CHLOROPLAST GENE EXPRESSION

1
CHLOROPLAST GENE EXPRESSION

• Transcription
• RNA processing (splicing, cleavages,
  modification)
• Translation
• Regulation
• Dependence on nuclear genes

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TRANSCRIPTION

• Many, but not all, cp genes are arranged in
  operon-like units and co-transcribed
• e.g., psbD-psbC gene cluster (see next slide)
• A unique feature of psbD-psbC gene transcription
  a different (closer) promoter is used in the
  light called the light-responsive promoter (LRP).
  

3
(No Transcript)
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How many promoters in cpDNA?

• 30 transcription units (promoters) in higher
  plant cp DNA
• determined experimentally by capping of cp RNA
  with guanylyl transferase and radioactive GT32P,
  and hybridization to cpDNA fragments.
• The transferase attaches GMP to the 5 end of
  RNAs that have 2 or 3 phosphates.
• Only primary transcription products have gt 1
  phosphate at the 5 end of the RNA.

5
A "transcription unit" is determined by the
position of the promoter (5') and terminator (3')
signals. Terminators not clearly defined, but
tRNA genes seem to be good transcription
terminators in chloroplasts.
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Cp Promoters

• Most resemble the major E. coli s70 (or -10,-35)
  promoter the consensus sequence is
• -35 -10 1
• TTGACA-------TATAAT------AAC--- (DNA)
• 5 UUG (RNA)
• Distance between -10 and -35 regions critical
• " " -10 and start (1)
  less critical
• Much variablility in the consensus sequence
• no -10, -35 for some cp genes (i.e. not always
  required, at least 1 other type of promoter)

7
Control of Cp transcription

• Transcription rate important
• mainly controlled at initiation step
• determined in part by "promoter strength
• also modulated for some genes (psbD) by
  upstream sequences that bind regulatory proteins
  
• Some genes have "alternative promoters"
• (e.g., psbD psbC)
• - also provides for regulation

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CP RNA polymerases

• Two main forms in vascular plants
• E. coli or eubacterial-like polymerase (also
  called PEP, plastid-encoded polymerase)
• Phage-like or NEP (nuclear-encoded polymerase)
  polymerase

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E. coli-like (PEP) polymerase

• composed of Core Sigma factor
• Core 4 subunits, a2 ßß'
• a is encoded by the rpoA gene
• ß is encoded by the rpoB gene
• ß' is encoded by the rpoC1 and rpoC2 genes
• Sigma factor needed to initiate transcription at
  the promoter (recognizes -10,-35 regions)
• Nuclear encoded, family of 6 genes in Arabidopsis
  
• Inhibited by rifampicin

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Fig. 6.31 in Buchanan et al.
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Phage-like (NEP) polymerase

• Catalytic subunit is similar to the 1-subunit
  phage (e.g., T7) and mitochondrial RNA
  polymerases
• Nuclear gene
• Enzyme insensitive to rifampicin
• Promoter is usually a single region of 7-10 bp
  (YRTA core), but other sequences stimulate
• Evolution
• Viral Origin?
• Mitochondrial origin?
• When did it get into plants?

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Why two chloroplast RNA polymerases? NEP is more
important early in plastid development when
plastid transcription (and translation) is
relatively low. - transcribes rRNA, rpo and
other genetic functions genes (GFG) PEP is
more important in mature chloroplasts. -
transcribes some GFG genes, but strongly
transcribes photosynthesis genes
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CP pre-mRNA PROCESSING

• Most, if not all primary transcripts are
  processed by cleavage(s) or splicing or both
• CP mRNAs are not polyadenylated, and are not
  "capped" (cap 7methylguanosine).
• Nucleolytic Cleavages
• Endonucleases - cut internally (e.g., between
  genes), fairly specific
• Exonucleases - trim at 3' or 5'-ends, processive,
  less specific

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Inverted repeats in cpRNA processing

• Inverted repeats occur at 3'-end of most cp
  protein-encoding genes.
• - processing sites, determine the 3'-end of
  mRNAs
• mechanisms
• proteins recognize the 3'-IR, bind and stop a
  processive exonuclease
• An endonuclease cleaves at the 3-IR
• Combination of the two above

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3- end processing and stabilization of
chloroplast mRNAs
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Pathways of Cp pre-mRNA Processing Degradation
in Chlamydomonas
(a) and (b) may use some of the same enzymes