chloroplast gene expression transcription rna processing (splicing, cleavages, modification)...
TRANSCRIPT
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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).
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J. Mullet, Aggieland
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Young (meristematic) cells w/proplastids
Older cells (etioplasts)
Barley (Hordeum vulgare) 7-10 days old
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• Etioplasts lack:
1. Chlorophyll
2. Photosynthetic capacity
3. Major thylakoid membrane proteins
Etioplastlight
Chloroplast
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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 > 1 phosphate at the 5’ end of the RNA.
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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 σ70 (or -10,-35) promoter; the consensus sequence is:
-35 -10 +1 TTGACA-------TATAAT------AAC--- (DNA)
5’ UUG… (RNA)
1. Distance between -10 and -35 regions critical2. " " -10 and start (+1) less critical3. Much variablility in the consensus sequence4. no -10, -35 for some cp genes (i.e. not always
required, at least 1 other type of promoter)
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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: 1. E. coli or eubacterial-like polymerase (also
called PEP, plastid-encoded polymerase)
2. 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, α2 ββ'
• α is encoded by the rpoA gene• β is encoded by the rpoB gene• β' is encoded by the rpoC1 and rpoC2 genes
– Sigma factor needed to initiate transcription at the bacterial 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:1. Endonucleases - cut internally (e.g., between genes),
fairly specific
2. 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:1. proteins recognize the 3'-IR, bind and stop a
processive exonuclease
2. An endonuclease cleaves at the 3’-IR
3. Combination of the two above
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3’- end processing and stabilization of chloroplast mRNAs
D. Stern, Cornell
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Pathways of Cp pre-mRNA Processing & Degradation in Chlamydomonas
(a) and (b) may use some of the same enzymes D. Stern, Cornell
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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)
Chloroplast polyribosomes will use E. coli soluble factors for elongation and termination phases.
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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 start5'----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.
In vitro translation w/chloroplast extract: Hirose and Sugiura, 1996.
EMBO J. 15, 1687–1695.
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mRNA recognition/binding using the Shine-Dalgarno sequence in plastid mRNAs
Fig. 9.17
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Differences with bacteria
1. Many chloroplast mRNAs have relatively long (~ 300 nt) 5' untranslated regions (UTR) that bind proteins.
2. Many chloroplast mRNAs don’t have a S-D sequence, and in 1 case, it
suppresses translation (Sugiura lab).3. Must be another initiation mechanism
• Scanning ?• Some of the proteins that bind the 5’ UTRs of
mRNAs promote translation
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Chloroplast tRNAs:Chloroplast translation relies heavily on wobble (or 2 out of 3) pairing between the tRNA anticodon and the mRNA
codon.