Transcription in the Eukaryotic Nucleus

1
Transcription in the Eukaryotic Nucleus

• RNA Polymerases
• Promoters for each polymerase
• General transcription factors
• Regulatory factors and combinatorial regulation

2
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

3
Roeder and Rutters separation of 3 nuclear RNA
polymerases from sea urchin embryos by ion
exchange chromatography on DEAE-Sephadex
Fig. 10.1
4
Nucleoplasmic fraction
Nucleolar fraction
Fig. 10.2
5
Determining roles for each polymerase

• Purified polymerases dont transcribe DNA
  specifically so used nuclear fractions.
• Also useful were two transcription inhibitors
• a-aminitin from a mushroom, inhibits Pol II,
  and Pol III at higher concentrations.
• Actinomycin D - general transcription inhibitor,
  binds DNA and intercalates into helix, prefers
  G-C rich regions (like rRNA genes).

6
Figure 10.3
7
a aminitin, from Amanita phalloides (death cap
mushroom).
8
Sensitivity of Purified RNA Polymerases to
?-amanitin
Figure 10.4
9

Actinomycin D, from Streptomyces
Intercalating Portion.
10
RNA Polymerase I

• Not inhibited by a-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 Pol I synthesizes the rRNA precursor
  (45S pre-rRNA ? 28S 18S 5.8S rRNAs)

11
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 Pol II synthesizes hnRNA (mostly
  mRNA precursors).

12
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 Pol III synthesizes many of the small
  abundant cytoplasmic and nuclear RNAs

13
Summary of RNAP roles and location
Also read Table 10.1
14
Subunit structure of purified nuclear RNA
polymerases (nRNAP)

• All 3 have 10-14 subunits.
• Subunits range from 10 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 Pol.

15
nRNAP II from Yeast

• Has 12 subunits, based on traditional enzyme
  purification and epitope tagging.
• Gene knockouts indicate that 10 subunits are
  essential, 2 are required under certain
  conditions.
• The 2 large subunits (genes Rpb1 and Rpb2) have
  regions of homology with the b and b subunits
  of the E. coli RNAP and seem to function
  similarly.
• RPB1 responsible for a-aminitin sensitivity.

16
Epitope tagging

• Add tag (small peptide) to a subunit gene.
• Transform in tagged gene.
• Use a specific antibody for the tag to
  immunoselect tagged protein.
• Analyze other proteins that come down with the
  immunoselected protein by SDS-polyacrylamide gel
  electrophoresis.

Figs. 10.6
17
RNAP II purified by the epitope tag on the Rpb3
protein 10 subunits.
RNAP II purified from wild type yeast 12
subunits.
Rpb1 subunit is phosphorylated.
Fig. 10.7
18
Table 10.3
19
Figure 10.8
Fig. 10.8 Partial subunit structure of mouse
plasmacytoma RNA Polymerase II
20
nRNAP II Heterogeneity

• Largest subunit - Rpb1 gene, a.k.a. Subunit II
  in mice, is phosphorylated on its
  carboxy- terminal domain (CTD).
• 2 forms of large subunit
• IIa - non-phosphorylated form
• IIo - phosphorylated form
• Functionally different IIa-containing enzyme
  binds promoter IIo-containing enzyme is in
  elongation phase.

21
Figure 10.9
Prposed Relationship Among the Different Forms of
the Largest Subunit of RNA Polymerase II
22
Model of yeast nRNAP II
DNA must bend as it passes through enzyme.
23
One of two small channels that may allow RNA to
exit. Too narrow for DS DNA.
24
Promoters for the 3 nuclear RNA polymerases
(nRNAPs)

• Order of lecture topics
• Class II promoters (for nRNAP II)
• Class I promoters (for RNAP I)
• Class III promoters (for RNAP III)
• Enhancers and Silencers

25
RNAP II Promoters (a.k.a. Class II)

• Class-II promoters usually have 5 components
• Upstream element
• TFIIB recognition element (BRE)
• TATA Box (at approx. 25)
• Initiation region
• Downstream element

BRE
1. 2. 3. 4. 5.
Many class II promoters lack 4 and 5.
Fig. 10.20
26
TATA Box of Class II Promoters

• TATA box TATAAAA
• Defines where transcription starts.
• Also required for efficient transcription for
  some promoters.
• Some class II promoters (e.g., for housekeeping
  genes or some developmentally regulated genes
  (e.g., homeotic)) dont have a TATA box.

27
Transcription starts at a purine 25-30 bp from
the TATA box.
Normal promoter.
SV40 early promoter analyzed in vivo.
28
S1 mapping of the 5 end of a RNA Transcript
A 5 end labeled single-stranded DNA probe is
prepared from the template strand. After
hybridization to RNA and digestion with S1, the
size of the protected probe tells approx. where
transcription started.
From Fig. 5.26
29
High resolution analysis of the 5end of an RNA
transcript by primer extension.
Primer is an end-labeled DNA oligonucleotide (20
nt) that is complementary to a sequence in the
RNA 150 nt from the expected 5 end.
Lane E- extended DNA product Lanes A,C, G, T
sequence ladder generated with the same oligo
primer, but on the corresponding cloned DNA.
From Fig. 5.29
30
Figure 10.21
31
TATA box also important for transcription
efficiency for some promoters.
Rabbit globin promoter, tested in Hela cells, and
assayed by S1 mapping of transcript 5 end.
32
Linker scanning mutagenesis of a stretch of DNA.
Replace 10 bp of natural sequence with 10 bp of
synthetic DNA.
Do this periodically throughout the stretch of
DNA you want to examine for important sequences.
From Fig. 10.22
33
Linker scanning mutagenesis of the Herpes virus
tk promoter identifies 2 important upstream
regions.
DNA was injected into frog oocytes, and the
transcribed RNA analyzed by primer extension.
-29 to -18 has TATA box-deletion abolished
transcription. Regions -47 to -61 and -80 to -105
contain GC boxes (GGGCGG and CCGCCC).
Fig. 10.23
34
(No Transcript)
35
Upstream Elements of Class II

• Can be several of these
• Two that are found in many class II promoters
• GC boxes (GGGCGG and CCGCCCC)
• Stimulate transcription in either orientation
• May be multiple copies
• Must be close to TATA box (different from
  enhancers)
• Bind the Sp1 factor
• CCAAT box
• Stimulates transcription
• Binds CCAAT-binding transcription factor (CTF) or
  CCAAT/enhancer-binding protein (C/EBP)