Selected topics in Transcription

Nir London.Computational Biology Seminar 2006

Overview

• Elongation– Pause; Arrest– Chromatin remodeling; Histones– CTD

• Mediator Complex– Mechanism model– Composition and Interaction network

• Initiation Mechanism– New findings

Elongation

• 17 BP Open bubble • 5’ to 3’ Elongation• 50-90 BP / second

Leninger 5’th edition

Elongation reaction

• 3 ASPs highly conserved across all species

Leninger 5’th edition

Elongation by RNA polymerase II: the short and long of it

Robert J. Sims, III, RimmaBelotserkovskaya and Danny Reinberg

Genes & Dev. 2004

What’s stopping elongation?

• Efficient elongation must overcome several blocks.– Transcriptional pause– Transcriptional arrest– Transcriptional termination

• Many elongation factors serve to counteract or remove one of the above.

Pause

• The RNA polymerase halts elongation for a time before resuming on its own.

• Pausing of bacterial RNA pol is caused by a structural rearrangement within the enzyme and DNA sequence.

Easy modulation of rate?

• Demonstrated for all three eukaryotic RNA polymerases, viral and prokaryotic.

• Pausing is self-reversible a natural mode of transcriptional regulation.

• Many factors modulate transcriptional pause and thus, the rate of elongation.

Pause to cap

• DSIF/NELF complex promotes pausing and enables capping

• TFIIF < Elongins < ELLs promote elongation at different places along the gene.

Arrest

• Irreversible halt to synthesis. Pol cannot resume without additional factors

• The polymerase “backtracking” relative to the DNA template

• Misalignment of the catalytic site and 3-OH of the transcript

• Pause decays into arrest in a time dependent fashion

Resume mechanism

• Resuming uses an evolutionarily conserved mechanism

• Requires cleavage of the RNA transcript in a 3’-to-5’ direction

• Cleavage allows the proper realignment of the active site and 3’-OH.

TFIIS – Arrest solver

• The cleavage reaction is intrinsic to the Pol. Enhanced in the presence of TFIIS.

TFIIS (cont.)

• An acidic hairpin coordinating a metal ion Re-aligns the RNA to the cleavage active site.

Kettenberger H. et al. 2003

Nucleosomes – another block

• How does the Pol. Traverses the nucleosomes ?

• Models:– Nucleosome mobilization– Histone depletion

Swi\Snf – ATP dependent chromatin remodeler

• Transcription pauses shortly after initiation.

• HSF1 alleviates the negative effect of chromatin structure.

• Recruits Swi\Snf to Hsp70 gene

• Both Activator and Swi\Snf are required for transcription on nucleosomal templates.

Mechanism?

Narlikar GJ. Et al. Cell 2002

FACT – histone chaperone

• Highly conserved

• ChIP showed it to be localized downstream to promoters of active genes upon induction

• Destabilize the nucleosome by removing one H2A/H2B dimer.

Spt6

• Promotes nucleosome assembly in vitro

• Spt6 mutants show alterations in chromatin structure

• Colocalized to transcribed regions

• Interacts with H3

Mechanism

Histone Modifications and elongation

• Histone acetylation destabilizes chromatin structure

• No evidence for a specific role of histone acetylation in elongation

Set1/2 - Methylation

• Methylation can co-map with silent or active regions – depend on Lys

• Linking CTD to histone modifications• Set2 - H3-K36-specific histone methyltransferase• Set2 associates with the hyperphosphorylated RNAPII• Deletion of the CTD, or the CTD-kinase Ctk1, results in a

loss of H3-K36 methylation• Set1 functions as a specific histone H3-K4

methyltransferase• Set1 interacts with the Ser-5 phosphorylated form of

RNAP II. the form associated with early transcriptional events

Chd1

Iws1

Swi/Snf

Spt6 FACT

DSIF

TFIIS

P-TEFb

Paf

ISWII

Elongator

Set1

Set2

TFIIF

Spt2

CTD

• CTD serves as a platform for many factors for mRNA maturation

• Different phosphorylation patterns creates different structures

Flexible

• A) Cgt1-CTD

• B) Pin1-CTD

• Heptad repeats are not identical

• Could explain specific factor binding

Conclusions?

• Why are there so many redundant EF’s ? – The answer might be that they are

promoter/gene specific

• How does elongation and chromatin remodeling work together ?

• How histone modifications translate to distinct functional outcomes ?

• Why is the rate of elongation in vitro, far less than the rates observed in vivo ?

The yeast Mediator complex andits regulation

Stefan Bjorklund and Claes M. Gustafsson

TRENDS in Biochemical Sciences, May 2005

Mediator

• Required for activator dependent stimulation of Pol2.

• Comprised of 25 subunits

• Can be found as free form or attached to Pol2.

Mediator interaction with Pol2

• CTD reminder: – Initiation – unphosphorylated – Elongation – phosphorylated

• Mediator complex interacts directly with the unphosphorylated form of the CTD

• Dissociation upon elongation

Transcriptional activation

• The model: Mediator acts as a bridge between activators and basal Pol2 machinery.

Activator Example – GAL4

Transcription Transcription Transcription Transcription

• Gal4 interacts directly with subunits Med15, Med17.

• ChIP showed association to be at an upstream activation sequence.

Separate recruitment

• 3 waves of TF recruitment:

• Separate recruitment has also been showed for other promoters.

• Demonstrated in higher eukaryotes• Mediator forms a scaffold for several rounds of

transcription

Galactose

0

SAGA

4-7

Mediator

6-10

Pol II

8-13

Transcriptional repression

• Srb8-11 identified as crucial for mediated repression– Tup1 repressor recruits Srb8-11

containing mediator– Srb10 kinase function is necessary

for repression– Srb8-11 genes showed in genetic

screens loss of repression

Transcriptional repression

• The model: repressors recruit mediator in a form in which interactions with Srb8-11 module are stabilized.

Example – C/EBPβ

• Switch phosphorylated by Ras

• Active form recruits mediator devoid of Srb8-11

• Repressive form recruits Srb8-11 containing mediator

Post translational modifications

• Irregularities in SDSpage migration for certain subunits.

• Treatment with phosphatase changed migration patterns

• ATP-analog experiments showed that Kin28 (part of TFIIH) phosphorylates not only the CTD but also the mediator

Modifications (cont.)

• Other kinases target mediator: (Srb10, ras, PKA)

• Another option for signaling pathways to modulate transcription

• The effects of modifications aren’t characterized – Lots more to investigate

Sub summary

• Mediator influences both recruitment of Pol. and initiation of transcription

• Might be involved in other transcription related processes (elongation, chromatin remodeling, splicing, RNA export)

• How does PT modifications affect mediator function ?

A high resolution protein interaction map of

the yeast Mediator complex

Benjamin Guglielmi, Nynke L. van Berkum,

Benjamin Klapholz, Theo Bijma, Muriel

Boube, Claire Boschiero, Henri-Marc

Bourbon, Frank C. P. Holstege and Michel

Werner

Nucleic Acids Research, 2004.

Pair-wise 2H analysis

• Each subunit was cloned as fusion protein with Gal4 DNA binding domain (GBD) or Gal4 Activation domain (GAD).

• Transformed into a GAL promoter-reporter genes strains.

• All possible matings were preformed.

Strains expressing GBD-Med2, Med3, Med4, Med13, Med15 showed strong expression of gal and were excluded from this analysis.

Results

Results (cont.)

• Identified interactions were retested by co-transformation to same strain

• 11 interactions found in middle-middle

• 7 interactions in head-head

• No interactions in tail

Screening genomic lib.

• Some interactions can’t be discovered using complete proteins

• Same screen only now attached to GAD are random S. cerevisiae genomic seqs.

• 17 interactions were found. (7 new ones)

Med31 – new subunit

• Med31 homologues found in mediator like complexes in higher eukaryots

• Fusion with GBD against all other 24 showed 2 interactions in middle section

• CoIP with Med17 confirmed it belongs to the mediator complex

"בואו נחבוש את כובע הביקורת..."

Interaction Domains

Truncation of conserved areas reveals different interaction domains for Med subunits.

Abortive Initiation and ProductiveInitiation by RNA Polymerase

Involve DNA ScrunchingAndrey Revyakin, Chenyu Liu, Richard H.

Ebright, Terence R. Strick

Science Nov. 2006

Initiation

• Transcription initiation is composed of:– RNAP binds to promoter (closed complex)– Unwinds 1 turn of DNA (open complex)– Abortive cycles of synthesis and release of

short RNA products (promoter initial transcribing complex)

– Upon synthesis of ~9-11 RNA nt enters into elongation (promoter escape)

Abortive initiation mystery

• Two contradicting observations:– RNA products of 8-10 nt are synthesized –

Thus the active center translocates relative to the DNA.

– Footprinting results indicates that the upstream DNA protected by RNAP is the same in RPo and RPitc – thus RNAP appears not to translocate relative to DNA.

Three models

Unwinding detection

Proving the scrunch

• The scrunching model is the only model that requires RNAP dependent DNA unwinding

• For each BP the RNAP pulls into itself, there another BP of DNA unwinding

Scrunching in abortive init.

• If no NTP are added we receive RPo

• If only some NTP’s are added we receive RPitc<8

Does scrunching requires RNA?

• Control I : only initiating A -> RPitc<=1

• Control II : rifampicin -> RPitc<=2

• Scrunching doesn’t occur -> requires an RNA product > 2 nt in length

RNA length and scrunching

• Tested on: – RPo (no NTP’s)– RPitc<4 (only A, U)– RPitc<8 (only A, U, C)

• Transition from 0 to 4 shows 2 bp unwinding

• Transitions from 0 to 8 shows 6 bp unwinding

• Simplest model : N-2

Productive initiation

• Constructs: Promotor-[400/100 bp]-Terminator.• Four transitions observed:

– Transition from initial state to RPo– Transition to scrunched RPitc– Transition to a “elongation state” – Transition to initial state again

Controls

• No NTP’s RPo transition 1• A,U,C RPitc<8 transition 1,2• All NTP’s, halted elongation transition

1,2,3• All NTP’s, no terminator transitions

1,2,3• Length of transcribed region varied

duration of phase between 3 and 4 changed

Conclusions

• Promoter escape requires RNA product ~9 to 11 nt in length.

• Thus requires scrunching of ~7 to 9 bp (N – 2), • Assuming an energetic cost of ~2 kcal/mol per

bp, a total of ~14 to 18 kcal/mol is accumulated in the stressed intermediate.

• RNAP-promoter interaction are ~7 to 9 kcal/mol• RNAP-initiation-factor interaction ~13 kcal/mol

(s70)

The energy accumulated in thatobligatory stressed intermediate drives the transition from initiation to elongation.

PAF – elongation complex

Gal11 module

• 3D EM reconstruction shows two conformations.

• Tail region doesn’t interact with Pol2

• Gal11 (Med[2,3,15,16]) module might function as a separate entity

• Associates with Gcn4 and promotes transcription of ARG1, SNZ1 genes

Gal11 (cont.)

• Option 1: interacts with SAGA or SWI/SNF complexes, which is enough for initiation complex

• Option 2: direct stimulatory effect on transcription machinery