Transcript Inquiry into Life Twelfth Edition

Lecture PowerPoint to accompany
Molecular Biology
Fourth 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 Three Nuclear
Polymerases
• Eukaryotic nuclei contain three RNA
polymerases
– These can be separated by ion-exchange
chromatography
• RNA polymerase I found in nucleolus
– Location suggests in 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
• Technique to help determine whether a
polypeptide copurifies or is a subunit is
called epitope tagging
10-6
Epitope Tagging
• Add an extra domain
to one subunit
• Other subunits normal
• Polymerase labeled
by growing in labeled
amino acids
• Purify with antibody
• Denature with
detergent and
separate on a gel
10-7
Polymerase II
Original 10 subunits are placed in 3 groups:
• Core – related in structure and function to
bacterial core subunits
• Common – found in all 3 nuclear RNA
polymerases
• Nonessential subunits – conditionally
dispensable for enzymatic activity
10-8
Core Subunits
• Three polypeptides, Rpb1, Rpb2, Rpb3 are
absolutely required for enzyme activity
• These are homologous to b’-, b-, and a-subunits
• Both Rpb1 and b’-subunit binds DNA
• Rpb2 and b-subunit are at or near the
nucleotide-joining active site
• Rpb3 does not resemble a-subunit
– There is one 20-amino acid subunit of great similarity
– 2 subunits are about same size, same stoichiometry
– 2 monomers per holoenzyme
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
• Suggests play roles fundamental in
transcription
10-10
Subunits Nonessential for
Elongation
• Rpb4 and Rpb7
–
–
–
–
–
Dissociate fairly easily from polymerase
Found in substoichiometric quantities
Might shuttle from one polymerase II to another
Rpb4 may help anchor Rpb7 to the enzyme
Mutants without Rpb4 and Rpb7 transcribes well, but
cannot initiate at a real promoter
• Rpb7 is an essential subunit, so must not be
completely absent in the mutant
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 ser in the
repeating heptad of the CTD
– Enzyme with IIa binds to the promoter
– Enzyme with IIo is involved in transcript
elongation
10-12
The Three-Dimensional
Structure of RNA Polymerase II
• Structure of yeast polymerase II (specifically pol
II 4/7) at atomic resolution reveals a deep cleft
that accepts a linear DNA template from one end
to another
• Catalytic center lies at the bottom of the cleft and
contains a Mg2+ ion
• A second Mg2+ ion present in low concentrations
• Geometry allows enough space for:
– TFIID to bind at the TATA box of the promoter
– TFIIB to link the polymerase to TFIID
– Places polymerase correctly to initiate transcription 10-13
3-D Structure - 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 has shifted
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
10-14
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
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
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
10-17
transcription
3-D Structure - RNA Polymerase
II in the Posttranslocation State
• X-ray crystallography has shown the lid of Rpb1
interacts with the DNA-RNA hybrid to force the
hybrid open after base pair -8
• The lid then interacts with bases of the nascent
RNA to keep the hybrid melted beyond base pair -8
• The rudder of Rpb1 collaborates with lid to keep the
hybrid melted by interacting with bases -9 and -10
• Fork loop 1 of Rpb2 interacts with bases -5, -6, and
-7 of the RNA to keep the RNA-DNA hybrid
together
10-18
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
– Rotation of nucleotide between the E and A sites may
play a role in base and sugar specificity
• 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
10-19
The Role of Rpb4 and Rpb7
• Structure of the 12-subunit RNA polymerase II
reveals that, with Rpb4/7 in place, clamp is
forced shut
• Initiation occurs, with its clamp shut, it appears
that the promoter DNA must melt to permit
template DNA strand to enter the active site
• The Rpb4/7 extends the dock region of the
polymerase, which makes binding of
transcription factors easier
10-20
10.2 Promoters
• Three eukaryotic RNA polymerases have:
– Different structures
– Transcribe different classes of genes
• Expect that the 3 polymerases would
recognize different promoters
10-21
Class II Promoters
• Promoters recognized
by RNA polymerase II
(class II promoters)
are similar to
prokaryotic promoters
• Considered to have
two parts:
– Core promoter having
4 elements
– Upstream promoter
element
10-22
Core Promoter Elements – TATA
Box
• TATA box
– Found on the nontemplate strand
– Very similar to the prokaryotic -10 box
– There are frequently TATA-less promoters
• Housekeeping genes that are constitutively active
in nearly all cells as they control common
biochemical pathways
• Developmentally regulated genes
10-23
Linker Scanning
• Systematically
substitute a 10-bp
linker for 10-bp
sequences
throughout the
promoter
• Found that mutations
within the TATA box
destroyed promoter
activity
10-24
Core Promoter Elements
• In addition to TATA box, core promoters are:
– TFIIB recognition element (BRE)
– Initiator (Inr)
– Downstream promoter element (DPE)
• 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
• Promoters for housekeeping genes tend to lack
them
10-25
Upstream Elements
• Upstream promoter elements are usually found
upstream of class II core promoters
• Differ from core promoters in binding to relatively
gene-specific transcription factors
– GC boxes bind transcription factor Sp1
– CCAAT boxes bind CTF (CCAAT-binding transcription
factor)
• Upstream promoter elements can be orientationindependent, yet are relatively positiondependent
10-26
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
10-27
Class III Promoters
• RNA polymerase III transcribes a set of
short genes
• These have promoters that lie wholly
within the genes
• There are 3 types of these promoters
10-28
Promoters of Some
Polymerase III Genes
• Type I (5S rRNA) has 3
regions:
– Box A
– Short intermediate element
– Box C
• Type II (tRNA) has 2
regions:
– Box A
– Box B
• Type III (nonclassical)
resemble those of type II
10-29
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
10-30