Lecture 5

Regulatory sequences in protein-coding genes.

Transcriptional activators and repressors.

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9.4 Regulatory Sequences in Protein-Coding
Genes and the Proteins Through Which They
Function
• Expression of eukaryotic genes is regulated through multiple
protein-binding transcription-control regions located at various
distances from the transcription start site.
• These control regions can be close to the transcription initiation
site (called promoter-proximal elements) or distal sites (called
enhancers)
• Promoter-proximal elements and enhancers are gene and cell-
type-specific regulatory elements on DNA.
• Transcription activators and repressors are modular proteins
containing a DNA-binding domain and one or more activation or
repression domains
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How do we find gene regulatory
sequences?
Linker scanning-reporter gene technique for
identifying gene regulatory elements on DNA

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Linker scanning mutagenesis and reporter genes
(You will study this technique in Tutorial 3)
• You buy a plasmid that contains a reporter gene, usually a gene that
encodes a enzyme (TK in figure 9-22)
• The TK ORF (Open Reading Frame) on the plasmid has no promoter.
• Upstream of the TK ORF you clone a large piece of DNA that you
know is controlling YOUR gene (not TK) of interest
• You systematically replace small pieces (10 bp) of the cloned
regulatory DNA by a “linker”. You produce multiple plasmids with
linker substitutions (9 in figure 9-22)
• You transform each individual plasmid in a cell culture and measure
the activity of the reporter (a simple enzyme reaction of cell extract)
• Loss of reporter activity means that you have hit a REGULATORY
SEQUENCE

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Reporter plasmids contain:
• an ORF for an enzyme (Luciferase (luc) in this case)
• NO promoter-proximal regulatory sequences
• A multiple cloning site (MCS) for the cloning a regulatory sequence
• Translation initiation elements upstream of the reporter gene
• Poly A+ signal downstream of the reporter gene

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Promoter-Proximal Elements Help Regulate Eukaryotic
Genes

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• Experiments like the one on the preceding slides have
generated detailed maps of the REGULATORY DNA
SEQUENCES of many genes
• Mammalian (and other complex eukaryotes) TATA/INT
containing promoters have multiple regulatory
elements that can be promoter-proximal or distal and
can be positioned upstream or downstream the
transcription initiation site.
• CpG island promoter do not have strong regulatory
elements and are expressed at a lower level
• Simple eukaryotes (like S. cerevisiae) have simple
regulatory elements

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Fig. 9-23: All boxes represent regulatory sequences

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Fig. 9-23: Eukaryotic genes contain multiple
promoter-proximal regulatory elements (close to
the transcription initiations site)

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Fig. 9-23: Enhancers are distal regulatory elements,
could be upstream or downstream of the initiation site

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 Properties of promoters and enhancers

Promoters Enhancers
Function within a short Can function over a long
distance; ~several hundred bps distance; maybe over tens of
from transcription initiation site kbps from transcription
initiation site
Immediately upstream from the Can be upstream,
initiation site (for RNA Pol II) downstream from the start or
within introns

Position dependent: usually Position independent: usually

non-functional if moved still functional when moved

Orientation dependent: drive Orientation independent:

transcription in one direction function in either normal or
only the inverted orientation
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Enhancers communicate with promotors via
bending DNA
We will revisit this figure in Lecture 6 to introduce the roles of Mediator,
Histone modifiers and Chromatin remodeling during gene activation
Here I discuss only the role of transcription activators via enhancers

Karp figure 12.48 12

Activatiors communicate with the
Mediator, Histone modifiers and
Chromatin Remodelers during gene
activation

BIG PICTURE !!
Bending of DNA and the
action of distal enhancers

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What kind of proteins bind to the
gene regulatory sequences?
Transcriptional activators and repressors

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 Transcriptional activators and repressors
are proteins with distinct domains
• A domain is a part of a protein that will acquire distinct 3D
structure and distinct activity within the whole protein
• Transcriptional activators or repressors often have
independent DNA-binding domains and effector (activation
or repression) domains
• These can be identified by deletion analysis of the protein
• Fig. 9-27 presents the deletion analysis of GAL4 – one of the
best characterised transcriptional activators from the model
organism S. cerevisiae (budding yeast)
• Read the figure legend and my notes in it.
• My notes are shown in Italic BOLD

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Read the figure
legend for
details. You need
to know them.

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• Assays like the one on the preceding slide have identified
multiple DNA-binding and Activation or repressor domains
• The majority of DNA binding domains and Activation domains
have conserved motifs
• Motif is a sequence in a protein that is likely to produce a
distinct 3D structure and a distinct activity in vivo.
• These motifs are easy to recognize by bioinformatic tools.

• In the next few slides we will study some highly conserved

DNA binding or Activation motifs and domains
• You need to know the name and recognize the function of the
motifs, not to memorize the structures

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 MOTIF: a frequently found sequence in proteins with
a similar function

Zn++ finger motif:

xxxCxxxxCxxxxHxxxxxHxx….
……xxxCxxxxHxxxxCxxxxxHxx

OR

xxxCxxxxCxxxxCxxxxxCxx….
……xxxCxxxxCxxxxCxxxxxCxx
Leu zipper motif :
xxxLxxxxxxLxxxxxxxLxxxxxxxLxx….
Helix-loop-helix
motif
 Zn-finger motif forms a domain
for binding to DNA

Leucine zipper motif forms a domain

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for dimerizing with another protein and then to DNA

Combinatorial binding to DNA of different
dimer combinations of proteins:
• Very often DNA-binding proteins of the same class (for
example Helix-Turn-Helix) work as dimers.
• Each monomer can recognize a specific DNA element, but
cannot work on its own. It will bind to this DNA only in a
complex with a similar protein.
• This means that if two proteins can combine, they will have
three binding sites, three proteins can have six binding
sites, etc…
• Examples are given on next four slides

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Protein dimerization
increases the complexity of
DNA-binding specificity

Binds to two Binds to two Binds to a combination

“green” DNA “orange” DNA of “orange” and “green”
elements elements DNA elements

3 different transcription factors from the same family (in this example 3 HLH
factors can be formed from two bHLH monomers)
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• If you have three monomers
that can form dimers, you can
have six combinations of these
proteins: AA, BB, CC, AB, AC, BC
• These six combinations can
recognize six DNA binding sites
• Adding an inhibitor for one of
the three proteins further alter
the possible combinations

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Combinatorial binding to DNA-binding proteins
from different classes: Enhancosome
• Eukaryotes have very complex promoters and enhancers.
• In these, various transcription activators and repressors can
bind
• The transcriptional activators or repressors often interact
with each other and cooperate to establish a strong
activator or repressor site to tightly control the expression
of the target genes.

• YOU DO NOT NEED TO REMEMBER THE NAMES OF THE

TRANSCRIPTIONAL ACTIVATORS SHIOWN ON THE NEXT
THREE SLIDES.

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BIG PICTURE !!!
Combinatorial binding of
various trans-activators at
enhancer elements

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