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Chapt. 10 Eukaryotic RNA Polymerases
and their Promoters
Student learning outcomes:
• Explain composition of 3 different nuclear RNAPs;
emphasis on pol II
• Explain which genes each pol transcribes
• Describe nature of 3 classes of promoters
• Generally appreciate how structural information of
pol II has informed mechanistic understanding
• Appreciate that mitochondria and chloroplasts also have their own RNA
polymerases (not discussed)
• Appreciate that Archaeal RNA polymerases share features with
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eukaryotes (not discussed)
• Important Figures: 1*, 2, 3, 4, 6*, 9*, 12, 14*, 19*, 20*, 22,
23, 24, 26; Tables: 1, 2, 3
• Review problems: 1, 4, 5, 6, 7, 8, 11, 15, 16, 17, 18, 21,
23, 26, 28; AQ problems: 1, 2, 3, 4
Roger Kornberg: crystal
structure of Pol II D4/7
with DNA/RNA active site
Nobel Prize
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10.1 Multiple Forms of nuclear RNAP
• Nuclei contain 3 RNAP
separated by ion-exchange
chromatography
• Pol I in nucleolus:
rRNA genes
• Pol II and pol III
in nucleoplasm
Fig. 1
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Roles of 3
RNA nuclear
Polymerases
• Pol I large rRNA precursor
• Pol II mRNA
– Heterogeneous nuclear RNA (hnRNA)
– Small nuclear RNA (snRNA)
• Pol III tRNAs, 5S rRNA, other small RNA
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Human and Yeast RNAP Subunit Structures
Note orthologs to bacterial subunits;
CTD = C-terminal domain
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RNA polymerases I, II, III distinguished by
sensitivity to inhibitor a-amanitin
Fig. 4 pol II is most sensitive
Figs. 3 Poisonous Amanita mushroom “death cap”
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Pol II Structure
Epitope tagging, purification,
helps distinguish true subunits
from associated proteins
• Add extra domain to one subunit; others
normal (e.g., His-tag)
• (RNAP labeled by growing cells in labeled
amino acids)
• Purify complex with antibody to epitope
• Denature, separate on SDS-PAG gel
• TAP tag common technique
(tandem affinity purification)
Fig.10-7
6
Pol II
Original 10 subunits:
• Core – related in structure
and function to bacterial
core subunits
• Common – found in all 3
nuclear RNAP
• Nonessential subunits –
conditionally dispensable
for enzymatic activity
• Numbered by size; later
subunits 11 and 12
Fig. 7; 32P shows phosphorylated subunits
A, Rick Young; b. Roger Kornberg
Core Subunits
• Rpb1, Rpb2, Rpb3 absolutely required for enzyme
activity: deletion mutants are dead; some ts mutants
– Homologous to b’-, b-, and a-subunits
– Both Rpb1 and b’-subunit bind DNA
– Both Rpb2 and b-subunit are at or near the nucleotidejoining active site
– Rpb3 does not really resemble a-subunit, but some aa
similarity, 2 subunits per holoenzyme
Common Subunits
Found in all 3 nuclear polymerases:
– Rpb5, Rpb6, Rpb8, Rpb10, Rpb12
• All are essential genes (deletion mutants are dead)
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Subunits Nonessential for Elongation
• Rpb4 and Rpb7
–
–
–
–
Dissociate fairly easily from polymerase
substoichiometric quantities
Rpb4 may help anchor Rpb7 to pol II
Mutants without Rpb4 and Rpb7 transcribe well, but
cannot initiate at real promoter
• Rpb7 is essential subunit, so must not be
completely absent in the rpbD4 mutant
• Assist binding of transcription factors
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Review Pol II subunits
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Heterogeneity of Rpb1 (Subunit II)
Subunit IIa is primary product in yeast
– Converted to IIb by proteolytic
removal of carboxyl-terminal domain
(CTD) which is 7-peptide (YSPTSPS)
repeated 26 times (heptad repeat)
– Kinase converts IIa to IIo by
phosphorylation of Ser2 in heptads
– RNAP with IIa binds promoter
– RNAP IIo involved in elongation
– Phosphatase converts IIo back to IIa
Fig. 8
C = Rpb2
3-D Structure of RNA Pol II - Kornberg Nobel prize
• Yeast pol II (pol II D4/7) at 2.8 A:
• Deep cleft accepts linear DNA
template
• Catalytic center at bottom
of cleft contains Mg2+ ion
• Second Mg2+ ion present
in low concentrations
• Geometry of linear DNA permits:
– TFIID to bind at TATA
box of promoter
– TFIIB link pol II to TFIID
– Places polymerase to
– initiate transcription
Fig. 10
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3-D Structure - Pol II in Elongation Complex
• pol II (D4/7) bound to DNA template, RNA product
• Clamp region of pol II closed over DNA and RNA
– Closed clamp ensures transcription is processive –
transcribe whole gene without falling off, early termination
Fig. 13
DNA template
strand in blue
DNA nontemplate
strand in green
RNA is in red
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Position of Critical Elements in Transcription
Bubble
Loops of clamp extend into
transcription bubble:
– Lid: maintains DNA
dissociation
– Rudder: initiates DNA
dissociation
– Zipper: maintains
dissociation of template
DNA
DNA template strand in blue
DNA nontemplate strand in green
RNA is in red
Fig. 14b
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Structure of 12-subunit pol II
• Initiating form of pol II; Clamp is shut
• Promoter must melt before template DNA inserts in active site
• Rpb4/7 extends dock region of pol II; makes binding of
transcription factors easier, for initiation
Fig. 19
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10.2 Eukaryotic Promoters
• Three eukaryotic nuclear RNA polymerases have:
– Different structures
– Transcribe different classes of genes
• Expect the RNAPs recognize different promoters
• Emphasis on Pol II (mRNA)
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Class II Promoters
Fig. 20
• Promoters recognized by pol II (class II promoters)
are similar to prokaryotic promoters:
• Considered to have two parts:
– Core promoter of 4 elements: TATAAA, TBP, BRE (IIB),
– Upstream promoter element
TATA box: on nontemplate strand (TATAAA consensus)
Very similar to prokaryotic -10 box (TAtAaT consensus)10-18
Linker Scanning
mutations clarify
promoter elements
• Systematically
substitute 10-bp linker
for 10-bp sequences
through promoter
• Found mutations
within TATA box
destroyed promoter
activity
Fig. 22
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Linker scanning
mutations of TK
promoter of herpes
simplex virus defined
critical elements
• mutations within TATA
box destroyed promoter
activity
Injected plasmids into frog
oocytes for transcription;
control had pseudo-WT
sequence
Fig. 10.23
Core Promoter Elements for pol II
• In addition to TATA box, core promoters have:
– TFIIB recognition element (BRE)
– Initiator (Inr)
– Downstream promoter element (DPE)
• At least one core element missing in most promoters
• TATA-less promoters tend to have DPEs
• Promoters for highly specialized genes tend to have
TATA boxes
• Promoters for housekeeping genes (constitutively
active in all cells) tend to lack TATA boxes
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Upstream Elements Regulate pol II
• Differ from core promoters in binding to relatively
gene-specific transcription factors (Chapt. 12)
– GC boxes bind transcription factor Sp1
– CCAAT boxes (‘cat boxes’) bind CTF (CCAAT-binding
transcription factor)
• Upstream promoter elements can be orientationindependent, yet relatively position-dependent
• Linker scanning mutations identified, clarified:
– Delete upstream elements -> less expression
– Ex. Mutation of GC boxes in SV40 early promoter
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Class I Promoters for rRNA - pol I
Fig. 24; linker scanning mutations define sites
• Class I promoters are not well conserved sequence
• Usually two elements:
– Core element surrounding transcription start site
– Upstream promoter element (UPE) 100 bp farther
– Spacing between elements is important
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Class III Promoters
• RNA pol III transcribes a set of short genes
• These have promoters that lie wholly within
the genes
• There are 3 types of these promoters
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Class III Promoters of Pol III Genes are
often within the gene
• Type I (5S rRNA) has 3 regions:
• Type II (tRNA) has 2 regions:
• Type III (nonclassical) resemble type II
Fig. 26
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10.3 Enhancers and Silencers
• Position- and orientation-independent DNA
elements stimulate or depress, respectively,
transcription of associated genes
• Often tissue-specific - rely on tissue-specific DNAbinding proteins for activities
• Some DNA elements can act either as enhancer or
silencer depending on what is bound to it:
– Ex. SV40 Early genes 2 x 72-bp GC boxes
Fig. 28 SV40
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Enhancer in
immunoglobulin genes
Fig. 29 Deletion of
enhancer reduces
transcription;
Fig. 30 enhancer
works in either
orientation, and
position
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Review questions
4. How many subunits does yeast pol II have? Which
are core subunits? Which are common to all 3
nuclear RNA polymerases?
7. What is the structure of the CTD of RPB1?
17. Diagram a pol II promoter, showing all of the types
of elements it could have.
AQ3. You are investigating a new class II promoter.
Design an experiment to locate promoter sequences
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