Molecular Biology

1. Molecular Biology
Fifth Edition
Chapter 10
Eukaryotic RNA
Polymerases and
Their Promoters
Lecture PowerPoint to accompany
Robert F. Weaver
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. 10-2
10.1 Multiple Forms of Eukaryotic
RNA Polymerase
• There are at least two RNA polymerases
operating in eukaryotic nuclei
– One transcribes major ribosomal RNA genes
– One or more to transcribe rest of nuclear genes
• Ribosomal genes are different from other nuclear
genes
– Different base composition from other nuclear genes
– Unusually repetitive
– Found in different compartment, the nucleolus

3. 10-3
Separation of the 3 Nuclear Polymerases
• Eukaryotic nuclei contain three RNA
polymerases
– These can be separated by ion-exchange
chromatography
• RNA polymerase I found in nucleolus
– Location suggests it transcribes rRNA genes
• RNA polymerases II and III are found in
the nucleoplasm

4. 10-4
Roles of the Three RNA Polymerases
• Polymerase I makes
large rRNA precursor
• Polymerase II makes
– Heterogeneous
nuclear RNA (hnRNA)
– small nuclear RNA
• Polymerase III makes
precursors to tRNAs,
5S rRNA and other
small RNA

5. 10-5
RNA Polymerase Subunit Structures

6. 10-6
Polymerase II Structure
• For enzymes like eukaryotic RNA
polymerases, can be difficult to tell:
– Which polypeptides copurify with polymerase
activity
– Which are actually subunits of the enzyme
• Epitope tagging is a technique to help
determine whether a polypeptide
copurifies or is a subunit

7. 10-7
Epitope Tagging
• Add an extra domain to
one subunit of RNA
polymerase
• Other subunits normal
• Immunopreciptate with
antibody directed
against epitope
• Denature with SDS
detergent and separate
via electrophoretic gel

8. 10-8
Core Subunits of RNA Polymerase
• Three polypeptides, Rpb1, Rpb2, Rpb3 are
absolutely required for enzyme activity (yeast)
• Homologous to b’-, b-, and a-subunits (E.coli)
• Both Rpb1 and b’-subunit binds DNA
• Rpb2 and b-subunit are at or near the
nucleotide-joining active site
• Similarities between Rpb3 and a-subunit
– There is one 20-amino acid subunit of great similarity
– 2 subunits are about same size, same stoichiometry
– 2 monomers per holoenzyme
– All above factors suggest they are homologous

9. 10-9
Common Subunits
• There are five common subunits
– Rpb5
– Rpb6
– Rpb8
– Rpb10
– Rpb12
• Little known about function
• They are all found in all 3 polymerases
which suggests they play roles
fundamental to the transcription process

10. 10-10
Summary
• The genes encoding all 12 RNA polymerase II
subunits in yeast have been sequenced and
subjected to mutational analysis
• Three of the subunits resemble the core subunits
of bacterial RNA polymerases in both structure
and function
• Five are found in all three nuclear RNA
polymerases, two are not required for activity and
two fall into none of these categories

11. 10-11
Heterogeneity of the Rpb1 Subunit
• RPB1 gene product is subunit II
• Subunit IIa is the primary product in yeast
– Can be converted to IIb by proteolytic removal
of the carboxyl-terminal domain (CTD) which
is 7-peptide repeated over and over
– Converts to IIo by phosphorylating 2 serine in
the repeating heptad of the CTD
– Enzyme with IIa binds to the promoter
– Enzyme with IIo is involved in transcript
elongation

12. 10-12
The Three-Dimensional Structure of
RNA Polymerase II
• Structure of yeast polymerase II (pol II
4/7) reveals a deep cleft that accepts a
DNA template
• Catalytic center lies at the bottom of the
cleft and contains a Mg2+ ion
• A second Mg2+ ion is present in low
concentration and enters the enzyme
bound to each substrate nucleotide

13. 10-13
3-D Structure of RNA Polymerase II in
an Elongation Complex
• Structure of polymerase II bound to DNA
template and RNA product in an
elongation complex has been determined
• When nucleic acids are present, the clamp
region of the polymerase is closed over
the DNA and RNA
– Closed clamp ensures that transcription is
processive – able to transcribe a whole gene
without falling off and terminating prematurely

14. 10-14
Position of Nucleic Acids in the
Transcription Bubble
• DNA template strand
is shown in blue
• DNA nontemplate
strand shown in
green
• RNA is shown in red

15. 10-15
Position of Critical Elements in the
Transcription Bubble
Three loops of the
transcription bubble are:
– Lid: maintains DNA
dissociation
– Rudder: initiating DNA
dissociation
– Zipper: maintaining
dissociation of
template DNA

16. 10-16
Proposed Translocation Mechanism
• The active center of the enzyme lies at the end of pore 1
• Pore 1 also appears to be the conduit for:
– Nucleotides to enter the enzyme
– RNA to exit the enzyme during backtracking
• Bridge helix lies next to the active center
– Flexing this helix may function in translocation during
transcription

17. 10-17
Structural Basis of Nucleotide Selection
• Moving through the entry pore toward the active
site of RNA polymerase II, incoming nucleotide
first encounters the E (entry) site
– E site is inverted relative to its position in the A site
(active) where phosphodiester bonds form
– E and A sites partially overlap
• Two metal ions (Mg2+ or Mn2+) are present at the
active site
– One is permanently bound to the enzyme
– The other enters the active site complexed to the
incoming nucleotide

18. 10-18
The Trigger Loop
• In 2006 a crystal structure with GTP rather than
UTP in the A site, opposite a C, revealed a part
of Rpb1 roughly encompassing residues 1070 to
1100 - a trigger loop
• The trigger loop only comes into play when the
correct substrate occupies the A site and makes
several important contacts with the substrate
that presumably stabilize the substrates
association with the active site and contribute to
the specificity of the enzyme

19. 10-19
The Role of Rpb4 and Rpb7
• Structure of the 12-subunit RNA polymerase II
reveals that, with Rpb4/7 in place, the clamp is
forced shut
• Initiation occurs, with its clamp shut, it appears
that the promoter DNA must melt to permit the
template DNA strand to enter the active site
• The Rpb4/7 extends the dock region of the
polymerase, making it easier for certain general
transcription factors to bind, thereby facilitating
transcription initiation
• Rpb7 can bind to nascent RNA and may direct it
toward the CTD

20. 10-20
10.2 Promoters
• Three eukaryotic RNA polymerases have:
– Different structures
– Transcribe different classes of genes
• We would expect that the three
polymerases would recognize different
promoters

21. 10-21
Class II Promoters
• Class II promoters are recognized by RNA
polymerase II
• Considered to have two parts:
– Core promoter - attracts general transcription factors
and RNA polymerase II at a basal level and sets the
transcription start site and direction of transcription
– Proximal promoter - helps attract general transcription
factors and RNA polymerase and includes promoter
elements upstream of the transcription start site

22. 10-22
Core Promoter Elements – TATA Box
• TATA box
– Very similar to the prokaryotic -10 box
– Promoters have been found with no
recognizable TATA box that tend to be found
in two classes of genes:
• 1 - Housekeeping genes that are constitutively
active in nearly all cells as they control common
biochemical pathways
• 2 - Developmentally regulated genes

23. 10-23
Core Promoter Elements
• The core promoter is modular and can contain
almost any combination of the following elements:
– TATA box
– TFIIB recognition element (BRE)
– Initiator (Inr)
– Downstream promoter element (DPE)
– Downstream core element (DCE)
– Motif ten element (MTE)
• At least one of the four core elements is missing
in most promoters
• TATA-less promoters tend to have DPEs
• Promoters for highly specialized genes tend to
have TATA boxes

24. 10-24
Elements
• Promoter elements are usually found
upstream of class II core promoters
• They differ from core promoters in binding
to relatively gene-specific transcription
factors
• Upstream promoter elements can be
orientation-independent, yet are relatively
position-dependent

25. 10-25
Class I Promoters
• Class I promoters are not well conserved in
sequence across species
• General architecture of the promoter is well
conserved – two elements:
– Core element surrounding transcription start site
– Upstream promoter element (UPE) 100 bp farther
upstream
– Spacing between these elements is important

26. 10-26
Class III Promoters
• RNA polymerase III transcribes a variety of
genes that encode small RNAs
• The classical class III genes have promoters that
lie wholly within the genes
• The internal promoter of the type I class III gene
is split into three regions: box A, a short
intermediate element and box C
• The internal promoters of the type II genes are
split into two parts: box A and box B
• The promoters of the nonclassical class III
genes resemble those of class II genes

27. 10-27
Promoters of Some Polymerase III Genes
• Type I (5S rRNA) has 3 regions:
– Box A, Short intermediate element, and Box C
• Type II (tRNA) has 2 regions:
– Box A and Box B
• Type III (nonclassical) resemble those of type II

28. 10-28
10.3 Enhancers and Silencers
• These are position- and orientation-
independent DNA elements that stimulate
or depress, respectively, transcription of
associated genes
• Are often tissue-specific in that they rely
on tissue-specific DNA-binding proteins for
their activities
• Some DNA elements can act either as
enhancer or silencer depending on what is
bound to it

29. 10-29
Enhancers
• Enhancers act through the proteins that are
bound to them, enhancer-binding proteins
or activators
• These proteins appear to stimulate
transcription by interacting with other
proteins called general transcription factors
at the promoter that promote the formation
of a preinitiation complex
• Enhancers are frequently found upstream
of the promoter they control although this is
not an absolute rule

30. 10-30
Silencers
• Silencers, like enhancers, are DNA
elements that can act at a distance to
modulate transcription but they inhibit,
rather than stimulate, transcription
• It is thought that they work by causing the
chromatin to coil up into a condensed,
inaccessible and inactive form thereby
preventing the transcription of neighboring
genes

31. 10-31
Vital theme
• The finding that a gene is much more active in
one cell type than another leads to an extremely
important point: All cells contain the same genes,
but different cell types differ greatly from one
another due to the proteins expressed in each
cell
• The types of proteins expressed in each cell
type is determined by the genes that are active
in those cells
• Part of the story of the control of gene
expression resides in the expression of different
activators in different cell types that turn on
different genes to produce different proteins