Isolated Spinach chloroplast envelope stroma thylakoid membrane 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.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

Fig 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 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

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.

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 Mg 2+ –Another less G-C rich RNA fraction was preferentially synthesized at higher ionic strength with Mn 2+

Roeder and Rutter’s separation of 3 nuclear RNA polymerases from sea urchin embryos by ion exchange chromatography on DEAE-Sephadex Fig. 10.1

Nucleolar fraction- Enriched in Pol I Nucleoplasmic fraction – enriched in Pol II Fig. 10.2

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:  -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. 10.3

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 + 5.8S rRNAs)

RNA Polymerase II 1.Actinomycin D, at low concentrations, did not inhibit synthesis of heterogenous nuclear RNA (hn RNA).  -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 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