EUKARYOTIC RNA

POLYMERASES & THEIR

PROMOTERS
Mary Grace DS. Bayot
 Multiple Forms of Eukaryotic RNA
Polymerase
■ There are at least two RNA polymerases operating in eukaryotic nuclei
1. One transcribes major ribosomal RNA genes
2. 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
 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 suggest in transcribes rRNA genes
■ RNA polymerases II and II are found in the nucleoplasm
 Roles of the Three RNA Polymerases

■ Polymerase I makes larger rRNA precursor

■ Polymerases II makes:
* Heterogenous nuclear RNA (hnRNA)
* Small nuclear RNA
■ Polymerase III makes precursors to tRNAs, 5S rRNA and other small
RNA
RNA Polymerase Subunit Structures
 Polymerase II Structure

■ For enzymes like eukaryotic RNA polymerases, can be difficult to tell:

* Which polypeptides co-purify with polymerase activity
* Which are actually subunits of the enzyme
■ Technique to help determine whether a polypeptide co-purifies or is a
subunit is called epitope tagging
 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
 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
■ Non-essential subunits – conditionally dispensable for enzymatic
activity
Subunit structure of yeast RNA
polymerase II
 Core Subunits

■ Three polypeptides, Rpb1 (named from RNA Pol B), Rpb2, Ppb3 are
absolutely required for enzyme activity
■ These are homologous to β’- β-, and α-subunits
■ Both PRpb1 and β’ –subunit binds DNA
■ Rpb2 and β-subunit are at or near the nucleotide-joining active site
■ Rpb3 does not resemble α-subunit
* There is one 20-amino acid subunit of great similarity
* 2 subunits are about same size, same stoichiometry
* 2 monomers per holoenzyme
 Common Subunits

■ There are five common subunits

1. Rpb5
2. Rpb6
3. Rpb8
4. Rpb10
5. Rpb12
■ Little known about function
■ They are all found in all 3 polymerases (Pol I, Pol II, Pol III)
■ Suggests play roles fundamental in transcription
 Subunits Non-essential for Elongation

■ Rpb4 and Rpb7

* Dissociate fairly easily from polymerase
* Found in sub-stoichiometric quantities
* 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.
 Heterogeneity of the Rpb1 Subunit

■ RPB1 gene product is subunit II

■ Subunit IIa is the primary product in yeast
* Can be converted to llb by proteolytic removal of the carboxyl-
terminal domain (CTD) which is 7-peptide repeated over and over
* Converts to Iio by phosphorylating 2 set in the repeating heptad
( 7 number of aa) of the CTD
* Enzyme with a binds to the promoter
* Enzyme with Iio is involved in transcript elongation.
 The Three-Dimensional Structure of
RNA Polymerase II
■ Structure of yeast polymerase II (specifically pol II Δ4/7) 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
Structure of yeast polymerase II
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 (cover) the DNA and RNA
* Closed clamp ensures that transcription is processive- able to
transcribe a whole gene without falling off and terminating
prematurely.
Crystal structure of the elongation
complex
 Position of Nucleic Acids in the
Transcription Bubble
■ DNA template strand is shown in
blue
■ DNA non-template strand shown
in green
■ RNA is shown in red
 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
 Position of Critical Elements in the
Transcription Bubble
■ The active center of the enzyme lies at the end of pore 1
■ Pore 1 also appears to be 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.
 Proposed Translocation Mechanism

■ During the translocation step, the RNA-DNA hybrid moves one base
pair to left, bringing new template strand nucleotide into the active
site. Simultaneously, the bridge helix bends (green dot), remaining
close to the end of the RNA.
■ When the bridge helix returns to the straight state (arrow at left), it
reopens the active site so another nucleotide can enter.
 3-D Structure – RNA Polymerase II in
the Post translocation State
■ X-ray crystallography has shown the lid of Rpb 1 interacts with the
DNA-RNA hybrid to force the hybrid open after base pair8
■ 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.
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 E and A sites may play a role in base
and sugar specificity
Structural Basis of Nucleotide Selection
(cont…)
■ 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.
 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.
 Promoters

■ Three eukaryotic RNA polymerases have:

* Different structures
* Transcribe different classes of genes
■ Expect that the 3 polymerases would recognize different promoters.
 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
 Core Promoter elements – TATA Box

■ TATA box
* Found on the non-template 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.
 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
 Core Promoter Elements

■ In addition to TATA box, core promoters are:

* TFIIB recognition element (BRE)
* Initiator (Inr)
■ 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
 Upstream Elements

■ Upstream promoter elements are usually found upstream of class II

core promoters
■ Differ form core promoters in binding to relatively gene-specific
transcription factors
* GC boxes bind transcription factor Sp 1
* CCAAT boxes bind CTF (CCAAT- binding transcription factor)
■ Upstream promoter elements can be orientation-independent, yet are
relatively position-dependent
 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
 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
 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
 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