Translation in Chloroplasts

1
Translation in Chloroplasts

• Translation machinery is bacteria-like
• Ribosomes
• -70S (composed of L (50S) and S (30S)
  subunits)
• -contain 23S (L), 16S (S), and 5S (L) rRNAs
• -each subunit (L and S) contains 30 proteins
• Initiation factors if1, if2, if3
• Elongation factors ef-Tu, ef-Ts, and G
• Translation is initiated with fmet (formylated
  Met)

2
How mRNAs selected for translation?

• Many cp mRNAs contain a Shine-Dalgarno sequence
  preceding the first codon it base-pairs to the
  3'-end of 16S rRNA.
• S-D start
• 5'----GGAGG-------AUG-----3 mRNA
• 3'----CCUCC--------5' 16S rRNA
• Start codon (AUG) very important for starting
  translation at right codon.
• Can translate internal ORFs of a polycistronic
  transcript.

3
mRNA recognition/binding using the Shine-Dalgarno
sequence in plastid mRNAs
Fig. 9.17
4
Differences with bacteria

• Many chloroplast mRNAs have relatively long (
  300 nt) 5' untranslated regions (UTR) that bind
  proteins.
• Many chloroplast mRNAs dont have a S-D sequence,
  or it is not important (redundant?).
• Must be another initiation mechanism
• Scanning ?
• Some of the proteins that bind the 5 UTRs of
  mRNAs promote translation

5
Chloroplast tRNAs Chloroplast translation relies
heavily on wobble (or 2 out of 3) pairing between
the tRNA anticodon and the mRNA codon.
6
Regulation of Chloroplast Gene Expression

• Studied principally during photomorphogenesis
  i.e., development of cotyledons and leaves during
  "greening" (etioplast -gt chloroplast).
• Also studied (in mature chloroplasts) during
  light-dark cycles, and in response to certain
  stresses (heat, cold, radiation).
• Multiple levels are regulated for most genes.
• Difficult to generalize, but some trends emerge.

7
Plastid Transcriptional Regulation

• Transcriptional regulation is often global or
  large-scale
• NEP functions early in development, PEP
  dominates later (etioplast ? chloroplast)
• PEP-transcribed genes increase or decrease
  together
• E.g. - overall transcription increases during
  "greening", but decreases during chloroplast ?
  chromoplast
• There are examples of gene-specific
  transcriptional regulation
• psbD/psbC promoter switching in response to light

8
Plastid development is plastic, mostly under
nuclear control. Shoots light
proplastids etioplasts chloroplasts
chromoplasts Roots proplastids
amyloplasts
PEP
NEP
9
psbD-psbC Light-responsive Promoter (LRP or BLRP)

• Preferentially utilized in the light (not dark)
  stimulated by blue and UV light.
• Also shows circadian rhythm of utilization.
• Evolutionarily conserved among higher plants.
• PEP-type promoter, but the -35 region not
  necessary.
• 2 upstream regions important for the
  light-response PGT and AAG boxes.
• Boxes bind proteins (PTF1, AGF) binding of PTF1
  is inhibited by ADP-dependent phosphorylation
  (ADP levels increase in darkness).

10
BLRP promoter
11
psbD BLRP
Schematic diagram of the barley psbD-LRP and
constructs used for plastid transformation. A.
The boxed regions identify conserved sequences
which include the PGT-box (-71 to -100), AAG-box
(-36 to -56) and the prokaryotic-like -10 (-7 to
-12) and -35 (-28 to -33) promoter elements. The
psbD open-reading frame is shown at the far
right. The direction of transcription is
represented as an arrow and the initiation site
is labeled as 1. A sequence alignment between
the barley (Sexton et al., 1990b) and tobacco
(Shinozaki et al., 1986) psbD-LRP was made with
the ClustalW 1.7 Multiple Sequence Alignment
Program. Aligned nucleotide sequences
corresponding to conserved sequences are boxed in
and labeled accordingly. Numbering of nucleotides
and designation of conserved promoter elements
are in accordance with the structure of the
barley psbD-LRP from the transcription initiation
site (1).
(From Thum et al. 2001)
12
Models of transcription complexes associated with
the psbD BLRP, rbcL and psbA promoters
- extra TATA box likely maintains high rate of
transcription in mature chloroplast
Kim, M. et al. J. Biol. Chem. 19992744684-4692
13
Regulation of RNA splicing stability

• Splicing of psbA introns (Group I) in Chlamy is
  strongly promoted by light ( redox).
• Splicing of some photosynthetic genes introns
  (Group II) is inefficient in maize roots
  (amyloplasts), but efficient in leaves.
• Stability of some plastid mRNAs increases during
  greening (psbA), but most decrease in mature
  chloroplasts in the light.

14
Translational Regulation

• Cp mRNAs are relatively long-lived (half lives of
  0.5 to 8 h or more)
• Translation is regulated by
• Global changes in rate (e.g., light-dark cycles)
• e.g. - high in daytime, low at night
• Preferential translation of specific mRNAs under
  certain conditions.
• e.g.- very high light intensity increases psbA
  translation and decreases rbcL translation

15
Light-activated translation of psbA mRNA

• Complex of proteins that bind to the 5 UTR of
  psbA mRNA in the light.
• Demonstrate with gel-shift (electrophoretic
  mobility shift) assay.

Lane 1 control (no protein extract) Lane 2 -
extract from light-grown cells Lane 3 - extract
from dark-grown cells
Box 9.4 in Buchanan et al.
16
Proteins in complex that bind to the 5 UTR of
psbA mRNA

• PABP - similar to a polyA-binding protein, binds
  A-U rich region in the 5 UTR, activates
  translation
• PDI - a protein disulfide isomerase (reduces
  disulfide bonds on certain proteins), activates
  PABP to bind RNA
• Kinase responds to ADP levels, at high ADP,
  kinase deactivates PDI by phosphorylating it

17
Model for Activation of psbA translation by Light
via photosynthesis.
Fig. 9.23 in Buchanan et al.
18
Translational regulation of RuBPCase LS by SS
Incoming SS somehow promotes translation of rbcL
mRNA!
Fig. 9.16 in Buchanan et al.
19
Rough Thylakoids

• Polyribosome (polysomes) can be observed bound
  to thylakoid membranes.
• At least some of these polysomes are attached to
  the membrane by the nascent (new) protein.
• Suggests these polysomes make thylakoid membrane
  proteins and simultaneously insert them into the
  membrane.
• Chloroplasts also contain a Signal Recognition
  Particle (SRP) homologue.

20
Thylakoid-bound polysomes from Chlamydomonas
Occur in the light period of
a light-dark cycle
polysome
polysome
A. Michaels, M. Margulies and G. Palade (1972)
21
Stabilization of nascent chlorophyll - binding
proteins of PSI and PSII with Chlorophyll
Fig 9.24 in Buchanan et al.
22
Regulation of protein stability in chloroplasts

• Protein stability is regulated by
• binding of cofactors (e.g., chlorophyll)
• assembly with other subunits in a multi-subunit
  enzyme complex

23
Photoinhibition inhibition of photosynthesis at
very high light flux
Photosystem II damage is critical.
Box 9.6 in Buchanan et al.
24
psbA encodes 32-35 kDa D1 polypeptide of PSII
Yamamoto, Plant Cell Physiol. 2001
D1 protein turns over rapidly because it becomes
damaged in the light.
25
D1 turnover and replacement is ongoing and
critical

• At photoinhibitory light intensity, D1 protein of
  PSII is damaged faster that it can be removed
  (and degraded).
• At most lower light intensities, degradation,
  synthesis and replacement of D1 keeps up with the
  damage rate.

26
Nuclear Control of Chloroplast Gene Expression

• Genetic studies have revealed considerable
  potential for nuclear control of specific cp
  genes.
• Many Mendelian (nuclear) mutants that are
  defective in plastid development or function
  dont express a specific Cp-encoded gene.

27
Cp genes and their required nuclear gene function
in Chlamydomonas.
Cp gene step affected no. of
nuclear loci
28
Does the cross-talk go both ways way? (Does the
chloroplast control nuclear genes?)

• Nuclear genes that encode proteins for
  photosynthesis are expressed poorly if plastids
  don't develop, or are damaged by photo-oxidation.
  
• - suggest signal from plastids to nucleus
• Signal?
• - small molecule?
• - intermediate in chlorophyll synthesis?