Monocistronic vs Polycistronic Genes

1
Isolated Spinach chloroplast
envelope
thylakoid membrane
stroma
From Hoober
2
Chloroplast 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

3
Chloroplast 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)

4
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

5
Some chloroplast genes have promoters for both
the PEP and NEP RNAPs..
Fig. 6.31 in Buchanan et al.
6
Why is chloroplast transcription much more
complex than mitochondrial transcription?
Chloroplasts are larger, more complex organelles,
that differentiate.
7
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

8
From U. Wisconsin Botany Dept.
9
Plastid types develop from proplastids Shoots
light-gt proplastids lt----gt etioplasts
lt----gt chloroplasts chromoplasts
Roots proplastids lt----gt amyloplasts
10
Complex 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

11
Monocistronic and/or Polycistronic Transcription

• Prokaryotes Both
• Eukaryotes
• Nucleus Monocistronic (polycistronic rare)
• Mitochondria
• Mammals Polycistronic (2 promoters)
• Other lower species Both
• 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

13
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

14
Roeder and Rutters separation of 3 nuclear RNA
polymerases from sea urchin embryos by ion
exchange chromatography on DEAE-Sephadex
Fig. 10.1
15
Nucleoplasmic fraction enriched in Pol II
Nucleolar fraction- Enriched in Pol I
Fig. 10.2
16
Determining 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).

17
Fig. 10.3
a aminitin, from Amanita phalloides (death cap
mushroom).
18

Actinomycin D, from Streptomyces
Intercalating Portion.
19
RNA 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)

20
RNA 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).

21
RNA 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)
23
Subunit 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