Slide 1Chloroplast RNA polymerases (RNAPs)Chloroplast Bacterial-like RNAPChloroplast phage-like polymerase (NEP)Slide 5Slide 6Chloroplasts are a type of PlastidSlide 8Slide 9Slide 10Monocistronic and/or Polycistronic TranscriptionTranscription in the Eukaryotic NucleusStudies of RNA synthesis by isolated nucleiRoeder and Rutter’s separation of 3 nuclear RNA polymerases from sea urchin embryos by ion exchange chromatography on DEAE-SephadexSlide 15Determining Roles for Each Nuclear RNA Polymerase (nRNAP)Slide 17Slide 18RNA Polymerase IRNA Polymerase IIRNA Polymerase IIISlide 22Subunit structure of purified nRNAPsIsolated Spinach chloroplastenvelopestromathylakoid membraneFrom HooberChloroplast 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) polymeraseChloroplast Bacterial-like RNAP•Inhibited by Rifampicin•composed of Core + Sigma factor1. 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. Similar to 1-subunit phage RNA polymerases2. Nuclear gene(s)3. Enzyme insensitive to rifampicin4. Recognize promoter of 7-10 bp 5. Specificity factor not yet identifiedFig. 6.31 in Buchanan et al.Some chloroplast genes have promoters for both the PEP and NEP RNAPs..Why is chloroplast transcription much more complex than mitochondrial transcription?Chloroplasts are larger, more complex organelles, that differentiate.Chloroplasts are a type of Plastid1. Proplastids – precursor form, in meristems2. Etioplasts - in shoots of dark-grown plants3. Chloroplasts - green tissues4. Amyloplasts - prominent in roots, store starch, colorless5. Chromoplasts - mature fruit, carotenoidsFrom U. Wisconsin Botany Dept.Plastid types develop from proplastids:Shoots: light->proplastids <----> etioplasts <----> chloroplasts chromoplasts Roots:proplastids <----> amyloplasts1. 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 sigmasComplex suite of RNAPs provides for developmental regulation.Monocistronic and/or Polycistronic Transcription•Prokaryotes – Both •Eukaryotes1. Nucleus – Monocistronic (polycistronic rare)2. Mitochondria–Mammals – Polycistronic (2 promoters)–Other lower species – Both3. Plastids - BothTranscription in the Eukaryotic Nucleus•RNA Polymerases•Promoters for each polymerase•General transcription factors•Regulatory factors and combinatorial regulationStudies 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 Rutter’s separation of 3 nuclear RNA polymerases from sea urchin embryos by ion exchange chromatography on DEAE-Sephadex Fig. 10.1Nucleolar fraction-Enriched in Pol INucleoplasmic fraction – enriched in Pol IIFig. 10.2Determining 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).α – aminitin, from Amanita phalloides (death cap mushroom).Fig. 10.3Actinomycin D,from StreptomycesIntercalatingPortion.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)RNA Polymerase II1. 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 RNAsSubunit 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