Objectives - Organelle gene expression
& signaling:
List the molecular processes or steps involved in
going from organelle gene to functional organelle
protein complex and briefly describe a technical
approach that can be used to assay each of these
steps
Discuss the ways in which various organelle gene
expression steps can be inter-dependent and give
examples
Describe molecular mechanisms that adapt plastid
gene expression to different light environments
Define retrograde regulation and describe the
nature of retrograde signaling molecules
Describe the nature and functions of plant
pentatricopeptide repeat (PPR) proteins
Discuss the reasons that PPR proteins are wellsuited to be a central player in multiple organelle
gene expression processes
Design a genetic screen to identify nuclear genes
that function in plastid gene expression and explain
how you will analyze mutants to determine which
plastid genes are affected
What are the processes needed to
take us from gene to fully functional,
multi-subunit, organelle protein
complex?
Plastid gene expression overview
Translation
(del Campo Gene Reg & Syst Biol 3:31)
Organelle DNA copy number can
influence gene expression levels
1 – Consider RUBISCO – the most
abundant protein on earth!
2 – Mitochondrial orf239 in Phaseolus
vulgaris
•
•
•
•
•
cytoplasmic male sterility (CMS) gene
locates on a subgenomic molecule
high copy number > CMS
reduced copy number > pollen fertility
copy number mediated by nuclear
gene – Fr
(Mackenzie and Chase Plant Cell 2:905)
RNA Polymerases and promoters
Polymerase
Subunits
Consensus promoter
αββ’ β’’& σ 70
-35/-10
GTGTTGACA/TATAA
TG
Plastid –
encoded
(PEP)
αββ’ &
nuclearencoded σ
specificity
-35/-10
-TTGACA/TATAAT
Phage T7
single core
no σ
overlaps initiation
ATACGACTCACTATA
GGGAGA
Nuclear encoded
plastid
(NEP)
T7-like core &
+/- specificity
factor
overlaps initiation
ATAGAAT A/G AA
Nuclear –
encoded
mit
T7-like core &
+/- specificity
factor
Bacterial
overlaps initiation
CRTA G/T
Differential plastid gene expression based
upon recognition of distinct promoters
by NEP and PEP
Most plastid genes have promoters for both
polymerases
Genes encoding expression machinery (e.g.
rpo, rrn, rps, rpl) primarily transcribed by
NEP
Photosynthetic genes primarily transcribed
by PEP
(from Hajdukiewicz et al. EMBO J 16:4041)
Organelle transcripts - initiated vs.
processed 5’ ends
initiated
5’ end - PPP
* processed 5’
end - P
PPP
*
Organelle transcripts - initiated vs.
processed 5’ ends
Processed transcripts 5’ mono-phosphate
Substrate for ligation
e.g. RNA adapter for 5’ RACE
e.g. Self-ligation -> Circularization
Initiated transcripts 5’ tri-phosphate
Ligate only after de-phosphorylation
tobacco acid pyrophosphatese (TAP)
Compare 5’ RACE products +/—TAP
initiated transcript –not a ligation substrate
5’PPP
RNA
3’
processed or TAP-treated transcript
RNA
adaptor
Adaptor
primer
RNA
P
cDNA
PCR product
3’
3’ Gene
3’ primer
PCR products containing initiated 5’ ends
appear only after TAP treatment
Identification of promoters in
Arabidopsis plastids
+ T: with tobacco acid pyrophosphatase treatment
- T: no pyrophosphatase treatment
g: green tissue
w: white tissue (seedlings grown on spectinomycin)
[Swiatecka-Hagenbruch Mol Genet Genomics 277:725]
Diversity of promoters in
Arabidopsis plastids
[Swiatecka-Hagenbruch Mol Genet Genomics 277:725]
Plasticity of promoters in
Arabidopsis mitochondria
+TAP
- TAP
[Kühn et al. Nucleic Acids Res. 33:337]
Plasticity of promoters in
Arabidopsis mitochondria
Consensus for 20 sequences supporting
initiation at A
Consensus of 11 sequences supporting
initiation at G
[Kühn et al. Nucleic Acids Res. 33:337]
Differential plastid gene
expression - polymerases and sigma
subunits
[from
Lopez-Juez and
Pyke Intl Int.
J Dev J.
BiolDev.
49:557]
[Lopez-Juez
& Pyke,
Biol. 49: 557 ]
Multiple sigma factors of A.
thaliana with different plastid
promoter targets
II(↑) over
expression
+Sig2 +Sig5
I (↓) under expression
−Sig2
−Sig4
trnEYD ndhF
trnV
trnM
psaJ
psbAa
constpsbDb
−Sig5
LRPpsbDb
−Sig6
atpBEtrnEYD psaA
2.6kbb
psbAc psbA
psbA
psbBc
psbB
psbCc
psbD
psbDc
psbHc
psbNc
psbTc
rbcLc
rrn16c
rrn23c
rrn5c
rrn4.5c
SIG2 and SIG6 are essential
– knock outs are chlorophyll deficient
[Lysenko, Plant Cell Rep. 26:845]
Redox regulation of
photosynthetic gene expression
is adaptive
PSI
PSII
PET
Light II
PSII most efficient
PSI less efficient
Additional PSI subunits
needed
PQ highly reduced
(as in + DBMIB)
Light I
PSI most efficient
PSII less efficient
Additional PSII
subunits needed
PQ highly oxidized
(as in + DCMU)
[Surpin, Plant Cell Supplement 2002:S327]
Regulation of plastid transcription
through plastid redox signals
PSI
PSII
Why do the curves for relative transcript amounts
and relative transcription activity differ? What do
these two things measure?
Complementary changes in transcription rate and
mRNA abundance for psaAB (photosystem I) and
psbA (photosystem II) during acclimation to light I
or light II
[Pfannschmidt et al. Nature 397:625]
Regulation of nuclear gene
transcription through plastid redox
signals
PSI or PET nuclear gene promoters
• Fused to GUS reporter gene
• GUS activity measured in response to light
changes
[Pfannschmidt et al. J Biol Chem. 276:36125]
Transduction pathways of
photosynthetic redox signals
[Pfannschmidt et al. Ann Bot 103:599]
Plant organelle RNA metabolism
Plant organelle genes are often cotranscribed
• Plastid operons
• Mitochondria – di-cistronic transcripts
In contrast to prokaryotic transcripts,
plant organelle transcripts:
• Are processed to di or mono-cistronic
transcripts
• Frequently contain introns
• Must undergo RNA editing
Plant organelle RNA metabolism:
psbB operon processing in maize
Plastid operons
Processed to di or mono-cistronic forms
• endo- and exo-nucleases
• termini stabilized by stem-loops
• termini stabilized by PPR protein
binding
[Barkan Plant Physiol 155:1524]
Plant organelle RNA metabolism:
psbB operon processing in maize
Plastid operons
Frequently contain introns
• Splicing mediated by different sub-sets
of nuclear-encoded RNA binding proteins
[Barkan Plant Physiol 155:1524]
Plant organelle RNA metabolism:
psbB operon processing in maize
RNA processing factors are discovered
through forward genetics!!!!!!!!!!!!
• APO1, APO2
• HCF107, HCF152
• CAF1, CAF2
• RNC1
• CFM3
• WTF1
• CRP1
[Barkan Plant Physiol 155:1524]
High chlorophyll fluorescence (hcf)
mutants (maize and arabidopsis)
Mutants in the nuclear genes required for
plastid biogenesis and function
hcf/hcf > pale-green, yellow, or albino
seedlings; some fluoresce in the dark due
to dysfunctional photosystems
hcf/hcf seedlings are lethal, but in maize
they grow large enough for molecular
analysis
[Jenkins et al. Plant Cell 9:283]
Nuclear mutation crp1
Disrupts processing of the psbB operon
 missing in crp1/crp1 mutant seedlings:
• 1.1 and 0.75 kb petB RNA
• 0.75 kb petD RNA
•How do we see this experimentally?
[Barkan et al. EMBOJ13:3170]
Nuclear mutation crp1
• Disrupts processing of the psbB-psbHpetB-petD operon
Which proteins are reduced in the crp1
mutant?
We saw RNA processing effects for petB &
petD transcripts.
Why might PSAA/B be affected?
Why are ALL of the PET protein subunits
missing? (Hint, there must be 50 ways to
lose a protein, name two!)
(Barkan et al. EMBOJ 13:3170)
Nuclear mutation crp1
PET A,B,C,D protein translation studies
35S-labeled
leaf
proteins
immunoprecipitated
35S-labeled
in
organello
synthesized
proteins
immunoprecipitated
Which proteins are translated in the crp1
mutant? Which are not?
We saw PETA, B,C & D proteins did not
accumulate in this mutant.
What explains the difference between
translation and accumulation?
[Barkan et al. EMBOJ 13:3170]
Nuclear mutation crp1
PET A,B,C,D protein translation studies
petB stop
codon
petD start
codon
Secondary structures of monocistronic
petD (left) and bi-cistronic petB-petD
(right) transcripts
Propose a model: How does and RNA
processing defect interfere with protein
synthesis?
[Barkan et al. EMBOJ 13:3170]
Inter-dependence of plant organelle
gene expression steps
No monocistronic petD transcripts and no
PETD translation
• The petD initiation codon is buried in
secondary structure in the petB / petD
transcript
• The petD initiation codon is free of
secondary structure in the monocistronic
petD transcript
But what about
• PETB and PETC
– Translated but no accumulation
– What is likely mechanism here?
• PETA
– Not translated !
– What possible mechanisms here?
CRP1 associates w/ the 5’ region of
the petA transcript
Immunoprecipitate CRP1 RNA-protein
complexes
Slot-blot and hybridize
• Immunoprecipitated RNA (pellet)
• Unbound RNA (supernatant)
PET1 protein associates with regions 5’ of
petA and 5’ of psaC
Does this show direct RNA binding?
[Schmitz-Linneweber et al. Plant Cell 17:2791]
CRP1- RNA interactions
Why is the identification of two interaction
sites much more powerful than one?
C – consensus RNA binging site for CRP1
based on two binding regions
D - model for
CRP1 protein –
RNA interaction
[Schmitz-Linneweber et al. Plant Cell 17:2791]
CRP1 is a Pentatricopeptide repeat
(PPR) protein
One of the largest multigene families in
plants
• 441 members in arabidopsis vs 7 in
humans
Plastid- or mitochondria-targeted
Most aspects of post-transcriptional RNA
metabolism
• e.g. crp1 locus in maize necessary for
plastid petB / petD RNA processing
•e.g. restorer-of-fertility loci for CMS
in petunia, radish and rice all influence
processing or stability of mitochondrial
CMS gene transcripts
• e.g. editing of plastid ndh gene
transcripts
Pentatricopeptide repeat (PPR) proteins
Why so many?
•? RNA editing
How do they function?
• Site-specific RNA binding proteins
• Endo and Exonucleases
• Recruit enzymatic protein complexes
• Simply melt RNA structures to allow
interaction with processing, splicing,
translation & editing factors
Pentatricopeptide repeat (PPR) proteins
Motif Structure of Arabidopsis PPR
Proteins
• Degenerate 35 amino acid repeats
• The number and order of repeats can
vary in individual proteins
• The number of proteins falling into each
subgroup is shown
[Lurin et al. Plant Cell 16:2089]
Plant organelle introns
Group I and Group II, defined by
characteristic secondary structures and
splicing mechanisms
[from Gillham 1994 Organelle Genes and Genomes]
Organelle introns
Group II intron structural domains are the
ancestor of the nuclear splicosomal RNAs
splicosomal RNAs
[from Gillham 1994 Organelle Genes and Genomes]
Organelle introns
In land plants almost all are group II
• Spoke-and-wheel structure
• Necessary for splicing
• Some fungal group IIs self-splice in vitro
• RNA &/or protein factors required in vivo
e.g. maize nuclear genes crs1 & crs2
encode proteins required for splicing
•Genome rearrangements have split some
group II introns
Require trans-splicing
Spoke-and-wheel structure can be
assembled from separate transcripts!
Organelle introns
How do we see whether introns are spliced
or not? There are lots of ways!
Reverse transcribe + PCR (RT-PCR)
DNA/
un-spliced RNA
<R 3’
cDNA 5’
<R 3’
PCR 5’ F>
spliced RNA
cDNA
PCR
5’
<R 3’
5’ F>
<R 3’
Others you may see:
• Ribonuclease protection
• Poison primer RT-PCR
• RNA blot hybridization
The maize crs1 and crs2 mutants
disrupt the splicing of different
group II introns
rps16
intron
[Jenkins et al. Plant Cell 9:283]
Plant organelle intron splicing
requires multiple nuclear-encoded
splicing factors
[Watkins et al. Plant Cell 23:1082
Trans-splicing Chlamydomonas
psaA transcripts
i1 5’ end
i1 3’ end
[Gillham 1994 Organelle Genes and Genomes]
Plant organelle RNA metabolism:
psbB operon processing in maize
What two features confer RNA stability?
For nuclear-encoded transcripts 3’ poly A
stabilizes
For organelle-encoded transcripts 3’ poly A
tract DE-stabilizes
• Also a de-stabilizing feature of
bacterial transcripts
• Enhances susceptibility to degradation
by exonucleases
[Barkan Plant Physiol 155:1524]
Plant organelle RNA editing
Post transcriptional enzymatic conversion
of C > U, or less commonly, U > C
Given a fully sequenced organelle genome,
how would the RNA editing process be
detected?
genomic coding strand
5’ ....... ACG.....
unedited RNA
5’ ....... ACG.....
edited RNA
5’ ....... AUG....
edited cDNA
5’ ....... ATG.....
Occurs in plastids and plant mitochondria
• many more mitochondrial sites
Primarily in coding sequences
•conserves predicted protein
Creates initiation codons
ACG > AUG
Creates termination codons
CGA > UGA
Removes termination codons UGA > CGA
Changes amino acid coding
CCA > CUA
(P > L)
Silent edits
CTT > CTC
(L > L)
Plant organelle RNA editing
Edit sites within the same gene vary
among species
• An edit site in one species may be
“pre-edited” (correctly encoded in the
genomic sequence) of another species
• e.g. plastid psbL gene initiation
codon:
maize
ATGACA.....
tobacco ACGACA..... must be
edited to AUG (RNA) = ATG (cDNA)
for translation initiation codon
Evolution of plant organelle RNA editing
Not in algae
Observed in
every land plant
lineage except
Marchantiid
liverworts
[Knoop , Curr Genet 46:123]
RNA editing improves evolutionary
conservation
Table 1. Evolutionary conserved amino acid residues
changed by C-to-U editing in ribosomal protein S12
(RPS12) of plant mitochondria
Amino acid residues encoded by unedited and edited
maize mitochondrial transcripts compared to amino
acid residues in RPS12 polypeptides from other taxa
[Mulligan and Maliga (1998) pp.153-161 In A look beyond
transcription
J Bailey-Serres and DR Gallie (eds) ASPB]
RNA editing by enzymatic de-amination
 32P UTP
V
 32P CTP >
32P CTP
[Rajasekhar and
Mulligan Plant Cell 5:1843]
[Russell, 1995, Genetics]
Short 5’ flanking sequences
define plant organelle RNA editing sites
[from Mulligan and Maliga (1998) pp.153-161 In A look
beyond transcription
J Bailey-Serres and DR Gallie (eds) ASPB]
RNA editing – genetic analysis
defines a trans-acting factor
[from Kotera et al. Nature 433:326]
Genetic analysis defines a PPRmotif RNA editing factor
[from Kotera et al. Nature 433:326]
Genetic analysis defines a PPRmotif RNA editing factor
The immunoblots implicating crr4 in NDH complex
biogenesis showed loss of the NDHH subunit, but
the affected RNA editing site is in the ndhD
transcript. What are some explanations for these
observations?
[from Kotera et al. Nature 433:326]
Translation of organelle genes
A significant regulatory process in
plastid gene expression
light-regulated chloroplast protein
accumulation increases 50-100 fold
w/out changes in mRNA accumulation
5’ UTR is key in regulating translation
~ 1/2 of plastid transcripts have a 5’
Shine-Delgarno sequence (GGAG)
homologous to small subunit rRNA in
this region
nuclear-encoded translation factors
bind 5’ untranslated region (UTR) (and
in some cases also the 3’ UTR)
Translation of organelle genes
Regulation of plastid gene translation by
light - mediated by pH, ADP, redox signals
e.g. translation of PSII D1 (PSBA) in
Chlamydomonas
• Accumulation of PSBA increased in light
• No change in steady-state level of mRNA
• Site-directed mutagenesis of 5’ UTR
5’ SD sequence
5’ stem-loop region
Required for translation
• 5’UTR binding proteins identified
Binding increased 10X in the light
Reduced thioredoxin required for
binding
Binding abolished by oxidation
Binding decreased by ADP-dependent
phosphorylation (ADP accumulates in
the dark)
The details of this mechanism are NOT
conserved in angiosperms
Redox regulation of PSBA protein
synthesis in Chlamydomonas
[Pfannschmit (2003) Trends Plant Sci 8:33]
Translation of organelle genes- PPR
protein RNA re-modeling enhances
ATPH translation
Translation of organelle genes
Control by Epistasy of Synthesis (CES)
Regulation of protein synthesis by
presence or absence of assembly
partners
e.g. Down-regulation of tobacco nuclear
rbcS gene by antisense
•Decreased translation of rbcL in plastid
e.g. Chlamydomonas plastid cytochrome f
(PET complex)
Absent other subunits, cytochrome f
cannot assemble
• Unassembled cytochrome f binds to
its own (petA ) 5’ UTR
• Down regulates translation
Organelle protein complex assembly
and protein turn-over
Failure to assemble a protein complex >
degradation of unassembled subunits
Assembly dependent upon availability of all
subunits and co-factors
Plastids contain several proteases that are
homologues of bacterial proteases
o Functions in protein turn-over
o ? Protease independent chaperone
functions (as seen in bacteria)
Bacterial – type proteases in plastids
Protease
Location and Function
in plastid
ClpP/ClpC
stroma
ATP-dependent serine
protease
degrades mis-targeted
proteins and cytb6/f
subunits
FtsH
stromal face of thylakoid
membranes
membrane-bound, ATPdependent metalloprotease
DegP
serine heat-shock
protease
degrades photo-damaged
PSI protein D1 from
stromal side
lumenal side of thylakoid
membranes
degrades photo-damaged
PSI protein D1 from
lumen side