Isolated Spinach chloroplast thylakoid membrane stroma envelope From Hoober 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 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) Chloroplast phage-like polymerase (NEP) 1. 2. 3.
4. 5. Similar to 1-subunit phage RNA polymerases Nuclear gene(s) Enzyme insensitive to rifampicin Recognize promoter of 7-10 bp Specificity factor not yet identified
Some chloroplast genes have promoters for both the PEP and NEP RNAPs.. Fig. 6.31 in Buchanan et al. Why is chloroplast transcription much more complex than mitochondrial transcription?
Chloroplasts are larger, more complex organelles, that differentiate. 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 From U. Wisconsin Botany Dept. Plastid types develop from proplastids: Shoots: light-> proplastids <----> etioplasts <----> chloroplasts chromoplasts
Roots: proplastids <----> amyloplasts Complex suite of RNAPs provides for developmental regulation. 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 Monocistronic and/or Polycistronic Transcription
Prokaryotes Both Eukaryotes 1. Nucleus Monocistronic (polycistronic rare) 2. Mitochondria Mammals Polycistronic (2 promoters) Other lower species Both 3. Plastids - Both
Transcription in the Eukaryotic Nucleus RNA Polymerases Promoters for each polymerase
General transcription factors Regulatory factors and combinatorial regulation 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+ Roeder and Rutters separation of 3 nuclear RNA
polymerases from sea urchin embryos by ion exchange chromatography on DEAE-Sephadex Fig. 10.1 Nucleoplasmic fraction enriched in Pol II
Nucleolar fractionEnriched in Pol I Fig. 10.2 Determining Roles for Each Nuclear RNA Polymerase (nRNAP) Purified polymerases dont 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).
Fig. 10.3 aminitin, from Amanita phalloides (death cap mushroom). Actinomycin D, from Streptomyces
Intercalating Portion. 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 + S + 18S + 18S + S + 5.8S + 18S + S rRNAs) 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). 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
Subunit structure of purified nRNAPs All 3 have 10-14 subunits. Subunits range from 8S + 18S + 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
