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Post-transcriptional Control of
Chloroplast Gene Expression
via RNA-Protein Complexes
Current
Josh Baecker
Patricia Merhige
Brandon Stilb
Department of Energy
Past
Dawn Both
Tracy Hotchkiss
Mark Robida
Chloroplasts are like big green prokaryotes
Spinach Chloroplast
genome: 150,725 bp
However, the majority of chloroplast polypeptides are
encoded by the nucleus.
Post-transcriptional Mechanisms Have
Important Effects on
Chloroplast Gene Expression
RNA processing
RNA stability
Translation
Mediated by
RNA-protein
Complexes
Protein modification/complex assembly
Protein turnover
RNA-protein Complexes Can Form in
5’Untranslated Regions (5’UTRs)
5’UTRs are sequences upstream of
open reading frames
In chloroplasts, they often encode
ribosome binding sites.
Proteins that bind to chloroplast 5’UTRs
have been shown to affect
RNA stability and/or translation initiation.
RNA-protein Complexes Can Be Analyzed
by Gel Mobility Shift Assays
+
RNAs
Potential binding proteins
(specific, radiolabeled)
(non-specific)
(specific)
(non-specific)
(non-binding)
Incubation
Self competition:
Chloroplast extract: RNA-protein
complexes
{
Native gel
Unbound RNA:
- +
+ +
Questions to Be Answered About Chloroplast
RNA-protein Complexes
What are the cis-acting elements that affect complex formation?
What are the proteins involved in the complexes?
What is the function of these complexes?
What are the cis-acting elements
that affect complex formation?
Is there conserved RNA structure?
Where do the proteins bind?
The atpI 5’UTR Is Our Baseline Binding Substrate
Arabidopsis
Calycanthus
Lotus
Maize
Nightshade
Pea
Primrose
Rice
Tobacco
Wheat
Spinach
Consensus
..........
.........G
GAAATTTTTG
..........
..........
..........
.........G
.........G
..........
.........G
..........
..........
..........
GTTACTATCC
ATTAATAATA
..........
CTTACTATTT
..GGGTATTA
GTTACTACTT
GGGAATATTG
..........
GGGAATATTG
......GATT
..........
..........
CTGAATCTTA
AAATAAAAAA
..........
CTGAATTTCA
TAGCATTACA
TTGAATCGCA
ATATATATTA
..........
ATATATATTA
TTGAATCTCA
....a....a
..........
.AAAAGAGAT
ATAAAGAGAA
....GGGTAT
AAAAAGAGAT
AAATTGTGTA
TAAAAGAGAT
..GAGGGTAT
..........
..GAGGGTAT
AAAACTAGTT
..........
.....GGGGA
AAAAATAAGT
TAAAGAACAA
TGATATATAT
AAAAATAACT
AAAAGAA...
CAAAATGGAT
TGATATATAT
..........
TGATATATAT
CAAAATAACA
......R...
Arabidopsis
Calycanthus
Lotus
Maize
Nightshade
Pea
Primrose
Rice
Tobacco
Wheat
Spinach
Consensus
TATTGTGTGA
GGGGATATTA
ACAGAAATAT
TATGATCTGA
GGGGATATTA
....ATATTT
GGAGATGCTG
TATCATCTGA
GGGGATATTA
TATGATCTGA
GGGGATATTA
....AT.T..
TTTGTTTAGT
TGCGATCGCT
TATG..TGAT
TGTGATTTCT
GGTGATTTAT
TGTGATTAGT
TGTGATTGGT
TGTGATTTCT
TGTGATTTAT
TGTGCTTTCT
TGTAATTAAT
TgTG.TT..T
TGGGATCCAA
TGGTATCCGA
TAGTAGATAA
TGGTATATTA
TAGTATTCGA
AGGTATTCAA
TAGTATACAA
TGATATCCTA
TAGTATTCTA
TGGTATCCTA
CAGTATCCGA
tRGTAT.c.A
AAC.TAAAAT
AATACACGAT
AATAGAAAAT
AATATAAGAT
AATCTTAG.T
AATAGAAAAT
AATAGAAAAT
AATATAAGAT
AATCTTAG.T
AATATAAGAT
AATAGAAAAT
AAT..aARaT
ATAAAATTTA
TAA.AGTAGG
CAA.AGTAGA
TAATACTTCA
TGGTATTCAA
CAA.......
TCA.ACTAAG
TAATACTTCA
TGGTATTCAA
TAATACTTCA
TTT.AATTAA
Y.R.a.T..R
Arabidopsis
Calycanthus
Lotus
Maize
Nightshade
Pea
Primrose
Rice
Tobacco
Wheat
Spinach
Consensus
AGTAAATAAG
GG....CGAG
......CAAG
AGTTGCTGAG
AATATCCGAT
........AG
......CAAG
CGTTGCTGAG
AATATCCGAT
AGTTGCTGAG
AGTAGACAAG
......YRAg
TAAAAAAAAA
CCGAGAA...
TCAAAAA...
TTGAGAA...
TCAAGTAGAC
TAAAATA...
TCAAAAA...
TTGAGAA...
TCAAGTAGAC
TTGAGAA...
TCGAGAA...
TyRAR.A...
GGGGGGGT.C
.AG.AGATGT
.AGGAGATGA
.AG.AGATGC
AAAGAGATGG
.AGGAAATGG
.AG.AGATGG
.AA.AGATGG
AAAGAGATGG
.AG.AGATGG
.AG.AGCTGA
.AR.AGaTG.
TTGAATCAAA
TTGAATCAAA
TTGAATCAAA
TTGAATCAAA
TTGAATCAAA
TTGAATCAAA
TTGAATCAAA
TTGAATCAAA
TTGAATCAAA
TTGAATCAAA
TTGAATCAAA
TTGAATCAAA
ATAATTTA..
ATAATTCCTT
ATAATTCCCT
AGAATTCCTT
AAATTTTGTT
ATAATTCCCT
ATAATTCCTT
AGAATTCTTT
AAATTTTGTT
AGAATTCCTT
ATAATT..TT
A.AaTTy.yT
Arabidopsis
Calycanthus
Lotus
Maize
Nightshade
Pea
Primrose
Rice
Tobacco
Wheat
Spinach
Consensus
........AA
TTT.....AA
TTC.....AA
TTT...TGAA
TA......AA
TTCAAGTTAT
TTA.....AA
TTT...TGAA
TA......AA
TTT...TGAA
TTT.....AA
T.......AA
GTTCTTATTT
GTTCT.ATTT
GTTCT.ATTT
GTTCA.ATTT
GTTCA.ATTT
ATT.T.TTTT
GTTCG.ATTT
GTTCA.TTTT
GTTCA.ATTT
GTTCA.ATTT
GTTAT.ATTT
GTTc..aTTT
CTGTCAGAGG
CTGTCAGAGG
CGTTTAGAGG
TTATCAGAGG
TT.TCAGAGG
ATTTTAGAGG
ATGTGAGAGG
TTATCAGGGG
TT.TCAGAGG
TTATCAGAGG
CTGTAAGAGG
yT.T.AGAGG
GC.....AAT
GC.....AAT
GCGTGGCAAT
AC.....AAT
GCAAGGCAAT
ACAGGGCAAT
GC.....A.T
AC.....AAT
GCAAGGCAAT
AC.....AAT
AC.....AAT
RC.....AAT
ATG
ATG
ATG
ATG
ATG
ATG
ATG
ATG
ATG
ATG
ATG
ATG
THERE ARE TWO BINDING SITES
IN THE atpI 5’UTR
ORF
-155
-52
-1 +15
C: + - E: + + *
*
ORF
-155
C: + - E: + + -
-58
-52
C: + - E: + + -
*
*
C: self-competitor
E: chloroplast extract
*: RNA-protein complex
-1 +15
What are the proteins involved
in the complexes?
Are they ATP synthase-specific?
Competition Binding Assays Revealed that
the Same Chloroplast Proteins Bind Diverse 5’UTRs
Good Competitors; similar binding proteins
atpI
ATP synthase subunit CFo-IV
atpA ATP synthase subunit CF1-α
atpE
ATP synthase subunit CF1-ε
Partial competitor; some shared proteins
atpF
ATP synthase subunit CFo- I
atpH ATP synthase subunit CFo-III rps16 small ribosome subunit 16
clpP
protease subunit
infA
translation initiation factor
Poor competitor; few shared proteins
ndhD NADH dehydrogenase subunit
psbA
Photosystem II D1 protein
petL
cytochrome b/f 3.5 kDa subunit
psaC photosystem I 9 kDa protein
rbcL large subunit, carboxylase
rpl22 large ribosome subunit protein 22
Supershift
rpoA RNA polymerase α-subunit
atpB
ATP synthase subunit CF1-β
rpoB RNA polymerase β-subunit
rps2
small ribosome subunit protein 2
rps11 small ribosomal subunit 11
Affinity purification of binding proteins
atpI 5’UTR
Streptavidinbinding motif
Chloroplast extract
Incubate
S
Isolate
mag
bead
SDS-PAGE of affinity-isolated 5’UTR-binding proteins
MW Unbound W1
66
55
42
37
27
20
14
W2
W3
Isolated
What is the function
of these complexes?
Do they affect translation?
Do they affect RNA stability?
Chloroplast Transformation
Target Plants (Week 2)
Second Selection (Week 10)
Antibiotic-resistant
transformants (Week 6)
Rooting (Week 12)
Roots
Variations on a single 5’UTR
Wild-type
D
Deletion
Chimeric
Complete
deletion
(except for RBS)
D
misc.
5’ UTR
atpI
sequence deletion 5’ UTR
uidA
reporter
rps16
3’ UTR
The atpI 5’UTR Affects Translation
and RNA Abundance
Conclusions & Projects
Cis-acting elements
There are two binding sites and extensive phylogentically conserved
sequence in the atpI 5’UTR
Structure probing studies will be used to identify specific
structure(s) necessary for binding.
Trans-acting factors
The same polypeptides bind many chloroplast 5’UTRs.
Affinity-based methods will be used to isolate the binding protein(s)
Once the genes encoding them are cloned, they will be mutated
and expressed in vivo
Function
Preliminary data demonstrate an effect of the atpI 5’UTR on
both RNA stability and translation.
In vivo experiments will be used to dissect 5’UTR function.