activities of rna polymerase sequence specific dna binding -promoters melts dna to reveal the...

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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Open complex formation

Abortive initiation

Promoter clearance

Promoter binding

Elongation

Stages of Transcription

[closed complex]

[open complex]

Robert Roeder

collecting sea urchin embryos 1968

today

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

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Roeder’s Assay, Incorporation of 32P-labeled UTP into RNA

note that the phosphate is incorporated into the RNA

he could measure the amount of radioactivity incorporated into RNA (vs that that remained associated with the UTP)

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Three Different RNA Polymerases in Eukaryotes(Roeder and Rutter, 1969)

DEAE-SephadexQuickTime™ and a

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protein(measured by UV light absorbance in a spectrophotometer)

polymerase activity(32P incorporation into RNA)

[salt](50mM KCl-400 mM)

functional group on column:

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-Amanitin (g/ml)

% M

axim

um

act

ivit

yRNA Polymerases I, II, and III Exhibit Different

Sensitivities towards -Amanitin



Amanita phalloides

Enzyme location relative -amanitinactivity 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

Eukaryotic Nuclear RNA Polymerases

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

Pol II

Pol III

45S rRNA

tRNA5S rRNA

RibosomeDNA mRNA Protein

this explains our emphasis on the mechanisms of Pol II transcription



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

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Crystal Structure of Yeast RNA Polymerase II at 2.8 Å Resolution (Cramer et al, 2001)





RNAP (T. aquaticus) RNA Pol II (S. cerevisiae)

’

Rpb2

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

• Deletion mutations

• Linker scanning

• Point mutations

Identifying Promoter Elements

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p o i n t m u t a t i o n s

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l i n k e r i n s e r t i o n m u t a g e n e s i s

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t x n

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s c a n n i n g d e l e t i o n m u t a g e n e s i s

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d e l e t i o n m u t a g e n e s i s t x n

Class II PromotersSeveral parts:– Core promoter– Upstream promoter

elements– Enhancers, may be

far from core promoters

sequence elements found in many core promoters

gene specific

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)

-2000 -1500 -1000 -200 -150 -100 -50 +1 50 100

core promoterupstreamelements

enhancers

CAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCCTCACCTT-30-20-10 +1

TATA-box Inr

CGTAGAGCCACACCCTGGTAAGGGCCAATCTGCTCAC -100 -90 -80 -70

CAAT-boxGC-box

CCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCT

AP4 AP1 AP3 AP2

A Typical RNA Pol II Promoter

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



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





Further Fractionation of S-100 Extract (Matsui et al, 1980)

Pol II 12

GTFs TFIID

TFIIB 1 TFIIE 2 TFIIH 9 TFIIF 2 TFIIA 3

Mediator 22

RNA polymerase II Transcription MachineryNumber of subunits

TBP 1TAFs 12*

*



Proposed Pathway of Initiation

helicases in TFIIH catalyze open complex formation

Pol II-TFIIF recruited by DNA-D/A/B complex

initiation and escape

Factor Role

TBP

TFIIB

TFIIE

TFIIF

TFIIH

Promoter recognition; configures DNA to the pol II surface

Promoter recognition; pol II recruitment; directs DNA path, stabilizes early transcribing complex; coupling of RNA synthesis to promoter clearance

Recognizes closed complex, recruits TFIIH

Captures nontemplate strand upon melting

Untwisting of promoter DNA (helicase), CTD phosphorylation

(kinase)

Mechanism of Initiationof RNA Polymerase IITranscription

H

Pol

TBPBC

H

Pol

TBP

BC

F BN

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 pausingTFIIS rescues arrested complexesothers

…. 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)

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

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

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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)

core

HIS3+1

UASH/core

+12

-Gal

UASG= GAL enhancer (binds Gal4)UASH= HIS3 enhancerUASC= CYC1 enhancer

decreased expression of reporter genes lacking Gal4 binding sites when Gal4 levels are high

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TATA box

activator A

UAS

Activator Interference or ‘Squelching’

activator B

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TATA box

activator A

UAS

Activator Interference or ‘Squelching’

activator B

hypothesis?

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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’

What is the Limiting Target of Activators?

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ChIP (Chromatin Immunoprecipitation)

Formaldehyde crosslink

Shear chromatin by sonication

Immunoprecipitation

Reverse crosslinks, PCR

TBP

TBP

TBP

TBP

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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Pol II 12

GTFs TFIID

TFIIB 1 TFIIE 2 TFIIH 9 TFIIF 2 TFIIA 3

Mediator 22

RNA Polymerase II Transcription MachineryNumber of subunits

TBP 1TAFs 12*

*

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The ‘TFIID Hypothesis’

in vitro assays suggest specific activator-TAF contacts

predictions?





1. TAFs provide surfaces for the interaction

of TFIID with activators. 2. TFIID recruits polymerase

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Activated Transcription in the Absence of TAFIIs

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

Oelgeschlager et al., 1998

no transcription after depletion of TFIID and TAFs 26

activities of rna polymerase sequence specific dna binding -promoters melts dna to reveal the...

Documents

eukaryotes rna polymerase

s rrna rna polymerase

rna strand

rna salt

rna vs

transcription slide

different rna polymerases

polymerase classes