Project

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
• Were adding DNA, but want Synechococcus to make
  a protein!

3

• Next Assignment
• Presentation on genome editing http//www.sciencem
  ag.org/content/339/6121/768.full
• 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

• 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 http//www.idtdna.com/analyzer/application
  s/oligoanalyzer/
• 5-TACTCGAAAGCAAAAGTCGTAG 62C
• 3-TTAGGCCGGGTAGCCACGC 60C

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
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
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 (5 3 5 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
• Regulated _at_ initiation frequency

20
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
RNA polymerase I promoter is 5' to "coding
sequence" 2 elements 1) essential core includes
transcription start site 2) UCE (Upstream
Control Element) at -100 stimulates
transcription 10-100x
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) gt100 different kinds of
genes 10 of all RNA synthesis
34
RNA Polymerase III makes ribosomal 5S and tRNA
( some snRNA scRNA) gt100 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) gt100 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
• Regulated _at_
• 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 gt 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 gt 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

• Processing tRNA
• tRNA is trimmed
• Transcript is spliced
• CCA is added to 3 end
• By tRNA nucleotidyl
• transferase (no template)
• tRNA CTP -gt tRNA-C PPitRNA-C CTP--gt tRNA-C-C
  PPitRNA-C-C ATP -gt 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! -gt 5 P
• (due to 5 RNAse P cut)