Transcript PowerPoint 2

Objectives - Organelle gene expression
& signaling:
List the molecular processes involved in going from
organelle gene to functional organelle protein
complex
Describe the technical approaches used to
investigate each of these processes
Compare and contrast organelle gene expression
processes with those of bacterial and eukaryotic
gene expression systems
Discuss molecular mechanisms that adapt organelle
gene expression to environmental signals
Define retrograde regulation and discuss possible
organelle signals that alter nuclear gene expression
Describe the plant pentatricopeptide repeat (PPR)
gene/protein family with respect to the nature and
functions of PPR proteins
Discuss why PPR proteins are well-suited to be
central in multiple organelle gene expression
Discuss the ways in which various organelle gene
expression steps can be inter-dependent and give
examples
Plastid gene expression overview
Translation
(del Campo Gene Reg & Syst Biol 3:31)
Organelle DNA copy number can regulate
gene expression
Cytoplasmic male sterility (CMS) in Phaseolus
vulgaris
• CMS gene (orf239) on a mitochondrial
subgenomic molecule
The nuclear fertility restoration gene Fr
• Depresses copy number of orf239
sub-genome
• Decreased accumulation of orf239
transcripts
• Prevents expression of CMS
(Mackenzie and Chase Plant Cell 2:905)
RNA Polymerases and promoters
Polymerase
Subunits
Consensus promoter
Bacterial
αββ’ β’’& σ 70
-35/-10
GTGTTGACA/TATAATG
Plastid –
encoded
(PEP)
αββ’ & nuclearencoded σ
specificity
-35/-10
-TTGACA/TATAAT
Phage T7
single core
no σ
overlaps initiation
ATACGACTCACTATAGG
GAGA
T7-like core &
+/- specificity
factor
overlaps initiation
ATAGAAT A/G AA
Nuclear encoded
plastid (NEP)
Nuclear –
encoded mit
T7-like core &
+/- specificity
factor
overlaps initiation
CRTA G/T
Differential plastid gene expression based
upon recognition of distinct promoters
by NEP and PEP
(from Hajdukiewicz et al. EMBO J 16:4041
Organelle transcripts - initiated vs.
processed 5’ ends
initiated
5’ end
*
* processed 5’
end
PPP
PPP
Organelle transcript initiated vs.
processed 5’ ends
Processed transcripts have 5’ monophosphate
Substrate for ligation
e.g. RNA oligo nucleotide for 5’ RACE
e.g. Self-ligation -> Circularization
Polymerase-initiated transcripts have 5’ PP
or 5’PPP termini
Substrate only after de-phosphorylation w/
tobacco acid pyrophosphatese (TAP)
Compare 5’ RACE products +/—TAP
initiated transcript –not a ligation substrate
3’
5’PPP
naturally processed or TAP-treated transcript
Ligate
RNA
Adaptor
P
cDNA
Adaptor
primer
3’
Gene
primer
Products containing initiated 5’ ends appear only
after TAP treatment
Organelle transcript initiated vs.
processed 5’ ends
Processed transcripts have 5’ monophosphate
Substrate for ligation
e.g. RNA oligo nucleotide for 5’ RACE
e.g. Circularization
Initiated transcripts have 5’ PP or 5’PPP
termini
Substrate only after de-phosphorylation w/
Tobacco acid pyrophosphatese (TAP)
Compare PCR products +/-TAP
Initiated transcript –not a ligation substrate
5’PPP
3’
Dilute, self – ligate & reverse transcribe a naturally
processed or TAP-treated transcript
3’ 5’P
cDNA
Gene
primer 2
Gene
primer 2
Gene
primer
1
3’ 5’P
Gene
primer
1
Amplify and sequence across ligation junction
to identify 5’ and 3’ end sequences
Identification of promoters in
Arabidopsis plastids
+ T = + tobacco acid pyrophosphatase treatment
- T = without 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
[Kühn et al. Nucleic Acids Res. 33:337]
Differential plastid gene expression
based upon polymerases and sigma
subunits
[from Lopez-Juez
and Pyke
Intl J Dev
BiolJ.49:557]
[Lopez-Juez
& Pyke,
Int.
Dev. Biol. 49: 557 ]
Multiple sigma factors of A. thaliana
with different plastid promoter
targets in vivo
I (↓)
−Sig2
−Sig4
trnEYD ndhF
trnV
trnM
psaJ
psbAa
constpsbDb
−Sig5
LRPpsbDb
II(↑)
+Sig2
−Sig6
+Sig5
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 in Arabidopsis
– 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 PETE nuclear gene promoters
• Fused to GUS reporter gene
• GUS activity measured in response to light
changes
[Pfannschmidt et al. J Biol Chem. 276:36125]
Possible 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
psbB operon processing in maize
[Barkan et al. EMBOJ 13:3170]
Plant organelle RNA processing
Polycistronic transcripts undergo extensive,
complex processing prior to translation
e.g. psbB operon in maize, encoding subunits
of two different plastid protein complexes:
psbB / psbH / petB / petD
The nuclear mutation crp1 disrupts
processing of the polycistronic message and
consequently, PETB and PETD protein
accumulation
High chlorophyll fluorescence (hcf)
mutants (maize and arabidopsis)
Mutants in the nuclear genes required for
plastid biogenesis and function
~15% of the Aarabidopsis nuclear genome
predicted to plastid 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]
psbB operon processing in maize
A
B
  missing in
crp1/crp1 mutant
seedlings
[Barkan et al. EMBOJ13:3170]
The crp1 mutant disrupts petB/petD RNA
processing and PETD protein accumulation
Which protein complexes are, and which are
not, affected by the crp1 mutant?
(Barkan et al. EMBOJ 13:3170)
PET A,B, C& D protein translation
in wild-type and crp1 mutant maize
35S-labeled
leaf
proteins
35S-labeled
in
organello synthesized
proteins
petB stop
codon
petD start
codon
Secondary structures of monocistronic petD
(left) and bi-cistronic petB-petD (right)
transcripts
[Barkan et al. EMBOJ 13:3170]
Inter-dependence of plant organelle gene
expression steps
Model:
Failure to accumulate monocistronic petD
transcripts results in failure to translate
petD
•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
• PET C
– Translated but ...
– Reduced accumulation
– What is likely mechanism here?
• PETA
– Not translated !
– What possible mechanisms here?
CRP1 interacts directly at the 5’ region of
the petA transcript to promote translation
Immunoprecipitate CRP1 RNA-protein complexes
Slot-blot and hybridize
• Precipitated RNA (pellet)
• Unbound RNA (supernatant)
PET1 protein associates with regions 5’ of petA and
5’ of psaC
? Does this approach demonstrate 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]
Pentatricopeptide repeat (PPR) proteins
One of the largest multigene families in plants
• 441 members in arabidopsis vs 7 in humans
Primarily plastid- or mitochondria-targeted
Implicated in post-transcriptional RNA
metabolism through single gene/mutant
analysis
• 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
• Recruit enzymatic protein complexes
that act on RNA
- or • Melt RNA structures to allow processing,
splicing, translation & stabilization
[Lurin et al. Plant Cell 16:2089]
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]
Plant organelle introns
Group I and Group II have distinct splicing
mechansims
Group II is the ancestor of the nuclear intron
•Characteristic group II intron structural domains
= ancestors of the nuclear splicosomal RNAs
[from Gillham 1994 Organelle Genes and Genomes]
Plant organelle introns
Land plant organelle introns primarily Group II
•Characteristic spoke-and-wheel structure
• Necessary for splicing
•Some fungal versions self-splice in vitro
• Trans-acting RNA and/or protein factors
required for splicing in vivo
o e.g. maize nuclear genes (crs1 & crs2)
encode proteins required for splicing
•Genome rearrangements have split introns
oRequire trans-splicing
o Spoke-and-wheel structure assembled
from separate transcripts
The maize crs1 and crs2 mutants disrupt
the splicing of different group II introns
atpF
intron
rps16
intron
[Jenkins et al. Plant Cell 9:283]
Trans-splicing Chlamydomonas psaA
transcripts
i1 5’ end
i1 3’ end
[Gillham 1994 Organelle Genes and Genomes]
Plant organelle transcript stability
Plant organelle transcripts are stabilized by 3’
stem-loop structures
Removal of the stem loop (by endonuclease
cleavage) makes the 3’ end accessible for polyA
addition
PPR proteins can substitute for stem loops!
In contrast to nuclear transcripts, plant
organelle transcripts are destabilized by the
addition of 3’ poly A tracts
•3’ polyA is also a de-stabilizing feature of
bacterial transcripts
•3’ polyA enhances susceptibility of
transcript to degradation by exonucleases
Model for plastid mRNA turn-over
[from Monde et al. Biochimie 82:573]
Plant organelle RNA editing
Post transcriptional enzymatic conversion of C > U
• less commonly, U > C
Given a fully sequenced organelle genome, how
would the RNA editing process be detected?
genomic coding strand
unedited RNA
edited RNA
edited cDNA
5’ ....... ACG.....
5’ ....... ACG.....
5’ ....... AUG....
5’ ....... ATG.....
Occurs in plastids and plant mitochondria
• many more mitochondrial sites
Primarily in coding sequences
• improves overall conservation of predicted
protein
Creates initiation codons
Creates termination codons
Removes termination codons
Changes amino acid coding
Silent edits
ACG > AUG
CGA > UGA
UGA > CGA
CCA > CUA
(P > L)
ATC > ATU
Plant organelle RNA editing
Edit sites within the same gene vary among
species
• An edit site in one species may be “preedited” (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 S 12 (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 occurs 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]
Further evidence for cis-guiding sequences in
plant mitochondrial RNA editing
Editing of naturally recombinant or
rearranged mitochondrial genes
•Recombination breakpoint immediately 3’ to
an editing site in rice atp6 did not abolish
editing
•Recombination breakpoint seven nucleotides
5’ to an editing site in maize rps12 did abolish
editing
•Recombination breakpoint 21 nucleotides 5’ to
an editing site in maize rps12 did not abolish
editing
Electroporation of genes into isolated
mitochondria & analysis of cDNA
•Editing of mutated coxII gene demonstrated
sequences from –16 to +6 required for editing
What about the trans-acting editing machinery?
RNA editing – genetic analysis defines a
trans-acting factor
[from Kotera et al. Nature 433:326]
RNA editing – genetic analysis defines a
trans-acting factor
[from Kotera et al. Nature 433:326]
RNA editing – genetic analysis defines a
trans-acting factor
The immunoblots implicating crr4 in NDH complex
biogenesis showed loss of the NDHH subunit, but the
affected 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’ ShineDelgarno 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) protein in
Chlamydomonas
•Accumulation of PSBA increased in light
• No change in steady-state level of mRNA
•Site-directed mutagenesis of psbA 5’ UTR
o 5’ SD sequence
o 5’ stem-loop region
o Required for translation
•A set of 4 major 5’UTR binding proteins
identified
o Binding increased 10X in the light
o PSI reduced thioredoxin required for
binding
o Binding abolished by oxidation
o Binding decreased by ADP-dependent
phosphorylation
(ADP accumulates in the dark)
• The details of this mechanism do not appear
to be conserved in angiosperms
Photosynthetic redox chemistry &
plastid gene expression
Light II
PSII most efficient
PSI less efficient
More PSI needed
PQ highly reduced
(as in + DBMIB)
Light I
PSI most efficient
PSII less efficient
More PSII needed
PQ highly oxidized
(as in + DCMU)
[Pfannschmit Trends Plant Sci 8:33]
Redox regulation of PSBA protein
synthesis in Chlamydomonas
[from Pfannschmit (2003) Trends Plant Sci 8:33]
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 to down regulate 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
ATP-dependent serine
protease
stroma
FtsH
membrane-bound, ATPdependent metallo
protease
stromal face of thylakoid
membranes
DegP
serine heat-shock
protease
lumenal side of thylakoid
membranes
degrades photo-damaged
PSI protein D1 from
lumen side
degrades mis-targeted
proteins and cytb6/f
subunits
degrades photo-damaged
PSI protein D1 from
stromal side