Transcript Document

Activities of RNA Polymerase
•sequence specific DNA binding
-promoters
•melts DNA to reveal the template strand
•selects ribonucleotide (not deoxynucleotides) that anneals to template strand
•polymerizes RNA strand
•translocates on DNA template, during which it must:
-unwind DNA in front of polymerase
-unwinds RNA:DNA hybrid
-rewinds DNA behind polymerase
•recognize termination signals in the nascent transcript (or on the DNA
template)
•In addition, the polymerase must be processive (have a high probability of
reaching the end of the gene)
Modular Organization of Regulatory Information via
Multiple Enhancers
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stripes of ftz and eve expression in
a Drosophila embryo
fragments of eve regulatory were
inserted upstream of a -Gal
reporter and inserted into flies.
different regulatory elements gave
distinct patterns of -Gal (dark
staining) expression. Normal eve
expression is shown in red.
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Stages of Transcription
Promoter binding
[closed complex]
Open complex formation
[open complex]
Abortive initiation
Promoter clearance
Elongation
Robert Roeder
today
collecting sea urchin embryos 1968
Keys to Successful Protein Purification
-an abundant source of material
-a quantitative assay
-must be able determine yield and purity
-a strategy for separation
-charge
-size
-hydrophobicity
-stability
-affinity reagents
Roeder’s Assay, Incorporation of 32Plabeled UTP into RNA
he could measure the amount of radioactivity
incorporated into RNA (vs that that remained
associated with the UTP)
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
note that the  phosphate is incorporated into the RNA
Three Different RNA Polymerases in Eukaryotes
(Roeder and Rutter, 1969)
functional group on column:
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DEAE-Sephadex
polymerase activity
(32P incorporation into RNA)
protein
(measured by UV light
absorbance in a
spectrophotometer)
[salt]
(50mM KCl
-400 mM)
RNA Polymerases I, II, and III Exhibit Different Sensitivities
% Maximum activity
towards -Amanitin
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Amanita phalloides
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-Amanitin (g/ml)
Eukaryotic Nuclear RNA Polymerases
Enzyme
location
relative
activity
-amanitin
sensitivity
Pol I
nucleolus
50-70%
not inhibited
Pol II
nucleoplasm
20-40%
inhibited
Pol III
nucleoplasm
≈10%
species-specific
All three polymerase classes....
weigh >500,000 D
contain 12-16 subunits
- some conserved across evolution
’ - like (~200,000 D)
- like (~140,000 D)
- like (~40,000 D)
- some shared among all 3 polymerases
- some unique
Three Classes of Transcription in Eukaryotes
RNA polymerase I (pol I)
ribosomal RNAs (5.8S, 18S, 28S rRNA)
RNA polymerase II (pol II)
mRNAs
some small nuclear RNAs (snRNAs)
non-coding RNAs (mostly of unknown function)
RNA polymerase III (pol III)
tRNAs
5S RNA
some snRNAs
small cytoplasmic RNAs (scRNAs)
RNA Polymerase II Underlies the Central Dogma of
Molecular Biology
Pol I
DNA
Pol II
Pol III
45S rRNA
mRNA
Ribosome
tRNA
5S rRNA
this explains our emphasis on the mechanisms of Pol II transcription
Protein
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Roger Kornberg (1947)
(With wife and sons,
Stockholm Dec. 2006)
Important things to know about Pol II
•
•
•
The general architecture of the polymerase, including the arrangement
of nucleic acids in the active site
That nucleotides likely enter through the “funnel”
That the polymerase is a catalyst that specifically accelerates the rate
at which the correctly paired ribonucleotide is added to a growing RNA
chain (the exact details of the proposed reaction mechanism are not
important to know)
– Review, but don’t feel obligated to memorize the exact details of the role of
the trigger loop in facilitating catalysis and substrate (I.e. nucleotide)
selection
•
That the polymerase must be able to translocate on the DNA template
after it has added a nucleotide to the RNA
Crystal Structure of Yeast RNA Polymerase II at 2.8 Å
Resolution (Cramer et al, 2001)
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RNA Pol II (S. cerevisiae)
RNAP (T. aquaticus)
Rpb2

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’
Rpb1
What is Required for Promoter Function?
cis (DNA sequences) vs trans (proteins)
identify cis elements by, conservation, mutagenesis and assays of
transcription
identify trans factors by biochemical (mainly) and genetic
(occasionally) approaches
Assays of Promoter Activity
in vitro
- use a specific promoter
- mix with NTPs and extract or purified factors
- measure RNA (directly, with 32P-labeled NTPs
or indirect assays, see Weaver pp 106-111)
in vivo
- introduce gene of interest into cells
transformation (yeast and bacteria)
transfection (cultured cells)
- fuse promoter to a reporter gene that can be differentiated
from normal genes in the recipient cells
“reporter gene”
- can collect and measure RNA from cells
- can measure activity of reporter gene
beta-Gal, selectable markers
Identifying Promoter Elements
•
•
•
Deletion mutations
Linker scanning
Point mutations
lin k e r i n s e r t io n m u ta g e n e s is
tx n
del et io n mu tag en es is
+
tx n
+
+
-
+
+
-
-
p o in t m u ta tio n s
+
sca nn in g de let io n mu ta ge nes is
X
+
X
+
X
+
X
-
+
-
Class II Promoters
Several parts:
– Core promoter
– Upstream promoter
elements
– Enhancers, may be
far from core
promoters
gene specific
sequence elements found in
many core promoters
Core Promoter Elements
• In addition to TATA box, core promoters are:
– TFIIB recognition element (BRE)
– Initiator (Inr)
– Downstream promoter element (DPE)
– note: the important thing to remember is the TATA box
• 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 from core promoters in binding to relatively
gene-specific transcription factors.
examples:
– GC boxes bind transcription factor Sp1
– CCAAT boxes bind CTF (CCAAT-binding transcription factor)
• Enhancers, function in a position and orientation
independent manner. (sometimes enhancers are
considered to be a distinct type of element)
A Typical RNA Pol II Promoter
-2000
-1500
-1000
enhancers
AP4
AP1
AP3
-200 -150 -100 -50
upstream
elements
+1 50
100
core promoter
AP2
CCA GCTGTGG AATG TGTGTCA GTT AGGGT GTGGAAAGTCCCCAGGC T
TATA-box
Inr
CAGAGC ATATAA GGTGAGGTAGGATCAGTTGCTC CTCACCTT
-30
-20
-10
+1
GC-box
CAAT-box
CGTAGA GCCACACCC TGGTAAG GGCCAATC TGCTCAC
-100
-90
-80
-70
Identifying the General Transcription Machinery
• establish robust in vitro assay with a strong core
promoter
– AdML adenovirus major late promoter
• purify proteins required for transcription
• goal is to identify a minimal set of purified proteins
with which to reconstitute transcription
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Fork loop 2
What is Required for Promoter Function?
Observation: RNA polymerase alone is not capable of accurately
initiated, gene-specific transcription.
cis (DNA sequences) vs trans (proteins)
identify cis elements by, conservation, mutagenesis and assays of
transcription
-core promoter (esp. TATA box)
-upstream elements
-enhancers
identify trans factors by biochemical (mainly) and genetic
(occasionally) approaches
-general transcription factors (GTFs)
-accessory factors required at all genes
transcribed by a polymerase
Identifying the General Transcription Machinery
• establish robust in vitro assay with a strong core
promoter
– AdML adenovirus major late promoter
• purify proteins required for transcription
• goal is to identify a minimal set of purified proteins
with which to reconstitute transcription
RNA Polymerase II Requires Additional Factors for
Accurate Transcription Initiation at Promoters
(Matsui et al, 1980)
Phosphocellulose chromatography
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Further Fractionation of S-100 Extract (Matsui et al, 1980)
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RNA polymerase II Transcription Machinery
Number of subunits
Pol II
12
GTFs
TFIID
TFIIB
TFIIE
TFIIH
TFIIF
TFIIA*
Mediator
TBP
TAFs *
1
12
1
2
9
2
3
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Proposed Pathway of Initiation
Pol II-TFIIF recruited by DNA-D/A/B complex
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helicases in TFIIH catalyze open
complex formation
initiation and escape
Mechanism of Initiation
of RNA Polymerase II
Transcription
Factor
Pol
Pol
H
H
F
TBP
BN
Role
BBCCTBP
TBP
Promoter recognition; configures DNA to the pol II surface
TFIIB
Promoter recognition; pol II recruitment; directs DNA
path, stabilizes early transcribing complex; coupling of
RNA synthesis to promoter clearance
TFIIE
Recognizes closed complex, recruits TFIIH
TFIIF
Captures nontemplate strand upon melting
TFIIH
Untwisting of promoter DNA (helicase),
CTD phosphorylation (kinase)
Transcription Elongation
...is slow compared to DNA replication
20-40 nucleotides / second
Typical 1° transcript is ≈20,000 nts., corresponds to ≈10 minutes /
transcript. Long transcripts can take hours to complete
… is regulated
TFIIF suppresses pausing
TFIIS rescues arrested complexes
others
…. polymerases stalled at the 5’ ends of genes appear to be common
...may involve proof-reading (observed in vitro)
...is coupled to DNA repair
Activating domains can be replaced by randomly selected
sequences (Ma and Ptashne, 1986)
beta-gal activity
Q. what are the essential features of
activation domains?
Approach: random e. coli sequences cloned
downstream of Gal4 DBD and expressed in
yeast containing a -gal reporter with Gal4
sites in its promoter
activator
-gal
+gal
Gal4
111
1895
Gal4DBD
<1
<1
none
<1
<1
B17
415
794
B42
542
756
B6
429
588
B9
21
9.3
B15
90
73
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Activating domains can be replaced by randomly selected
sequences (Ma and Ptashne, 1986)
1.
random e. coli sequences cloned
downstream of the Gal4 DBD and expressed
in a yeast strain containing a -gal reporter
with Gal4 sites in its promoter
2.
~1% of all the clones activated transcription
3.
activating sequences did not resemble
known proteins, no catalytic domains etc.
-activation domains unlikely to have
enzymatic activity
4.
negatively charged residues common
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Overexpression of Gal4 from the strong ADH promoter
inhibits promoters that lack Gal4 binding sites
(Gill and Ptashne, 1988)
HIS3
+1
core
+12
UASH/core
-Gal
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UASG= GAL enhancer (binds Gal4)
UASH= HIS3 enhancer
UASC= CYC1 enhancer
decreased expression of
reporter genes lacking Gal4
binding sites when Gal4 levels
are high
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Activator Interference or ‘Squelching’
activator B
activator A
UAS
TATA box
19
Activator Interference or ‘Squelching’
activator B
activator A
UAS
TATA box
hypothesis?
20
What is the Limiting Target of Activators?
1.
Eukaryotic activators do not bind to RNA pol II polymerase and
therefore do not directly recruit polymerase to promoters.
2.
Activators may, however, indirectly recruit RNA polymerase by
recruiting factors (often called co-activators) that serve as a physical
bridge between activator and polymerase.
‘TFIID
hypothesis’
‘Holoenzyme
hypothesis’
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ChIP (Chromatin Immunoprecipitation)
TBP
Formaldehyde crosslink
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Shear chromatin by sonication
Immunoprecipitation
TBP
TBP
TBP
Reverse crosslinks, PCR
input control: reverse
crosslinks and analyze
sample prior to IP
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Extent of TBP binding correlates with promoter activity
(Li et al, 1999)
TBP crosslinks to active
promoters
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RNA Polymerase II Transcription Machinery
Number of subunits
Pol II
12
GTFs
TFIID
TFIIB
TFIIE
TFIIH
TFIIF
TFIIA*
Mediator
TBP
TAFs *
1
12
1
2
9
2
3
22
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The ‘TFIID Hypothesis’
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1. TAFs provide surfaces for the interaction
of TFIID with activators.
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2. TFIID recruits polymerase
in vitro assays suggest specific activator-TAF contacts
predictions?
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Activated Transcription in the Absence of TAFIIs
Oelgeschlager et al., 1998
western blot demonstrating
depletion of TAFIIs
see p97 of Weaver or p769 of
Watson for a description of
the Western blot technique
in vitro transcription shows that
- transcription is abolished in the
TFIID depleted extract
- TBP is sufficient to restore
activated transction
- 4 different activators were tested
no transcription after depletion of TFIID and TAFs
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