Title: Monocistronic vs Polycistronic Genes
1Isolated Spinach chloroplast
envelope
thylakoid membrane
stroma
From Hoober
2Chloroplast RNA polymerases (RNAPs)
- Two different RNAPs in vascular plant
chloroplasts - Bacterial-like polymerase (also called PEP,
plastid-encoded polymerase) - Phage-like or NEP (nuclear-encoded polymerase)
polymerase
3Chloroplast Bacterial-like RNAP
- Inhibited by Rifampicin
- composed of Core Sigma factor
- Core 4 subunits, ?2 ? ?'
- ? (rpoA)
- ? (rpoB gene is sometimes split)
- ?' (rpoC1 and rpoC2)
- Sigma factor (recognizes -10, -35 promoters)
- Nuclear-encoded, 6 genes in Arabidopsis (3 of
which have non-overlapping targets)
4Chloroplast phage-like polymerase (NEP)
- 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
5Some chloroplast genes have promoters for both
the PEP and NEP RNAPs..
Fig. 6.31 in Buchanan et al.
6Why is chloroplast transcription much more
complex than mitochondrial transcription?
Chloroplasts are larger, more complex organelles,
that differentiate.
7Chloroplasts 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
8From U. Wisconsin Botany Dept.
9Plastid types develop from proplastids Shoots
light-gt proplastids lt----gt etioplasts
lt----gt chloroplasts chromoplasts
Roots proplastids lt----gt amyloplasts
10Complex suite of RNAPs provides for developmental
regulation.
- NEP more important in proplastids (needed to make
the rpo genes). - PEP more important in chloroplasts.
- PEP also regulated by sigma factors
- - selective transcription by different sigma
genes - - phosphorylation of sigmas
11Monocistronic and/or Polycistronic Transcription
- Prokaryotes Both
- Eukaryotes
- Nucleus Monocistronic (polycistronic rare)
- Mitochondria
- Mammals Polycistronic (2 promoters)
- Other lower species Both
- Plastids - Both
12Transcription in the Eukaryotic Nucleus
- RNA Polymerases
- Promoters for each polymerase
- General transcription factors
- Regulatory factors and combinatorial regulation
13Studies 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
14Roeder and Rutters separation of 3 nuclear RNA
polymerases from sea urchin embryos by ion
exchange chromatography on DEAE-Sephadex
Fig. 10.1
15Nucleoplasmic fraction enriched in Pol II
Nucleolar fraction- Enriched in Pol I
Fig. 10.2
16Determining Roles for Each Nuclear RNA Polymerase
(nRNAP)
- Purified polymerases dont transcribe DNA
specifically so used nuclear fractions. - Also useful were two transcription inhibitors
- a-aminitin from a mushroom, inhibits RNAP II,
and RNAP III at higher concentrations. - Actinomycin D - general transcription inhibitor,
binds DNA and intercalates into helix, prefers
G-C rich regions (like rRNA genes).
17Fig. 10.3
a aminitin, from Amanita phalloides (death cap
mushroom).
18 Actinomycin D, from Streptomyces
Intercalating Portion.
19RNA Polymerase I
- Not inhibited by aminitin, but inhibited by low
concentrations of actinomycin D. - RNA produced in the presence of a-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)
20RNA Polymerase II
- Actinomycin D, at low concentrations, did not
inhibit synthesis of heterogenous nuclear RNA (hn
RNA). - a-aminitin inhibited synthesis of hnRNA in
nucleoplasmic fraction. - Conclusion nRNAP II synthesizes hnRNA (mostly
mRNA precursors).
21RNA Polymerase III
- Synthesis of small abundant RNAs inhibited only
at high a-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
22(No Transcript)
23Subunit structure of purified nRNAPs
- All 3 have 10-14 subunits.
- Subunits range from 8 to 220 kDa.
- All 3 have 2 very large (gt125 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