2. Control of initiation of transcription in E. coli
Repressor (trans-acting factor) will block transcription initiation when
bound to the operator (cis-acting element) – negative control
Sites for RNAP (promoter) and repressor (operator) could overlap -
repressor binding blocks RNAP from interacting with DNA at the start site
Presence of auxiliary operators – additional repressor binding sites
Affinity of repressor for operator depends on presence/absence of effector
(repressor / inducer and corepressor / aporepressor) – its binding changes
repressor’s conformation change in activity
Activators (trans-acting factor) that could increase binding of RNAP
to promotor- positive control
Binding sites for activators are called enhancers (elements)
Affinity of activator for enhancer also sometimes depends on
presence/absence of effector
2
3. Some proteins are regulators (could execute positive or negative
regulation depending on presence/absence of effector
different conformation = affinity for different cis elements
Regulation through two component regulatory system
(two proteins : sensor-transmitter AND response regulator)
DNA bending (as a consequence of binding of a trans factor) could lead to
negative or positive regulation - regulatory protein can directly contact
RNAP - preventing or helping RNAP to interact with DNA at the start site
(promoter)
Direct contact could be achieved even when promoter and regulatory
protein’s binding sites are far apart – DNA looping
Direct contact of RNAP and activator protein causes conformational
changes in RNAP which promote formation of open complex
Control of initiation of transcription in E. coli
3
4. Overview of Eukaryote RNAPs
Learning objectives:
Describe the criteria used to separate and purify RNAPs
Describe the subunit structure of each RNAP
• discuss similarities between subunit structures of eukaryotic and
prokaryotic RNAPs
• discuss the complexity of eukaryotic RNAPs
Describe the structure of RNAP II including CTD tail
Describe how the knowledge of RNAP II crystal structure has
improved the understanding of transcription mechanism
4
5. Eukaryotic vs. Prokaryotic Transcription
Transcription and translation occur in separate compartments
Eukaryote pre-mRNAs are subject to extensive post-transcriptional
modification – processing
Chromatin structure in eukaryotes limits accessibility (Transcription is
tightly regulated. Only 0.01% of genes in a typical eukaryotic cell are
undergoing transcription at any given moment )
Eukaryotic RNAP does not recognize binding site by itself – needs
general transcription factors to help
Eukaryotes are mostly multicellular organisms (diff. cells/tissues)
Three RNA polymerases – different roles (other RNAPs…)
• RNA Polymerase IV Directs Silencing of Endogenous DNA J. Herr,1
M. B. Jensen,1
T. Dalmay,2
D. C.
Baulcombe1
Science, Vol 308, Issue 5718, 118-120, 1 April 2005
• Roles of RNA polymerase IV in gene silencing Craig S. Pikaard, Jeremy R. Haag, Thomas Ream and
Andrzej T. Wierzbicki Trends Plant Sci. 2008 May 30
5
6. Regulation of
Transcription
in Eukaryotes
Regulation of
Transcription
in Eukaryotes
Control of gene expression
similar to the mechanisms
used in prokaryotic cells:
Expression is often controlled at
the level of transcription
initiation
Regulation by proteins that
recognize specific regulatory
sequences and modulate
binding and activity of RNAP
6
7. Three main (and different) RNAPs in eukaryotes…
Separation and purification of RNAPs
Early studies (1) - rRNA genes:
rRNA genes have high GC content (60% vs. 40%)
rRNA genes are repetitive (up to 20 000 copies of the gene/cell)
rRNA genes found in nucleolus
Early studies (2) - RNA synthesis under various conditions:
High ionic concentration – RNA with low GC content
Low ionic concentration – RNA with high GC content (similar to rRNA)
Mg2+
+ Low ionic strength – most of transcription in nucleolus
Mn2+
+ High ionic strength – transcription throughout the nucleus
Þ conclusion: more then one RNAP
One would work in nucleolus, stimulated by low salts and Mg2+
One would work in nucleoplasm, stimulated by high salts and Mn2+
7
8. Early studies (3)
protein fractions
(lots of them)
Purification of
different protein
fractions extracted
from the nucleus
Use proteins from
each fraction for
in vitro transcription
Salt concentration in elution
buffer will be higher in each
subsequent elution
– higher salt conc. displaces
proteins with positive
charge
– different fractions will have
different proteins based on
charge differences
8
Protein properties and behaviors in
Ion-exchange chromatography
9. RNAP I – active at low ionic
strength, works with both
Mg2+
and Mn2+
RNAP II – more active at
high ionic strength, works
better with Mn2+
RNAP III – active over a
broad range of ionic
strengths, works better with
Mn2+
LEGEND:
Green – total protein
Red – activity = transcription measured
by incorporation of labeled UMP
Blue – concentration of ammonium
sulfate used as an elution buffer
Conclusion: there are
three distinct nuclear
RNA polymerases
that transcribe three
different sets of genes
9
Protein properties and behaviors in
Ion-exchange chromatography
10. Early studies (4)
Mouse cell nuclei incubated with
increasing concentrations of a-amanitin
(product of mushroom Amanita
bisporigera) and transcripts submitted to
electrophoresis; RNAP II is the most
sensitive to a-amanitin
(toxin binds to rpb1 subunit – prevents
RNAPII translocation. Amanita RNAPII
not affected. Plant RNAPII less
sensitive).
Similar experiments with actinomycin D
(highly toxic antibiotic produced by
Streptomyces; earlier used in
chemotherapy); intercalates into GC rich
regions and inhibits transcription
– RNAP I is the most sensitive one
(recall: genetic information for rRNA is
GC rich)
10
Biochemical experiments
Differing sensitivity to chemicals
Actinomycin D
11. Roles of eukaryotic RNA polymerases
Different Polymerases produce different transcripts
11
Ω
Ω
~ to b and b’
~ to a
~ to Ω
Rpb = RNA Polymerase B = RNAP II
1
2
3
4
5
6
7
8
9
10
12. Each one of three RNAPs is a complex enzyme:
8-14 subunits depending on the RNAP
5 subunits are common to all three RNAPs (Rpb 5, 6, 8, 10 and 12)
Share several functional properties
Large subunits similar to β and β’ of E. coli RNAP
Completely different binding sites = promoters
12
13. Comparison of 3-D structures of
bacterial and eukaryotic RNA polymerases
(subunits 4 and
7 are missing)
13
14. Structure of
RNAP II
X-ray crystallography of 3D yeast
RNAP II crystals (Rpb4 and Rpb7
cannot be included) and posed on
synthetic DNA (no promoter)
25Å channel (width of dsDNA?)
in the face of the RNAP II
– can accommodate 20 bp of DNA;
channel formed by Rbp1 and Rbp2
At the opening of the channel
– “jaws” role: grabbing of dsDNA
Sliding clamp - composed of parts
of Rbp 1, 2 and 6
the active site is on Rbp2; includes
single Mg 2+
and conserved
aspartate motif – RNA synthesis
Two pores – one should be the
exit for the growing RNA
(other nucleotide entry?)
14
