Monocistronic vs Polycistronic Genes - PowerPoint PPT Presentation

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Monocistronic vs Polycistronic Genes

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Isolated Spinach chloroplast envelope thylakoid membrane stroma From Hoober Chloroplast RNA polymerases (RNAPs) Two different RNAPs in vascular plant chloroplasts ... – PowerPoint PPT presentation

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Title: 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

12
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
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