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                     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, Rimma
Belotserkovskaya 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                  Swi/Snf          Elongator        TFIIF




                                                                  Set1



Spt2          ISWII
                                TFIIS                             Set2

                      DSIF                        Paf




       Iws1              Spt6             FACT               P-TEFb
                   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 and
         its 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
• Gal4 interacts directly with subunits
  Med15, Med17.
• ChIP showed association to be at an
  upstream activation sequence.




Transcription   Transcription   Transcription   Transcription
             Separate recruitment
• 3 waves of TF recruitment:
  0            4-7   6-10       8-13

 Galactose      SAGA Mediator    Pol II



• Separate recruitment has also been showed for
  other promoters.
• Demonstrated in higher eukaryotes
• Mediator forms a scaffold for several rounds of
  transcription
   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


       Strains expressing GBD-Med2,
       Med3, Med4, Med13, Med15
       showed strong expression of b-
• Each subunit was cloned as fusion protein with
                     excluded from this
       gal and weredomain (GBD) or Gal4
  Gal4 DNA binding
       analysis.
  Activation domain (GAD).
• Transformed into a GAL promoter-reporter
  genes strains.
• All possible matings were preformed.
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 Productive
 Initiation by RNA Polymerase
    Involve DNA Scrunching
Andrey 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 tothat (N – 2),
    The energy accumulated in 9 bp
• Assuming an stressed cost of ~2 kcal/mol per
    obligatory   energetic intermediate drives
  bp, a total of ~14 to 18 kcal/mol is accumulated
     the transition from initiation to
  in the stressed intermediate.
     elongation.
• RNAP-promoter interaction are ~7 to 9 kcal/mol
• RNAP-initiation-factor interaction ~13 kcal/mol
  (s70)
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

				
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posted:9/25/2012
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