Isolated Spinach chloroplast envelope stroma thylakoid membrane From Hoober.
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Transcript of Isolated Spinach chloroplast envelope stroma thylakoid membrane From Hoober.
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Isolated Spinach chloroplast
envelope
stroma
thylakoid membrane
From Hoober
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Chloroplast RNA polymerases (RNAPs)
Two different RNAPs in vascular plant chloroplasts:
1. Bacterial-like polymerase (also called PEP, plastid-encoded polymerase)
2. Phage-like or NEP (nuclear-encoded polymerase) polymerase
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Chloroplast Bacterial-like RNAP
• Inhibited by Rifampicin• composed of Core + Sigma factor
1. Core = 4 subunits, 2 ' (rpoA) (rpoB gene is sometimes split) ' (rpoC1 and rpoC2)
2. Sigma factor (recognizes -10, -35 promoters) • Nuclear-encoded, 6 genes in Arabidopsis (3
of which have non-overlapping targets)
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Chloroplast phage-like polymerase (NEP)
1. Similar to 1-subunit phage RNA polymerases
2. Nuclear gene(s)
3. Enzyme insensitive to rifampicin
4. Recognize promoter of 7-10 bp
5. Specificity factor not yet identified
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Fig. 6.31 in Buchanan et al.
Some chloroplast genes have promoters for both the PEP and NEP RNAPs..
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Why is chloroplast transcription much more complex than mitochondrial transcription?
Chloroplasts are larger, more complex organelles, that differentiate.
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Chloroplasts are a type of Plastid
1. Proplastids – precursor form, in meristems
2. Etioplasts - in shoots of dark-grown plants
3. Chloroplasts - green tissues
4. Amyloplasts - prominent in roots, store starch, colorless
5. Chromoplasts - mature fruit, carotenoids
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From U. Wisconsin Botany Dept.
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Plastid types develop from proplastids:
Shoots: light->
proplastids <----> etioplasts <----> chloroplasts
chromoplasts
Roots:proplastids <----> amyloplasts
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1. NEP more important in proplastids (needed to make the rpo genes).
2. PEP more important in chloroplasts.3. PEP also regulated by sigma factors:
- selective transcription by different sigma genes
- phosphorylation of sigmas
Complex suite of RNAPs provides for developmental regulation.
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Monocistronic and/or Polycistronic Transcription
• Prokaryotes – Both
• Eukaryotes1. Nucleus – Monocistronic (polycistronic
rare)
2. Mitochondria– Mammals – Polycistronic (2 promoters)– Other lower species – Both
3. Plastids - Both
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Transcription in the Eukaryotic Nucleus
• RNA Polymerases
• Promoters for each polymerase
• General transcription factors
• Regulatory factors and combinatorial regulation
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Studies of RNA synthesis by isolated nuclei
• RNA synthesis by isolated nuclei indicated that there were at least 2 polymerases; one of which was in the nucleolus and synthesized rRNA– rRNA often has a higher G-C content than other
RNAs; a G-C rich RNA fraction was preferentially synthesized with low ionic strength and Mg2+
– Another less G-C rich RNA fraction was preferentially synthesized at higher ionic strength with Mn2+
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Roeder and Rutter’s separation of 3 nuclear RNA polymerases from sea urchin embryos by ion exchange
chromatography on DEAE-Sephadex
Fig. 10.1
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Nucleolar fraction-Enriched in Pol I
Nucleoplasmic fraction – enriched in Pol II
Fig. 10.2
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Determining Roles for Each Nuclear RNA Polymerase (nRNAP)
• Purified polymerases don’t transcribe DNA specifically – so used nuclear fractions.
• Also useful were two transcription inhibitors:
1. -aminitin – from a mushroom, inhibits RNAP II, and RNAP III at higher concentrations.
2. Actinomycin D - general transcription inhibitor, binds DNA and intercalates into helix, prefers G-C rich regions (like rRNA genes).
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α – aminitin, from Amanita phalloides (death cap mushroom).
Fig. 10.3
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Actinomycin D,from Streptomyces Intercalating
Portion.
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RNA Polymerase I
1. Not inhibited by aminitin, but inhibited by low concentrations of actinomycin D.
2. RNA produced in the presence of -aminitin could be competed by rRNA for
hybridization to (rat) DNA.
Conclusion: nRNAP I synthesizes the rRNA precursor (45S pre-rRNA 28S + 18S + 5.8S rRNAs)
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RNA Polymerase II
1. Actinomycin D, at low concentrations, did not inhibit synthesis of heterogenous nuclear RNA (hn RNA).
2. -aminitin inhibited synthesis of hnRNA in nucleoplasmic fraction.
• Conclusion: nRNAP II synthesizes hnRNA (mostly mRNA precursors).
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RNA Polymerase III
• Synthesis of small abundant RNAs inhibited only at high [-aminitin]
– Small RNAs: tRNA precursors, 5S rRNA, U6 (involved in splicing), and 7SL RNA (involved in protein secretion through the ER, part of the signal recognition particle).
• Conclusion: nRNAP III synthesizes many of the small abundant cytoplasmic and nuclear RNAs
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Subunit structure of purified nRNAPs
• All 3 have 10-14 subunits.
• Subunits range from 8 to 220 kDa.
• All 3 have 2 very large (>125 kD) subunits and several smaller ones.
• Several of the smaller subunits (5 in yeast) are common to all 3
RNAPs.
Human RNAP II, Table 10.2