Promoters and Enhancers

1
Promoters and Enhancers

• Chapter 24

2
24.1 Introduction

• Significant difference between transcription of
  eukaryotic and prokaryotic mRNA
• Initiation at eukaryotic promoter involves many
  factors that bind to a variety of cis-acting
  elements
• Eukaryotic RNA polymerase bind around the
  startpoint, but NOT directly contact the extended
  upstream region of the promoter
• Eukaryotic Promoter the region containing all
  these binding sites
• Major feature defining promoter for eukaryotic
  mRNA location of binding sites for transcription
  factors
• Bacterial promoter binding site for RNA
  polymerase in the immediate vicinity of startpoint

3
24.1 Introduction

• Transcription factors are needed for initiation,
  but not required subsequently
• In eukaryotes, the transcription factors are
  principally responsible for recognizing promoter
• Bacterial RNA polymerase recognize promoter
• RNA Polymerase II require a large group of
  transcription factors basal factors
• RNA Pol I and III relatively simple
• Basal transcription apparatus basal factors
  RNA polymerase

4
24.1 Introduction

• The sequences farther upstream of promoter
  determine whether the promoter is expressed in
  all cell types or specifically regulated
• Constitutively expressed promoter (for
  housekeeping genes) have upstream sequence
  elements recognized by ubiquitous activators
• Beta-actin, glucose-6-phosphate dehydrogenase
• Promoters expressed only in certain times/places
  have sequence elements that require activators
  available only at those times/places

5
Enhancers

• Another type of site involved in initiation
• Sequences that stimulate initiation, but located
  a considerable distance from startpoint
• Often targets for tissue-specific or temporal
  regulation
• Components of enhancer resemble those of promoter
• Consist of a variety of modular elements
• Proteins bound at enhancer interact with proteins
  bound at promoter
• Eukaryotic transcription usually under positive
  regulation
• Less by repression regulation

6
24.1 Introduction
Figure 24.1
7
24.2 Eukaryotic RNA Polymerases Consist of Many
Subunits

• RNA polymerase I synthesizes rRNA in nucleolus.
• RNA polymerase II synthesizes mRNA in
  nucleoplasm.
• RNA polymerase III synthesizes small RNAs in the
  nucleoplasm.
• All eukaryotic RNA polymerases have 12 subunits
  and are aggregates of gt500 kD.
• Largest subunit in RNA Pol II has
  carboxy-terminal domain (CTD), which consists of
  multiple repeats of a consensus sequence of 7 AA
  (YSPTSPS)
• Some subunits are common to all three RNA
  polymerases.
• RNA polymerase in mitochondria and chloroplasts
• Smaller
• Resemble bacterial RNA polymerase

8
Figure 24.2
9
(No Transcript)
10
24.3 Promoter Elements Are Defined by Mutations
and Footprinting

• Promoters are defined by their ability to cause
  transcription of an attached sequence in an
  appropriate test system in vitro or in vivo.

Figure 24.3
11
24.4 RNA Polymerase I Has a Bipartite Promoter

• The RNA polymerase I promoter consists of
• a core promoter -45 to 20
• an upstream control element (UPE) -180 to -107

Figure 24.5
12
24.4 RNA Polymerase I Has a Bipartite Promoter

• Requires 2 ancillary factors
• The factor UBF1 wraps DNA around a protein
  structure to bring the core and UPE into
  proximity.
• UBF upstream binding factor
• SL1 (core-binding factor) includes the factor TBP
  that is involved in initiation by all three RNA
  polymerases.
• TBP TATA-binding protein
• RNA polymerase binds to the UBF1-SL1 complex at
  the core promoter.

13
24.5 RNA Polymerase III Uses Both Downstream and
Upstream Promoters

• RNA polymerase III has two types of promoters.

Figure 24.7
14
24.5 RNA Polymerase III Uses Both Downstream and
Upstream Promoters

• Internal promoters
• have short consensus sequences located within the
  transcription unit
• cause initiation to occur a fixed distance
  upstream
• Upstream promoters contain three short consensus
  sequences upstream of the startpoint that are
  bound by transcription factors.

Figure 24.6
15
24.6 TFIIIB Is the Commitment Factor for Pol III
Promoters

• TFIIIA and TFIIIC bind to the consensus sequences
  and enable TFIIIB to bind at the startpoint.
• TFIIIA and TFIIIC assembly factors whose only
  role is to assist binding of TFIIIB at right
  location
• TFIIIB has TBP as one subunit and enables RNA
  polymerase to bind.

Figure 24.9
16
Figure 24.08 Type 2 internal promoters use
TFIIIC.
17
(No Transcript)
18
24.7 The Startpoint for RNA Polymerase II

• RNA polymerase II requires general transcription
  factors (called TFIIX) to initiate transcription.
• RNA pol II promoters have a short conserved
  sequence Py2CAPy5 (the initiator, Inr) at
  startpoint.
• The TATA box is a common component of RNA
  polymerase II promoters
• It consists of an A-T-rich octamer located 25 bp
  upstream of the startpoint.
• The DPE a common component of RNA pol II
  promoters that do not contain a TATA box.
• down-stream promoter element, 28 - 32
• A core promoter for RNA polymerase II includes
  TATA box Inr, or Inr DPE

19
Figure 24.10
20
24.8 TBP Is a Universal Factor

• TATA-binding protein (TBP) is a component of the
  positioning factor that is required for each type
  of RNA polymerase to bind its promoter.
• The factor for RNA polymerase II is TFIID, which
  consists of
• TBP
• 11 TAFs (TBP-associated factors)
• The total mass is 800 kD.
• Positioning factors containing TBF and TAFs
  responsible for identifying all classes of
  promoters

Figure 24.11
21
24.9 TBP Binds DNA in an Unusual Way

• TBP binds to the TATA box in the minor groove of
  DNA.
• It forms a saddle around the DNA and bends it by
  80.
• Some of the TAFs resemble histones and may form a
  structure resembling a histone octamer.

22
24.10 The Basal Apparatus Assembles at the
Promoter

• Binding of TFIID to the TATA box is the first
  step in initiation.
• Other transcription factors bind to the complex
  in a defined order
• This extends the length of the protected region
  on DNA.
• When RNA polymerase II binds to the complex, it
  initiates transcription
• TBP binds to the TATA box in the minor groove of
  DNA.

Figure 24.14
23
Figure 24.16 TFIIB helps position RNA polymerase
II.
24
24.11 Initiation Is Followed by Promoter
Clearance

• TFIIE and TFIIH are required to melt DNA to allow
  polymerase movement.
• Phosphorylation of the CTD may be required for
  elongation to begin.

Figure 24.17
25
Roles of CTD

• CTD may be a general focus for connecting other
  processes with transcription
• Directly or indirectly involved in processing RNA
  after it synthesized by RNA polymerase
• Bind to capping enzyme
• Bind to SCAFs, then binding to splicing factors
• Bind to components of cleavage/polyadenylation
  apparatus

Figure 24.18
26
(No Transcript)
27
24.13 Short Sequence Elements Bind Activators

• Short conserved sequence elements are dispersed
  in the region preceding the startpoint.
• The upstream elements increase the frequency of
  initiation.
• Control the efficiency and specificity with which
  a promoter is recognized
• TATA box -30
• CAAT box -75
• GC box -90

28
Figure 24.21 The ß-globin promoter has three
short sequence elements. Correspond to TATA,
CAAT, and GC boxes
29
24.14 Promoter Construction Is Flexible but
Context Can Be Important

• Promoters organized of mix and match principle
• A variety of elements can contribute to promoter
  function
• No individual upstream element is essential for
  promoter function
• Although one or more elements must be present for
  efficient initiation.
• Some elements are recognized by multiple factors.

Figure 24.22
30
24.15 Enhancers Contain Bidirectional Elements
That Assist Initiation

• An enhancer activates the nearest promoter to it.
• It can be any distance either upstream or
  downstream of the promoter.

Figure 24.23
31
24.15 Enhancers Contain Bidirectional Elements
That Assist Initiation

• A UAS (upstream activator sequence) in yeast
  behaves like an enhancer but works only upstream
  of the promoter.
• Similar sequence elements are found in enhancers
  and promoters.
• Enhancers form complexes of activators that
  interact directly or indirectly with the promoter.

32
24.16 Enhancers Contain the Same Elements That
Are Found at Promoters

• Enhancers are made of the same short sequence
  elements that are found in promoters.
• The density of sequence components is greater in
  the enhancer than in the promoter.

Figure 24.24
33
24.17 Enhancers Work by Increasing concentration
of Activators Near Promoter

• Enhancers usually work only in cis configuration
  with a target promoter.
• Enhancers can be made to work in trans
  configuration by
• linking the DNA that contains the target promoter
  to the DNA that contains the enhancer via a
  protein bridge or
• catenating the two molecules