cells are spatially ordered assemblies of molecular mainly

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Reconfiguring the connectivity of transcription
proteins E. Peter Geiduschek

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• Cells are spatially ordered assemblies of
  molecular (mainly protein) machines.
• Molecular machines are assemblies of multiple
  protein components/subunits.
• Many of these subunits are themselves evolved
  assemblies of multiple domains with separate
  functions joined together on a single polypeptide
  chain.
• The evolutionary record of the assembly process
  can be discerned by examining conservation of
  amino acid sequences and structural
  domains/motifs of multi-domain proteins.
• Thus, the functional components of protein
  machines are held together in part covalently and
  in part by non-covalent protein-protein
  interactions.

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• One can wonder what the consequences are of
  switching covalently joined domains
• and
• replacing non-covalent with covalent linkages
  (and vice versa).
• Questions of this kind can be addressed globally
  (by generating a more-or-less random, and
  more-or-less global reassortment, followed by
  selection)
• or
• they can focus on a specific protein or protein
  complex in order to address specific questions
  about mechanism.
• The focus of my laboratory is on mechanisms of
  transcriptional regulation
• I shall present experiments in which the
  connectivity of transcription factors has been
  rearranged, generating new properties.

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TBP
240
Zn
2
3
282
596
439
Zn
1
2
3
Brf1
SANT domain
240
487
312
355
269
416
472
594
Bdp1
253
325
372
Interaction domains (protein footprinting)
II
I
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Yeast TFIIIB and Brf1
Zn
2
3
282
596
439
Does not bind stably to the TBP-DNA complex Forms
an unstable TFIIIB-DNA complex Active in
transcription
Binds stably to the TBP-DNA complex Forms a
stable TFIIIB-DNA complex Inactive in
transcription
Yeast TFIIIB and Bdp1
352
Forms an unstable TFIIIB-DNA complex Active in
transcription
352
Forms an unstable TFIIIB-DNA complex Active in
transcription
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Z. Sean Juo et al. (2003)
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A TBP-Brf1 Fusion Protein
Brf1-439
TBP-240
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A TBP-Brf1 Triple Fusion Protein
Brf1-439
TBP-61
Brf1-273
Brf1n
Brf1c
TBPc
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The triple fusion substitutes for Brf1 in vivo
Chr-BRF
pGal-BRF
pGal-TF
pGal-Split
Strain MATa, leu2D, his3D, lys2D,ura3D,
brf1DKanMx pGal-BRF - pRS315GUBRF1 pGal-TF -
pRS315GUbrf1N-tbpC-brf1C pGal-Split -
pRS315GUbrf1N-tbpC pRS313GUbrf1C
Western analysis with antibodies specific to
Brf1n or Brf1c The Brf1n-TBPc-Brf1c triple
fusion functions as an intact protein, in vivo
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• The Brf1n-TBPc-Brf1c triple fusion creates a TBP
  paralogue that is privatized for Pol III
  transcription
• Its modular design makes it a useful platform
  for further mutational analyses
• Unlike Brf1, it is easy to purify the
  recombinant protein to homogeneity

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The Bdp1-binding site in the C-terminal half of
Brf1
All but 5 Brf1c side chains not in contact with
TBP were substituted
Starting with the fusion protein TBPcBrf1c,
mutagenize the Brf1 segment, purify the mutant
proteins
Screen for defects of Bdp1 recruitment to the
promoter by site-specific protein-DNA
photochemical cross-linking
C-terminal truncations were used to analyze
residues not resolved in the structure
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Brf1 mutations that diminish Bdp1 assembly
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Part II
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The T4 Phage Genome
Genome
168903 bp
300 probable protein-encoding genes (40
uncharacterized)
8 tRNAs
2 other RNAs
Transcription
Early, middle and late promoters
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50 T4 Late Promoters Simple Promoters with
Complex Rules
Late Promoter Consensus TATAAATA Required for
Expression in vivo gp55 (T4-encoded s-family
promoter recognition) DNA replication gp33 (a
T4-encoded RNA polymerase-binding protein) In
vitro, transcription of negatively supercoiled
DNA requires only E.gp55 for promoter
recognition. (E is the E. coli RNA polymerase
core.)
(-10)
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Three viral proteins are the principal
players in this regulation. One replication
protein - gp45, the DNA polymerase's "sliding
clamp" - is the transcriptional activator Two
proteins convert the E. coli RNA polymerase to T4
late gene-specific transcription gp55
recognizes the (50) late T4 promoters gp33
makes late transcription dependent on the
activator (i.e. it's an "enforcer" of
activation)
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• Sliding clamps are universal components of
  cellular DNA replication machineries.
• molecular rings that slide freely along DNA
• and can confine other proteins to the DNA thread
  by binding to them including DNA polymerases,
  for which they serve as processivity factors
  (speeding up DNA polymerization).
• Getting sliding clamps onto DNA requires the help
  of clamp-loading factors (and ATP).
• The clamp-loaders do their work at specific DNA
  sites places at which one strand is
  interrupted/broken (i.e. at sites for DNA
  replication, repair, recombination).

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Requirements for in vitro T4 late
transcription Basal
Activated Transcription
Transcription Linear or supercoiled DNA DNA
with a sliding-clamp loading site E. coli RNA
polymerase core E. coli RNA polymerase
core gp55 (T4 late s factor) gp55
?????????? gp33 (co-activator of
transcription) gp45 (sliding clamp
activator of transcription)
gp44/62 (gp45 clamp loader) ATP/dATP
(for clamp-loading)
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• What does activation of late genes by the gp45
  sliding clamp achieve?
• a several-hundred-fold (gt300 x) increase in
  the rate
• constant for initiation of transcription,
  contributed
• by a combination of two effects
• attachment to the sliding clamp tightens
• binding of polymerase to its promoters
• and accelerates promoter opening
• AND
• relief of repression generated by gp33, the
  "enforcer, in the absence of gp45
• ALTOGETHER
• a greater than thousand-fold effect!
• generated by a combination of separate
  relatively

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Hanging onto the sliding clamp by their C-ends
gp55 - T4 late sigma factor S L D F
L Y E A N D gp33 - T4 co-activator of
transcription T L D F L L gp43 - T4 DNA
polymerase S L D F L F G
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Sliding Clamp
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adapted from Murakami et al., Science 296 1280
(2002)
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• The Task- to make a sliding clamp activator of
  eukaryotic transcription.
• The Design- a bacterial sliding clamp fused to
  a eukaryotic RNA polymerase II activating domain.
• The Components
• - E. coli sliding clamp, ß, a dimeric ring with
  pseudo-six-fold symmetry, fused to the
  transcriptional activation domain of VP16, an
  HSV1 (herpesvirus) protein.
• - in vitro systems yeast pol II holoenzyme
  (retaining mediator) and initiation factors
  (purified) Drosophila embryo extract (crude).

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Transcription templates
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Activation of yeast RNA polymerase II depends on
the E. coli sliding clamp-loading factor
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Comparison of the activity of topologically and
physically bound VP16 activators
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The topologically bound activator slides along
DNA from its loading site to the promoter (as the
phage T4 sliding clamp does)
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ACKNOWLEDGEMENTS
Sliding Clamps (T4 late genes) George
Kassavetis Kelly Williams Dan Herendeen Rachel
Tinker-Kulberg Tsu-Ju Fu Jean-Paul
Léonetti Scott Kolesky Mohamed Ouhammouch Kevin
Wong Masood Kamali Sergei Nechaev Vikas Jain
RNA Polymerase III George Kassavetis Elisabetta
Soragni Robert Driscoll David Steiner