gene encodes
different protein
in different tissues
Stressed plants use
AS to make variant
Stress-response
proteins
Splice-regulator
proteins control AS:
regulated by cell-specific
expression and phosphorylation

27. Splicing:
Why splice?
1) Generate diversity
2) Modulate gene expression
introns affect amount of mRNA produced

28. mRNA Processing: RNA editing
Two types: C->U and A->I

29. mRNA Processing: RNA editing
Two types: C->U and A->I
Plant mito and cp use C -> U
>300 different editing events have been detected in plant mitochondria: some create start & stop codons

30. mRNA Processing: RNA editing
Two types: C->U and A->I
Plant mito and cp use C -> U
>300 different editing events have been detected in plant mitochondria: some create start & stop codons: way to prevent nucleus from stealing genes!

31. mRNA Processing: RNA editing
Human intestines edit APOB mRNA C -> U to create a stop aa 2153 (APOB48) cf full-length APOB100
APOB48 lacks the CTD LDL receptor binding site

32. mRNA Processing: RNA editing
Human intestines edit APOB mRNA C -> U to create a stop aa 2153 (APOB48) cf full-length APOB100
APOB48 lacks the CTD LDL receptor binding site
Liver makes APOB100 -> correlates with heart disease

33. mRNA Processing: RNA editing
Two types: C->U and A->I
Adenosine de-aminases (ADA) are ubiquitously expressed in mammals
act on dsRNA & convert A to I (read as G)

34. mRNA Processing: RNA editing
Two types: C->U and A->I
Adenosine de-aminases (ADA) are ubiquitously expressed in mammals
act on dsRNA & convert A to I (read as G)
misregulation of A-to-I RNA editing has been implicated in epilepsy, amyotrophic lateral sclerosis & depression

35. mRNA Processing: Polyadenylation
Addition of As to end of mRNA
Why bother?
helps identify as mRNA
required for translation
way to measure age of mRNA
->mRNA s with < 200 As have short half-life

36. mRNA Processing: Polyadenylation
Addition of As to end of mRNA
Why bother?
helps identify as mRNA
required for translation
way to measure age of mRNA
->mRNA s with < 200 As have short half-life
>50% of human mRNAs have alternative polyA sites!

37. mRNA Processing: Polyadenylation
>50% of human mRNAs have alternative polyA sites!

38. mRNA Processing: Polyadenylation
>50% of human mRNAs have alternative polyA sites!
result : different mRNA, can result in altered export, stability or different proteins

39. mRNA Processing: Polyadenylation
>50% of human mRNAs have alternative polyA sites!
result : different mRNA, can result in altered export, stability or different proteins
some thalassemias are due to mis-poly A

40. mRNA Processing: Polyadenylation
some thalassemias are due to mis-poly A
Influenza shuts down nuclear genes by preventing poly-Adenylation (viral protein binds CPSF)

41. mRNA Processing: Polyadenylation
1) CPSF (Cleavage and Polyadenylation Specificity Factor) binds AAUAAA in hnRNA

42. mRNA Processing: Polyadenylation
1) CPSF binds AAUAAA in hnRNA
2) CStF (Cleavage Stimulatory Factor) binds G/U rich sequence 50 bases downstream
CFI, CFII bind in between

43. Polyadenylation
1) CPSF binds AAUAAA in hnRNA
2) CStF binds; CFI, CFII bind in between
3) PAP (PolyA polymerase) binds & cleaves b 3’ to AAUAAA

44. mRNA Processing: Polyadenylation
3) PAP (PolyA polymerase) binds & cleaves b 3’ to AAUAAA
4) PAP adds As slowly, CFI, CFII and CPSF fall off

45. mRNA Processing: Polyadenylation
4) PAP adds As slowly, CFI, CFII and CPSF fall off
PABII binds, add
As rapidly until 250

46. Coordination of mRNA processing
Splicing and polyadenylation factors bind CTD of RNA Pol II-> mechanism to coordinate the three processes
Capping, Splicing and Polyadenylation all start before transcription is done!

47. Export from Nucleus
Occurs through nuclear
pores
anything >
40 kDa needs
exportin
protein
bound
to 5’ cap

48. Export from Nucleus
In cytoplasm nuclear proteins fall off, new proteins bind
eIF4E/eIF-4F bind cap
also new
proteins bind
polyA tail
mRNA is
ready to be
translated!

49. Cloning Orthologs
Identify sequence from an organism that we can obtain
For eubacteria DNA will do
For plants, animals, fungi or archaebacteria need mRNA
2) Identify coding sequence:
Most sites identify Start and Stop codons
We need 5’ and 3’ UTR!

50. Cloning Orthologs
Identify sequence from an organism that we can obtain
For eubacteria DNA will do
For plants, animals, fungi or archaebacteria need mRNA
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”

51. Cloning Orthologs
Identify sequence from an organism that we can obtain
For eubacteria DNA will do
For plants, animals, fungi or archaebacteria need mRNA
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

54. Cloning Orthologs
Identify sequence from an organism that we can obtain
For eubacteria DNA will do
For plants, animals, fungi or archaebacteria need mRNA
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

55. Cloning Orthologs
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!

56. Cloning Orthologs
Design primers to obtain entire CDS + minimal flanking sequence from suitable source!
Get Primer 3 (or other program) to design primers that bind upstream of start and downstream of stop

57. Cloning Orthologs
Design primers to obtain entire CDS + minimal flanking sequence from suitable source!
Get Primer 3 (or other program) to design primers that bind upstream of start and downstream of stop: exclude the CDS

58. 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

59. 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

60. 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

61. 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

62. 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
4) Do the two PCR rxns: if rxn 2 gives band of
expected size clone it into pSyn_1/D-TOP