Biol 568 Advanced Topics in Molecular Genetics

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Biol 568Advanced Topics in Molecular Genetics
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Initiation of Transcription -Euk.

• General Overview
• Eukaryotic RNA Polymerases
• Transcription in Eukaryotes
• RNA pol II promoters
• Promoters / Enhancers

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Overview

• Three eukaryotic RNA Polymerases
• RNA Pol I transcribes rRNA
• RNA Pol II transcribes mRNA
• RNA Pol III transcribes tRNA other

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Overview

• RNA Pol II cant initiate Txn alone
• need accessory factors
• General Factors
• Upstream Factors
• Inducible Factors

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General Factors

• Required at all promoters
• Aid in determining txn start point
• Basal transcription factors

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Upstream Factors

• Bind with specific short consensus sequences
  upstream of start site
• Activity not regulated
• Ubiquitous
• Required for promoters to function at adequate
  levels

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Inducible Factors

• Similar to Upstream Factors
• Regulatory role
• Synthesized or activated as needed

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Fig 21.1 Overview of control regions for a
eukaryotic gene
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Transcription Control Regions

• Promoter
• required for initiation
• generally located immediately upstream of start
  site
• Enhancer
• sequences that stimulate initiation
• may be located upstream or downstream of gene
• may be in either orientation

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RNA Pol II

• Transcribes genes for mRNAs encoding proteins
• Highly sensitive to a-amanitin
• RNA Pol I not inhibited, RNA Pol III variable
  inhibition (depending on source)
• Large protein - 500kDal - 8-14 subunits

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RNA Pol II

• Purified enzyme can transcribe
• Can not initiate selectively at promoters

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Fig 21.2 Yeast RNA Pol II subunits
CTD - carboxy terminal domain 7 aa repeat
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Promoter Analysis

• Defined by ability to initiate transcription in
  test system
• Define the overall length
• Define the cis-acting element
• Once promoters are defined, the interacting
  proteins can be identified

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Promoter Analysis

• Three test systems
• Oocyte system
• Transfection systems
• Transgenic systems

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Oocyte system

• Injection of DNA template into nucleus of Xenopus
  laevis oocyte
• RNA recovered and analyzed
• Limited to conditions in X.laevis oocyte
• Can be used for characterization of promoter
  sequences
• The specific interacting proteins may not be
  identified

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Transfection Systems

• Exogenous DNA is introduced into cultured cells
  and expressed
• Conditions similar to in vivo
• Can be done with more than one gene
• Cotransfection

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Transgenic Systems

• Addition of gene to germline of animal
• Transgene expression can be followed in any
  tissue of animal
• Gene may be present in multiple copies
• Gene may have integrated at different chromosomal
  location

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Fig 21.3 Promoter Analysis (deletion)
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Definition of promoter boundaries
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Promoter Functional Analyses

• Deletion analyses
• Mutation analyses (bp changes)
• Protein footprinting
• Promoter sequence comparison
• Look for consensus sequence

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Initiation of Transcription -Euk.

• General Overview
• Eukaryotic RNA Polymerases
• Transcription in Eukaryotes
• RNA pol II promoters
• Promoters / Enhancers

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RNA Pol II promoter

• Initiator (Inr)
• First base generally an A
• Flanked by pyrimidines
• C/T Ys
• Y2CAY5 (-3 to 5)

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RNA Pol II promoter

• TATA box
• usually at -25
• found in all eukaryotes
• 8bp consensus sequence
• generally all A/T
• analogous to bacterial -10 sequence

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RNA Pol II promoter

• TATA box
• Mutations down mutations
• Surrounded by GC sequences, function not known
• TATA-less promoters
• DPE Downstream promoter element
• 28-32 bp downstream of the start point

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Fig. 21.10 RNA pol II promoter

• Initiatior region InR
• TATA InR
• InR DPE

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RNA Pol II basal apparatus

• RNA Pol II cant initiate txn itself
• Additional protein factors required
• Transcription factors
• For RNA Pol II - TFII X
• X identifies the individual factor

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RNA Pol II Initiation

• First factor to bind to promoter is TF II D
• binds at TATA box
• contains TATA binding protein subunit (TBP)
• other subunits are TAFs -TBP associated factors
• TAFs may differ, and thus confer specificity
• TAFII43, TAFII230, etc.

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Fig 21.11 Positioning RNA Pol II
TF II D protects -45 to -10 TBP protects - 37
to -25
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Fig 20.9 TBP bound to DNA
Binds to minor groove Forms saddle on helix Bends
DNA by 80
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Fig 21.13 TBP bound to DNA
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TBP binds to DNA

• Protected DNA region ( in vitro)
• -37 to -25, 1 turn
• TAFII230 competes for TBP DNA binding region

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• Add discussion about histone interactions?
• Some TFs have histone-like structures

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Fig 21.15 Additional TFIIs bind
TFIID TFIIA TFIIB TFIIF and RNA Pol
II TFIIE TFIIH J
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Fig 20.12 TF II B Interactions
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Fig 20.12 TF II B Interactions
TBP
TF II B subunits
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(No Transcript)
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TFIIB interacts with RNA pol II
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Fig 21.15 Additional TFIIs bind
TFIID TFIIA TFIIB TFIIF and RNA Pol
II TFIIE TFIIH J
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TFIIF

• Two subunits
• RAP74 helicase activity
• RAP38 similarities with s factors
• Binds tightly to RNA pol II
• TFIIF may bring RNA pol II to the complex

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RNA pol II holocomplex

• TF II D
• TBP binds and bends DNA
• TF II A
• Binds upstream of TBB
• TF II B
• Binds downstream and upstream of TATA
• TF II F and RNA Pol II
• RNA pol II interacts with TBP and TAFs via CTD
• RNA pol II interacts with TF IIB
• TF IIF melts DNA

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TATA-less promoters

• Inr, DPE binding elements for specific TAFs
• Same general transcription factors
• TFIID
• TBP
• Bending and binding maybe carried out by specific
  TAFs

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Comparison with txn in prokaryotes

• Bacterial RNA pol
• Intrinsic DNA binding activity
• s factors provide specificity
• First form complex with the core enzyme

• RNA pol II
• Cannot bind to DNA
• Txn factors provide specificity
• First bind to DNA then form complex with RNA pol
  II
• Independent s factors

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• TFIID
• TFIIA
• TFIIB
• TFIIF and RNA Pol II
• TFIIE
• TFIIH J

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Txn Promoter Clearance

• TFIIE
• Helicase activity
• TFIIH J
• Helicase activity
• Kinase activity

DNA Melting
Phosphorylation of CTD
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Fig 21.17 Release of RNA Pol II from promoter
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Release of RNA Pol II from promoter

• Most of TFs are released prior to RNA Pol II
  continuing elongation
• CTD tail is phosphorylated by TFII H and other
  factors
• (YSPTSPS)

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Phosphorylation of CTD

• CTD is involved in post-txn modifications
• Capping
• Guanylyl transferase binds to phosphorylated CTD
• Splicing
• SCAFs bind to CTD, may in turn bind to splicing
  proteins
• Phosphorylation of CTD may also be a mean to
  coordinate txn and processing

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Initiation of Transcription -Euk.

• General Overview
• Eukaryotic RNA Polymerases
• Transcription in Eukaryotes
• RNA pol II promoters
• Promoters / Enhancers

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Promoter Analysis

• Start site
• TATA box
• Short sequence elements upstream of TATA box
• Recognized by activators
• Provide txn efficiency and specificity

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Fig 21.21 Saturation mutagenesis of a b-globin
promoter
Most mutations do not affect function of promoter
-30 TATA Box
-75 CAAT Box
-90 GC Box
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Modularity of Eukaryotic Promoters

• Short sequence elements are functional
• Binding sites for TFs
• Mix and match of elements provides level of
  control needed

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Modularity of Eukaryotic Promoters

• Type of element
• Number of elements
• Position of elements
• Determine whether a gene is expressed and level
  of expression

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Fig 21.22 Promoters contain different
combinations of elements
Upstream factors
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Element - TF interactions

• GC box
• factor SP1 binds
• upstream factor
• position and number vary in promoters

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Element - TF interactions

• CAAT box
• upstream factor
• recognized by several TFs
• CP1, CP2 (CTF family)
• C/EBP
• ACF
• boxes in different promoters recognized by
  different factors

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Element - TF interactions

• Octamer element
• upstream element
• bound by two TF
• Oct-1
• ubiquitous TF (non-lymphoid cells)
• Oct-2
• only lymphoid cells (activates k-light gene)

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Element - TF interactions

• Context of elements must be important
• Oct 1 doesnt activate k-light genes in
  non-lymphoid cells
• Presence of specific elements does not mean that
  the specific TF activates txn
• May need to interact with other TFs
• Protein - protein interactions

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Role of Enhancers

• Boost level of transcription
• Act in conjunction with promoter
• Cis- acting elements
• Position relative to promoter is not fixed
• upstream or downstream
• Can work in either orientation

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Fig 20.19 Enhancer mutational analysis
SV40 virus enhancer
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Enhancers

• Modular nature, like promoters
• Higher density of TF binding sites
• Some elements found in both enhancers and
  promoters
• Increase Txn up to 200 fold!

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What is the function of the enhancer?

• Brings transcription factors close to the
  promoter
• DNA looping
• May initiate chromatin remodeling
• Insulators may inhibit an enhancer function

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Initiation of Transcription -Euk.

• General Overview
• Eukaryotic RNA Polymerases
• Transcription in Eukaryotes
• RNA pol II promoters
• Promoters / Enhancers