Transcript Inquiry into Life Twelfth Edition

Lecture PowerPoint to accompany
Molecular Biology
Fifth Edition
Robert F. Weaver
Chapter 10
Eukaryotic RNA
Polymerases and
Their Promoters
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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
10-2
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
10-3
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
10-4
RNA Polymerase Subunit Structures
10-5
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
10-6
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
10-7
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
10-8
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
10-9
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
10-10
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
10-11
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
10-12
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
10-13
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
10-14
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
10-15
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
10-16
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
10-17
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
10-18
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
10-19
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
10-20
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
10-21
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
10-22
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
10-23
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
10-24
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
10-25
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
10-26
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
10-27
10.3 Enhancers and Silencers
• These are position- and orientationindependent 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
10-28
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
10-29
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
10-30
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
10-31