Multiple Classes of Eukaryotic Cellular RNAs

1

• Multiple Classes of Eukaryotic Cellular RNAs
• messenger RNA (mRNA)
• ribosomal RNA (rRNA)
• 18S (small subunit)
• 28S (large subunit)
• 5.8S (large subunit)
• 5S (large subunit)
• transfer RNA (tRNA)
• small nuclear RNA (snRNA)
• U1, U2, U3, U4, U5, U6, U7, U8, U9, U10...
• small cytoplasmic RNA (scRNA)
• 7SL RNA
• What are the polymerases responsible for the
• synthesis of these RNAs?

2
Eukaryotes possess 3 RNA polymerases
Polymerase Location What RNA does this
polymerase synthesize? RNA polymerase I
(A) nucleolus 18S, 28S, 5.8S rRNA RNA
polymerase II (B) nucleoplasm mRNA,
hnRNA U1, U2, U4, U5 snRNA RNA polymerase
III (C) nucleoplasm tRNA,5S RNA, U6
snRNA,7SL and 7SK RNA Mitochondrial
mitochondrion all mitochondrial RNA RNA
polymerase ______________________________
______________________
S is for sedimentation coefficient separating by
size on sucrose gradient
3
How do we know?
Input protein fraction of entire cell extract Or
specific to certain cellular compartments
(nuclear)
1969 Roeder and Rutter fractionated sea urchin
embryo extracts Activity assay incorporation of
UMP into RNA
Gel filtration column contains porous bead/resin
that resembles a slurry (sephadex is a
carbohydrate polymer)
See fig 5.7
Ion-exchange filtration column contains resin
with a charge (DEAE positive charge attracts
negative charged proteins) Wash the column with
increaseing ionic strength (salt conc.) to
compete with the charge on the proteins
A280 to measure protein content
UMP incorporation
0 20 40 60 80
RNA pol I
RNA pol II
RNA pol III (esp conc. In nucleolus)
Fraction collections based on size Individual
fractions concentrated for specific proteins
tested for activity
Fig 10.1
4
RNA Polymerases contain multiple
subunits conserved across species as diverse as
yeast and humans

• All structures are complex
• All contain two large (gt100kD) subunits analogous
  to prokaryotic ? and ?
• All contain a variety of smaller subunits
• Some of the smaller subunits are common to all
  three polymerases

5
RNA Polymerase I transcribes genes for ribosomal
RNA
Polymerase Location What RNA does this
polymerase synthesize? RNA polymerase
II (B) nucleoplasm mRNA, hnRNA U1, U2, U4,
U5 snRNA RNA polymerase III (C)
nucleoplasm tRNA,5S rRNA, U6 snRNA,7SL and
7SK RNA Mitochondrial mitochondrion all
mitochondrial RNA RNA polymerase
__________________________________________________
__
RNA polymerase I (A) nucleolus 18S, 28S, 5.8S
rRNA (large rRNA precursor)
S is for sedimentation coefficient separating by
size on sucrose gradient
6
RNA Polymerase I recognizes Class I Promoters

• Two elements of the Class I promoter are well
  conserved
• Core element surrounding transcription start site
• Upstream promoter element (UPE) 100 bp farther
  upstream
• Spacing between these elements is important

Linker scanning to mutate short stretches of DNA
in rRNA promoter identifies important regions
Fig. 10.24
7
RNA Polymerase I recognizes Class I Promoters
Sl1

• UBF (Upstream binding factor) protein promotes
  initiation by bringing UPE and promoter in
  proximity
• It works by bending the DNA dramatically
• Degree of reliance on UBF varies considerably
  from one organism to another
• This transcription factor is an assembly factor
  that recruits the core binding factor SL1 (a 4
  protein complex) to bind to the core promoter
  element

8
RNA Polymerase I recognizes Class I Promoters
Sl1
UBF (Upstream binding factor) protein promotes
initiation by bringing UPE and promoter in
proximity
UBF
RNA Pol
TBP
Sl1
UBF recruits Sl1 core binding factor that
recruits TATA Binding Protein (TBP) to position
RNA Pol I and SL1 for start site selection
9
Structure and Function of SL1

• The core-binding factor, SL1, was originally
  isolated on the basis of its ability to direct
  polymerase initiation
• This factor is the fundamental transcription
  factor required to recruit RNA polymerase I
• SL1 also shows species specificity
• Human SL1 is composed of TBP and TAFs which bind
  TBP tightly
• TAFI110
• TAFI63
• TAFI48
• These TAFs are completely different from those
  found in TFIID
• Yeast and other organisms have TAFIs that are
  different from the human group

10
RNA Polymerase III transcribes genes for a
variety of RNA species
Polymerase Location What RNA does this
polymerase synthesize? RNA polymerase I
(A) nucleolus 18S, 28S, 5.8S rRNA RNA
polymerase II (B) nucleoplasm mRNA,
hnRNA U1, U2, U4, U5 snRNA
Mitochondrial mitochondrion all mitochondrial
RNA RNA polymerase ___________________
_________________________________
RNA polymerase III (C) nucleoplasm tRNA,5S
rRNA, U6 snRNA,7SL and 7SK RNA
S is for sedimentation coefficient separating by
size on sucrose gradient
11
RNA Polymerase III recognizes Class III Promoters

• There are three types of Class III promoters
• Type I includes only the 5S rRNA genes that have
  promoters that lie wholly within the genes.
• Type II includes most of the RNA Pol III targets
  including the tRNA genes. These promoters lie
  wholly within the genes.
• Type III are nonclassical and more similar to
  Pol II promoters

12
RNA Polymerase III types I and II
useTranscription FactorIIIC for initiation

• TFIIIC (6 subunits) binds to internal promoter
  (with or without TFIIIA)
• TFIIIC promotes binding of TFIIIB with its TBF
• TFIIIB promotes polymerase III binding and
  positioning at start site
• Transcription begins

13
Class III Factors

• TFIIIB and TFIIIC are required for transcription
  of the classical polymerase III genes. They
  depend on each other for their activities.
• TFIIIC is an assembly factor that allows TFIIIB
  to bind to the region just upstream of the
  transcription start site
• TFIIIB can remain bound and sponsor initiation of
  repeated transcription rounds
• TFIIIA was the first member of the family of
  DNA-binding proteins that feature a zinc finger
  domain that is roughly finger-shaped protein
  domain with 4 amino acids that bind a zinc ion

14
RNA Pol II acts on the majority of genes
Polymerase Location What RNA does this
polymerase synthesize? RNA polymerase I
(A) nucleolus 18S, 28S, 5.8S rRNA RNA
polymerase III (C) nucleoplasm tRNA,5S
RNA, U6 snRNA,7SL and 7SK RNA
Mitochondrial mitochondrion all mitochondrial
RNA RNA polymerase ___________________
_________________________________
RNA polymerase II (B) nucleoplasm mRNA,
hnRNA U1, U2, U4, U5 snRNA
S is for sedimentation coefficient separating by
size on sucrose gradient
15
RNA Polymerase II contains 12 subunits
Human
Yeast
Size (kD)
Function
Despite this complexity RNA pol II must be
recruited to promoter by another transcription
factor complex!
16
How do we know? Epitope tagging and co-IP Work by
Young and colleagues and Kornberg and colleagues
Introduce engineered gene into deficient
cells Such that gene contains extra domain that
will bind Strongly to antibody (his ,myc,HA,FLAG
are common)
RBP3
Grow cells in medium with 35S labelled methionine
to label all protein subunits so they will be
visible on a gel
Immunoprecipitate with Antibody against the
engineered subunit to known component to isolate
complex
Add detergent (SDS) to separate components Run
products on SDS-PAGE gel (protein gel)
17
Structure of RNA polymerase II
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
RBP3 and RBP4 not present Mg2 ion (magenta
sphere) marks the position of the catalytic
center.
18
Structure of RNA polymerase II
Original 10 subunits are placed in 3 groups Core
subunits are related in structure and function to
bacterial core subunits Common subunits are found
in all 3 nuclear RNA polymerases Nonessential
subunits are conditionally dispensable for
enzymatic activity
RBP3 and RBP4 not present Mg2 ion (magenta
sphere) marks the position of the catalytic
center.
19
Core Subunits
Rpb1, Rpb2, Rpb3 are absolutely required for
enzyme activity These are homologous to bacterial
?, ?, and ??subunits Rpb1 (?) binds DNA Rpb2
(?) is at or near the nucleotide-joining active
site Rpb3 acts functionally similar to ??subunit
but does not resemble ?-subunit except for same
size and one 20-amino acid subunit of great
similarity
RBP3 and RBP4 not present Mg2 ion (magenta
sphere) marks the position of the catalytic
center.
20
Common Subunits
There are five common subunits Rpb5 Rpb6 Rpb8 Rpb1
0 Rpb12 Little known about function They are all
found in all 3 polymerases which suggests they
play roles fundamental in transcription or
stabilization
RBP3 and RBP4 not present Mg2 ion (magenta
sphere) marks the position of the catalytic
center.
21
Nonessential for elongation subunits Rbp4 and
Rbp7
Dissociate fairly easily from polymerase and
might shuttle from one PolII to another Rpb4 may
help anchor Rpb7 to the enzyme Mutants without
Rpb4 and Rpb7 transcribe well, but cannot
initiate at a real promoter with Rpb4/7 in place,
clamp is forced shut Rpb4/7 extends the dock
region of the polymerase, which makes binding of
transcription factors easier Rpb7 is an essential
subunit, so must not be completely absent in the
mutant
RBP3 and RBP4 not present Mg2 ion (magenta
sphere) marks the position of the catalytic
center.
22
Position of Critical Elements in the
Transcription Bubble
Upstream DNA

• Three loops of the transcription bubble are
• Lid maintains DNA dissociation
• Rudder initiating DNA dissociation
• Zipper maintaining dissociation of template DNA

RNA exit
Downstream DNA
Fig. 10.14
23
Position of Critical Elements in the
Transcription Bubble
4 unpaired nucleotides on template DNA

• 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

24
Class II Promoters

• Core promoter having 4 elements
• TFIIB recognition site (BRE)
• TATA box
• Initiator (Inr)
• Downstream promoter element (less common)
• Upstream element

25
promoter elements for RNA polymerase II
1
upstream element
Distance can be several kb
exon
exon
UE
P
Inr
Promoter (about 200 bp)
(enhancer)

• Promoter (DNA sequence upstream of a gene)
• determines start site (1) for transcription
  initiation
• located immediately upstream of the start site
• allows basal (low level) transcription
• Upstream element (DNA sequence that regulates the
  gene)
• determines frequency or efficiency of
  transcription
• located upstream, downstream, or within genes
• can be very close to or thousands of base pairs
  from a gene
• includes enhancers (increase transcription
  rate), silencers (decrease
• transcription rate), response elements (targets
  for signaling molecules)
• genes can have numerous transcription elements

26
Sequence elements within a typical eukaryotic
gene promoter1
octamer transcription element
1
GC
TATA
CAAT
GC
Inr
ATTTGCAT
-25
-50
-80
-95
-130

• TATA box (TATAAAA)
• GC box (CCGCCC)
• binds Sp1 (Specificity factor 1)
• CAAT box (GGCCAATCT)
• binds CTF (CAAT box transcription factor)
• Octamer (ATTTGCAT)
• binds OTF (Octamer transcription factor)

Exact and position of these elements varies
from gene to gene
1 thymidine kinase gene is shown
27
Promoter elements for RNA polymerase II
1
exon
GC
TATA
CAAT
GC
P
Inr
ATTTGCAT
-25
-50
-80
-95
-130

• TATA box (TATAAAA)
• Similar to the prokaryotic -10 box and determines
  the exact start site
• located approximately 25-30 bp upstream of the 1
  start site
• Deletion leads to erratic initiation site
  selection
• binds the TATA Binding Protein (TBP) which is a
  subunit of TFIID

• TATAless promoters may have the DPE at 28 or 32
• Housekeeping genes that are constituitively
  active in nearly all cells
• Developmentally regulated genes

28
Promoter elements for RNA polymerase II
1
exon
GC
TATA
CAAT
GC
P
Inr
ATTTGCAT
-25
-50
-80
-95
-130
TATA Binding Protein (TBP) which is a subunit of
TFIID

• Binds to minor groove of DNA saddling DNA and
  bending 80
• Binding may be inconsistent with nucleosomes
  which tend to shield minor groove
• Allows better contacts of other factors to DNA

29
Sequence elements within a typical eukaryotic
gene promoter1
1
GC
TATA
CAAT
GC
Inr
ATTTGCAT
-25
-50
-80
-95
-130

• GC box (CCGCCC)
• binds Sp1 (Specificity factor 1)
• CAAT box (GGCCAATCT)
• binds CTF (CAAT box transcription factor)
• Octamer (ATTTGCAT)
• binds OTF (Octamer transcription factor)

• Differ from core promoter in binding to
  relatively gene-specific transcription factors
• GC boxes bind transcription factor Sp1
• CCAAT boxes bind CTF (CCAAT-binding transcription
  factor)
• Exact and position of these elements varies
  from gene to gene
• Upstream elements can be orientation-independent,
  yet are relatively position-dependent

30
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

31
Enhancer elements can be upstream or downstream
or within. Enhancers can be in either
orientation Enhancers can be tens of kb distant
from the gene Enhancers are dense with protein
binding sites and have a cooperativity between
sites. These proteins then interact with the
basal complex.
exon
P
enhancer
Inr
exon
exon
P
Inr
exon
(enhancer)
Immunoglobulin genes
exon
P
Inr
exon
(enhancer)
32
Insulators
gene
enhancer
1. Insulator will block action of an enhancer
gene
enhancer
2. Insulator may ensure gene specific action of
an enhancer
gene
enhancer
gene
3. Insulator may provide barrier to
heterochromatin propoagation
gene
enhancer
33
Insulators What are they?
gene
enhancer
Insulators themselves are neither positive nor
negative regulators, But block influence of
domains next door. An insulator will function
anywhere it is placed/inserted into the
genome. Typical insulator sequence includes
several hundred bp DNAse I resistant flanked by
sensitive sequence. Locus control region (LCR)
can also act as insulator.
34
Insulators How do they act?
gene
enhancer
Proteins recognize and bind to insulator
sequences. In Drosophila, insulator sequence
scs includes repeats of CGATA that binds
BEAF-32. Antibodies to this indicate that
Drosophila polytene chromosome bands are
insulator units.
35
Insulators may act via higher order chromatin
structure
Su(Hw) and mod(mdg4) in Drosophila are proteins
that recognize and bind insulator
regions. Su(Hw) and mod(mdg4) bind about 500
sites in Drosophila genome and co-localize at 25
discrete sites at nuclear periphery. Proteins
may come together creating a complex with about
20 loops, each with its own enhancers.
DNA loops
Su(Hw)
mod (mdg4)
Nuclear periphery
36
Formation of the Pre-initiation complex in four
distinct stages

• TFIID with help from TFIIA binds to the TATA box
  forming the DA complex
• TFIIB binds next generating the DAB complex
• TFIIF helps RNA polymerase bind to a region from
  -34 to 17, now it is DABPolF complex
• Last the TFIIE then TFIIH bind to form the
  complete preinitiation complex DABPolFEH
• In vitro the participation of TFIIA seems to be
  optional

37
Structure and Function of TFIID complex

• TATA-box binding protein (TBP)
• Highly evolutionarily conserved
• Binds to the minor groove of the TATA box
• Saddle-shaped TBP lines up with DNA to force open
  the minor groove
• The TATA box is bent into 80 curve
• 8 to 10 copies of TBP-associated factors (TAFIIs)
  specific for class II

38
Binding of the general transcription factors
F
TAFs
TFIID
B
A
TBP
1
-25

• TFIID complex along with TFIIA (upstream), binds
  to the TATA box
• the TATA binding protein (TBP) directly binds the
  TATA box
• TBP associated factors (TAFs) bind to TBP
• TFIIB (downstream) binds to TBP at the TATA box
  via its C-terminal domain and to RNA PolII via
  its N-terminal domain. The protein provides a
  bridging action that effects a coarse positioning
  of polymerase active center about 25 30 bp
  downstream of the TATA box.

39
Binding of RNA polymerase II
F
TFIID
B
TBP
A
RNA pol II

• RNA polymerase II binds to
• the promoter region by interacting with the
  TFIIs
• TFIIF (helicase) binds RNAPolII and may help
  recruit it to DABD complex
• TFs recruit additional proteins and histone
  acetylase to the promoter

40
Binding of Additional factors

• TFIIE and TFIIH are not essential for formation
  of an open promoter complex or elongation BUT are
  required for promoter clearance
• TFIIE extends the protected boundary of complex
  another 2 turns
• TFIIH is a very complex protein of 9 subunits
• Protein kinase complex of 4 subunits
• Core TFIIH complex of 5 subunits with 2 DNA
  helicase/ATPase activities
• TFIIH is a helicase and ATPase to unwind DNA at
  the transcription start site to create the
  transcription bubble, kinase that phosphorylates
  the CTD of RNA PolII to promote elongation, and
  acts in transcription coupled repair of DNA
  damage

F
E
TFIID
J
B
TBP
A
H
RNA pol II
41
Binding of specialized TFs
F
E
TFIID
J
B
TBP
A
H
RNA pol II

• transcription factors binding to
• other promoter elements and transcription
  elements interact
• with proteins at the promoter and further
  stabilize (or inhibit)
• formation of a functional preinitiation
  complex
• this process is called transactivation

42
Formation of a stable pre-initiation complex
F
E
J
TFIID
B
TBP
H
1
RNA pol II

• the stability and frequency with which complexes
  are formed
• determines the rate of initation of
  transcription
• the rate of initiation of transcription is of
  major importance in
• determining the abundance of an mRNA
  species

43
Initiation of transcription and promoter clearance
F
E
B
H
TFIID
initiation
TBP
J
1
RNA pol II
CTD
P
P
P

• TFIIH phosphorylates serines 2 and 5 in the
  heptad repeat in the carboxyl-terminal domain
  (CTD) of the largest RNA polymerase subunit (this
  is called the initation reaction) releasing it
  from the preinitiation complex and allowing it to
  initiate RNA synthesis and move down the gene

44

• Expansion of the Transcription Bubble
• DNA helicase of TFIIH causes unwinding of the DNA
• Expansion of the transcription bubble releases
  the stalled polymerase
• Polymerase is now able to clear the promoter

TFIID with TFIIB, TFIIF and RNA polymerase II
form a minimal initiation complex at the initiator

• Elongation complex
• Polymerase CTD is further phosphorylated by TEFb
• NTPs are continuously available
• TBP and TFIIB remain at the promoter
• TFIIE and TFIIH are not needed for elongation and
  dissociate from the elongation complex

• Addition of TFIIH, TFIIE and ATP allow DNA
  melting at the initiator region and partial
  phosphorylation of the CTD of largest RNA
  polymerase subunit
• These events allow production of abortive
  transcripts as the transcription stalls at about
  10

45
Promoter clearance and elongation
1
RNA pol II
CTD
P
P
P

• The initation reaction releases RNA Pol II from
  the preinitiation complex and allows RNA
  synthesis and movement down the gene
• Factors then dissociate

46
Elongation and TFIIS

• RNA polymerases do not transcribe at steady rate
• Short stops in transcription are termed
  transcription pauses
• Pauses are for variable lengths of time
• Pauses tend to occur at defined pause sites where
  DNA sequence at those sites destabilize the
  RNA-DNA hybrid, causing polymerase to backtrack
• If backtracking goes too far, polymerase cannot
  recover on its own Transcription arrest
• Polymerase needs help from TFIIS during a
  transcription arrest

47
TFIIS Stimulates Proofreading of Transcripts

• Proofreading is the correction of misincorporated
  nucleotides
• TFIIS stimulates proofreading, likely by
  stimulating RNase activity of the RNA polymerase
• This would allow polymerase to cleave off a
  misincorporated nucleotide and replace it with a
  correct one

48
Coupling of Transcription, capping and splicing
1
RNA pol II
CTD
P
P
P

• Multiple proteins bind to phosphorylated CTD of
  RNA PolII at initiation including RNA capping
  enzyme, and SCAFs which bind to splicing factors