CHAPER 10

EUKARYOTIC RNA
POLYMERASES AND
THEIR PROMOTERS
Presented by ALLAN JAY MAATA
10.1 Multiple Forms of Eukaryotic RNA Polymerase
a. Separation of the Three Nuclear Polymerases
b. The Roles of the Three RNA Polymerases
c. RNA Polymerase Subunit Structures

10.2 Promoters
a. Class II Promoters
b. Class I Promoters
c. Class III Promoters

10.3 Enhancers and Silencers

a. Enhancers
b. Silencers
MULTIPLE FORMS OF EUKARYOTIC RNA POLYMERASE
Eukaryotic nuclei contain multiple RNA polymerases.
At least two RNA polymerases are identified in eukaryotic nuclei: one
for transcribing major ribosomal RNA genes (28S, 18S, and 5.8S rRNAs)
and one or more for transcribing other nuclear genes.
Ribosomal genes have distinct features
1. Different base composition (e.g., rat rRNA genes have 60% GC
content, while other genes have 40% GC content)
2. High repetitiveness (several hundred to over 20,000 copies per cell)
3. Localized in the nucleolus, a separate compartment within the
nucleus.
These differences imply the presence of specialized RNA polymerases in
eukaryotic nuclei.
One RNA polymerase operates in the nucleolus, synthesizing rRNA, while
another operates in the nucleoplasm, transcribing other types of RNA.
SEPARATION OF THE THREE NUCLEAR POLYMERASES
Eukaryotes have three distinct RNA polymerases: RNA polymerase I, RNA
polymerase II, and RNA polymerase III.
These enzymes were separated and identified by Robert Roeder and
William Rutter in 1969 using DEAE-Sephadex ion-exchange
chromatography.
The three polymerases have different properties and behaviors, such
as responses to ionic strength and divalent metals.
Each polymerase has specific roles in transcription, synthesizing
different kinds of RNA:
1. RNA polymerase I is primarily responsible for synthesizing ribosomal
RNA (rRNA) and is primarily located in the nucleolus.
2. RNA polymerase II and RNA polymerase III are found in the
nucleoplasm and are involved in the synthesis of other types of RNA.
-
 THE ROLES OF THE THREE RNA POLYMERASE
RNA Polymerase I
Synthesizes the large rRNA precursor.
In mammals, the precursor (45S) is processed into the mature rRNAs:
28S, 18S, and 5.8S.

RNA Polymerase II
Produces heterogeneous nuclear RNA (hnRNA), precursor molecules
for microRNAs (miRNAs), and most small nuclear RNAs (snRNAs).
hnRNAs act as precursors for messenger RNAs (mRNAs), while snRNAs
are involved in the maturation of hnRNAs to mRNAs.
miRNAs regulate gene expression by causing mRNA degradation or
limiting translation.
 THE ROLES OF THE THREE RNA POLYMERASE

RNA Polymerase III

Generates precursors for transfer RNAs (tRNAs), 5S rRNA, and some
other small RNAs.
 THE ROLES OF THE THREE RNA POLYMERASE

Alpha-amanitin - a toxin found in certain mushrooms (Amanita

species), was used in experiments to study the effects on RNA
polymerases.

RNA Polymerase Inhibition

1. RNA Polymerase II: Highly sensitive to low concentrations of alpha-
amanitin; completely inhibited even at low doses.
2. RNA Polymerase III: Inhibited at higher concentrations, supporting its
role in synthesizing small RNAs like 5S rRNA and tRNA precursors.

Experimental Approach:
- Mouse cell nuclei were incubated with increasing concentrations of
alpha-amanitin.
 RNA POLYMERASE STRUCTURES

Eukaryotic RNA polymerases are complex, consisting of large and

small subunits.
Structures of RNA polymerases I, II, and III are complex, each
comprising two large (greater than 100 kD) subunits and various
smaller subunits.
Similarities exist between eukaryotic polymerases and prokaryotic
core polymerases, indicating evolutionary relationships.
 RNA POLYMERASE STRUCTURES

Polymerase II Structure and Subunits

Methodology: Scientists used epitope tagging to identify authentic
polymerase II subunits in yeast cells.
Subunits: Twelve subunits were identified for yeast polymerase II,
named Rpb1 to Rpb12.
Commonality: Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12 are common
subunits found in all three yeastnuclear polymerases, suggesting
fundamental roles in transcription.
 RNA POLYMERASE STRUCTURES
Functional Relationships:
Rpb1 and Rpb2: Rpb1 is homologous to the E. coli b9-subunit, binds
DNA, and is at or near the active site. Rpb2 shares functional
similarities with the E. coli b-subunit.
Rpb3: Although not closely resembling E. coli a-subunit, Rpb3 shares a
20-amino-acid region of similarity, has similar size and stoichiometry,
and exhibits similar assembly defects, indicating homology.

Common Subunits
Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12: These subunits are present in all
three yeast nuclear polymerases, indicating their fundamental roles in
the transcription process. Their specific functions are not fully
understood.
 10.2 PROMOTERS
Promoters for RNA Polymerase II (Class II Promoters):

Core Promoter and Proximal Promoter:

Core Promoter: Located within about 37 bp of the transcription

start site, contains elements like TATA box, TFIIB recognition
element (BRE), initiator (Inr), downstream promoter element (DPE),
downstream core element (DCE), and motif ten element (MTE).
Proximal Promoter: Extends from about 37 bp up to 250 bp
upstream of the transcription start site, includes elements like TATA
box, Inr, DPE, etc.
 10.2 PROMOTERS
TATA Box:
Consensus Sequence: TATAAA (in the nontemplate strand).
Function: Important for positioning the start of transcription. Some
promoters require the TATA box for function, while others need it
only to position the transcription start site.
 10.2 PROMOTERS
Initiators, Downstream Promoter Elements, and TFIIB Recognition
Elements:
Initiators: Conserved sequences around transcription start sites (e.g.,
PyPyAN(T/A)PyPy in mammals, TCA(G/T)T(T/C) in Drosophila).
Function: Essential for optimal transcription. Can constitute a
functional promoter alone or work in conjunction with other elements.
Downstream Promoter Elements (DPEs): Common in Drosophila,
located about 30 bp downstream of the transcription initiation site,
includes the consensus sequence G(A/T)CG.
Function: Can compensate for the lack of a TATA box in promoters,
bind to TFIID, and participate in the formation of the preinitiation
complex.
TFIIB Recognition Elements (BREs): DNA elements upstream of that
TATA box that assist TFIIB binding to the DNA.
 10.2 PROMOTERS
IRNA Polymerase I Promoters (Class I Promoters)
Target Gene: Predominantly the rRNA precursor gene, with high copy
numbers in each cell.

Promoter Elements:
Core Element (rINR/Initiator): Located at the start of transcription,
between positions 245 and 120.
Upstream Promoter Element (UPE): Positioned between positions
2156 and 2107.
 10.2 PROMOTERS
IRNA Polymerase I Promoters (Class I Promoters)
Importance of Spacing Between Elements:
Spacing Significance: The spacing between the core element and
the upstream promoter element is crucial for promoter strength.
Effect of Deletions: Deletions between the elements significantly
impact promoter strength, with shorter deletions having a more
substantial effect. For example, a 16-bp deletion reduces promoter
strength to 40% of wild-type, while a 44-bp deletion reduces it to
10%.
Effect of Insertions: Insertions between the elements also affect
promoter strength, but the impact is less significant than deletions.
For instance, adding 28 bp has no effect, while adding 49 bp
reduces promoter strength by 70%.
 10.2 PROMOTERS
IRNA Polymerase III Promoters (Class III Promoters)
Target Genes:
Classical Class III Genes: Include the 5S rRNA and tRNA genes, as
well as the adenovirus VA RNA genes.
Nonclassical Class III Genes: Include genes such as the U6 snRNA
gene, the 7SL RNA gene, the 7SK RNA gene, and the Epstein–Barr
virus EBER2 gene.
Promoter Types:
Type I (5S rRNA Genes): Promoters located entirely within the gene
itself.
Sensitive Regions: Identified regions between bases 50 and 83
are crucial for promoter function.
Critical Elements: Box A, Intermediate Element, and Box C are
essential components of the promoter.
 10.2 PROMOTERS

Type II (Most Class III Genes): Promoters resembling tRNA and VA

RNA promoters.
Components: Box A and Box B are key elements.
Spacing Sensitivity: Proper spacing between the boxes is
crucial for efficient transcription.
Type III (Nonclassical Promoters): Promoters with control
elements restricted to the 5'-flanking region of the gene.
Examples: Human 7SK RNA promoter and human U6 RNA
promoter.
Hybrid Promoters (Types II/III): Contain both internal and external
elements necessary for promoter activity.
Example: Human 7SL RNA promoter.
 10.2 PROMOTERS

Important Observations:
Internal Promoter Location: Classical class III promoters, like the 5S
rRNA promoter, are located within the genes they regulate, unlike
class I and class II promoters.
Sensitivity to Sequence Changes: Specific regions, such as Box A,
Intermediate Element, and Box C, cannot be altered without
significantly affecting promoter function.
Hybrid Promoters: Some promoters exhibit characteristics of both
type II and type III promoters, containing both internal and external
elements essential for transcription initiation.
 10.2 PROMOTERS

Important Observations:
Internal Promoter Location: Classical class III promoters, like the 5S
rRNA promoter, are located within the genes they regulate, unlike
class I and class II promoters.
Sensitivity to Sequence Changes: Specific regions, such as Box A,
Intermediate Element, and Box C, cannot be altered without
significantly affecting promoter function.
Hybrid Promoters: Some promoters exhibit characteristics of both
type II and type III promoters, containing both internal and external
elements essential for transcription initiation.
 KEY DIFFERENCE OF PROMOTERS
Associated
Genes Promoter
Class RNA Promoter Elements
Transcribed Location
Polymerase

RNA Internal
rRNA precursor Core element (rINR) and Upstream
Class I Polymerase within
genes Promoter Element (UPE)
I genes

RNA Protein-coding
Upstream TATA box, Initiator (Inr), Downstream
Class II Polymerase genes, some
of genes Promoter Elements
II RNAs

RNA Internal
tRNAs, 5S rRNA, Type I: Internal elements (e.g.,
Class III Polymerase within
small RNAs Xenopus 5S rRNA promoter)
III genes

Type II: Box A, Box B (e.g., tRNA

promoters)

Type III: External elements (e.g.,

human U6 RNA promoter)
 10.3 ENHANCERS AND SILENCERS

Enhancers
Discovery: The first enhancer was discovered in the 5'-flanking region
of the SV40 early gene.
Characteristics:
Position and Orientation Independence: Enhancers stimulate
transcription even when inverted or relocated far from the promoter,
demonstrating their position and orientation independence.
Proteins Involved: Enhancers act through proteins called transcription
factors, enhancer-binding proteins, or activators, which interact with
general transcription factors at the promoter.
 10.3 ENHANCERS AND SILENCERS
Enhancers Within Genes:
Example: An enhancer was found within an intron of the g2b gene,
encoding a mouse antibody subunit.
Effect of Deletions: Deletions within the intron caused a decrease in
gene product production, indicating the importance of the suspected
enhancer region.
Position and Orientation Independence: The enhancer functioned
even when inverted or relocated upstream of the promoter,
confirming its enhancer properties.
Cell Type-Specific Activity: Gene expression was more active in
plasmacytoma cells (antibody-producing cells) compared to
fibroblasts, emphasizing the cell type-specificity of enhancer activity.
 10.3 ENHANCERS AND SILENCERS

Significance of Enhancers:

Differential Gene Expression: Enhancers play a crucial role in

regulating gene expression patterns in different cell types.
Cell Type-Specificity: Different cell types express distinct activators
that bind to enhancers, leading to the activation of specific genes and
the production of cell type-specific proteins.
 10.3 ENHANCERS AND SILENCERS
Silencers:

Function: Silencers are DNA elements that inhibit transcription, acting

at a distance to modulate gene expression negatively.
Example: Mating System in Yeast:
Loci: In yeast chromosome III, there are three loci—MAT, HML, and
HMR—of very similar sequence.
Inactivity of HML and HMR: HML and HMR are not expressed, and
silencers located at least 1 kb away are responsible for their
genetic inactivity.
Response to External Influence: Active yeast genes can be
substituted for HML or HMR, but they become inactive, indicating a
response to an external negative influence, namely, a silencer.
 10.3 ENHANCERS AND SILENCERS
Silencers:
Mechanism of Action:
Chromatin Condensation: Silencers cause chromatin to coil up
into a condensed, inaccessible form, preventing transcription of
nearby genes.
Detailed Mechanism: The process of silencing involves complex
chromatin modifications and the recruitment of specific proteins,
leading to the establishment of repressive chromatin states.
Dual Activity: Some DNA elements can exhibit both enhancer and
silencer activity depending on the proteins bound to them. For
example, the thyroid hormone response element can act as a silencer
when bound by the thyroid hormone receptor without its ligand.
However, it functions as an enhancer when the thyroid hormone
receptor binds along with thyroid hormone.
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