Summary of Transcriptional Regulation

1
Some important definitions
coding or sense strand
Up-stream (minus)
Down-stream (plus)
1
template or anti-sense strand
- the coding strand of a gene has the same
sequence as the RNA produced BUT it is the other
(template) strand that RNA polymerase reads - RNA
polymerases (unlike DNA polymerases) do NOT
require a primer and they cannot proof-read so
errors are likely - The first nucleotide has a 5
triphosphate Usually RNAs start with a 5ppp-A or
5ppp-G
2
Two lines of evidence define key elements in
prokaryotic promoters
I. Consensus elements from sequencing data
mRNA
nucleotide nomenclature 1 first
transcribed nucleotide
1
2
3
-1
5-8
15-20
Figure 26.11 (Biochemistry)
3
DNase Footprinting
End labelled
A method to detect where a protein binds to DNA.
Used extensively for mapping contact points
between promoter sequences and RNA polymerase
and/or regulatory proteins
4
Why have gene regulation? 
In multicellular eukaryotic organisms        
all genes are passed on to subsequent
generations         only the products of some
genes are needed or wanted at any given
time         only the products of some genes are
needed in particular cell types   e.g.
hemoglobin in red blood cells  
keratin in skin and hair follicle cells  
muscle actin and myosin in muscle cells  
glycogen metabolizing enzymes in
liver cells  
5
This pattern of different products in different
cells/tissues is generated by differential gene
expression i.e. different cell types must express
different genes in order to carry out particular
tasks.  In turn, this is accomplished by the
selective activation of the desired genes and the
repression of the other genes.
6

• Single celled organisms, including prokaryotes-
  bacteria e.g. E. coli, must also regulate genes
•  cell must carry out all tasks required for
  survival and therefore, it must be able to adapt
  to changes (often rapid) in the environment
•  it does this by turning genes on or off in
  response to environmental changes
•  e.g. bacteria can synthesize enzymes that allow
  them to break down (catabolize) a variety of
  sugars.
•  

7
Most of these sugars will only be present in the
environment from time to time and the cell
requires only the enzymes capable of digesting
the available sugars the production of other
unwanted enzymes would be a major waste of
energy. It would even be more wasteful for the
cell to synthesize building blocks it requires
e.g. amino acids, if it can obtain these from the
environment.
8
To solve this problem, single celled organisms
have evolved two mechanisms  1)  they are able
to recognize or sense when a particular enzyme
is needed (or not needed).  2)  they are able to
turn the gene for that enzyme on or off in
response to need- they use elegant genetic
switches or control mechanisms to accomplish this.
9
Summary of Transcriptional Regulation in
Prokaryotes
- transcription is the primary control point for
gene expression ? In all organisms but
particularly in bacteria - control is most often
effected by modulation of promoter
activity Promoters can be turned on and off by
binding regulatory proteins at sites near the
promoter on the DNA near in prokaryotes 100
200 bp in eukaryotes up to 50,000 bp
typically lt1000 bp
Positive Regulation Binding of regulatory
proteins increases transcription Negative
Regulation Binding of regulatory proteins
reduces transcription
10
Operons

• - most commonly found in prokaryotes, with a few
  examples now known in eukaryotes.
• - in bacteria, when one promoter serves a series
  of clustered genes, the gene cluster is called an
  operon
• - all of these genes are transcribed into a
  single mRNA
• - each section of these mRNAs (called
  polycistronic mRNA) may then be translated
  independently
• - the genes in a given operon often encode for
  several enzymes active in a single metabolic
  pathway

gene a
b
c
promoter
11
The occurrence of genes in operons allows the
expression of the genes to be controlled
coordinately-transcription produces a
polycistronic mRNA. - a very economical situation
(although translation rates of individual genes
(cistrons) in the operon may vary) For example
On
Off
Figure 26.2 of Mathews
12
Induction Repression of Transcription
The expression (RNA production) of operons can be
induced or repressed by specific effector
molecules (e.g., chemicals, nutrients, etc.)- an
extremely important genetic switch in bacteria ?
not all promoters in a cell are constantly
accessible to RNA polymerase ? which promoters
are accessible depends on what specific molecules
are present in the medium (and in the cell) ?
specific enzymes required for metabolism of
particular molecules can thus be induced or
repressed on demand
13
Common Examples of Operons enzymes that convert
various sugars to glucose (a readily-metabolized
energy source) or are able to use other sugars
for energy
Lac Operon Lactose Gal Operon Galactose Ara
Operon Arabinose
Induction Repression
lactose removed
Lac Operon
RNA
Concentration (normalized)
Enzyme
lactose added
Time
0
14
Regulation of operons to optimize the use of
compounds to metabolize for energy etc. are not
the only examples. Operons coding for proteins
involved in many biosynthetic pathways are also
under stringent control.
e.g. Tryptophan (Trp) Operon synthesis of
tryptophan
Trp suddenly available
No Trp
- when tryptophan becomes available to the cell
it shuts off all the machinery used to
synthesize the amino acid - why waste energy
making something that is freely available from
the environment?
Concentration (normalized)
Time
15
Proteins Involved in Gene Control
I. Repressors Negatively acting regulatory
proteins, that bind to DNA AT or NEAR a promoter
site this binding prevents RNA polymerase from
associating with the promoter The repressor
binding site on DNA is called the Operator -A
specific DNA sequence recognized by the repressor
GENE
16
Repressor proteins can combine with Effectors
(Small molecules), that greatly affect their
ability to bind their operator DNA. -look back
at allosteric proteins in your notes
2 Types of Effectors 1) Inducers Decrease
repressors binding affinity to operator
DNA (e.g., lactose binding to lac
repressor) 2) Co-Repressors Increase affinity -
repressor not active when co-repressor
absent (e.g., tryptophan for trp repressor)

DNA


17

• II. Activators
• Positively acting regulatory proteins, that bind
  TO or NEAR a promoter site. This binding helps
  increase the frequency with which RNA polymerase
  binds to the promoter or to help it to initiate
  transcrption
• -best known example is CAP (catabolite
  activator protein) or CRP (cyclic AMP receptor
  protein)
• Two Types of activator protein
• Activator proteins that need an effector molecule
• Do not need an effector, but high levels of
  activator protein are needed for it to be
  effective

18
Structure of the CRP-cAMP complex bound to DNA.
This protein is involved in the lac operon as we
will see along with several others in E. coli
DNA
CRP
cAMP
19
Summary of Bacterial Gene Regulation

• A) Different genes/operons may be expressed at
  different levels according to how well their
  promoter matches an optimal promoter sequence
  (consensus sequence)
• B) The expression of genes and operons may also
  be modulated through various signals the main
  means of induction and repression of operons and
  individual genes include the use of
• Repressors - which inhibit transcription by
  binding to an operator DNA sequence near the
  promoter
• - which can be modulated by
• Effectors
• Co-repressors
• Activators - which enhance transcription by
  binding a DNA sequence near the promoter sequence
• - these may or may not be modulated by an effector

20
(No Transcript)
21
Regulation of the Lac Operon paradigm of
bacterial gene regulation
Figure 26.17 of Mathews

• Three structural genes in the operon z, y and a
• - these code for enzymes involved in lactose
  metabolism
• - these genes are expressed a very low levels all
  of the time
• - induced x1000 when lactose is present
• - also influenced by the level of glucose in the
  environment
• An adjacent gene (but not part of the lac
  operon), is lac i which codes the protein lac
  repressor or lac I protein.

22

• The lac repressor binds to its operator, which
  overlaps with the lac promoter
• ? Lactose binds to the repressor
• This results in a conformational change in the
  repressor, which disrupts in DNA binding domain
• Repressor no longer can bind its operator
  sequence

lactose
Lac operator DNA sequence
Active Repressor
23
- the lac operator is 35 bp long - it is
downstream overlapping with the promoter - it
has a two-fold sequence symmetry (imperfect)
i.e., a palindromic sequence 22 bp out of 35 are
protected from nucleases during footprinting
experiments - the affinity of the repressor for
the operator sequence is 4x106 higher than for
other DNA sequences
Operator Sequence (spans region shown)
Figure 26.19 (Biochemistry)
24
Structure of the Lac Repressor Bound to DNA
The crystal structure of the lac repressor of
Escherichia coli (LacR) at 2.6 A resolution. The
quaternary structure consists of two
dyad-symmetric dimers that are nearly parallel to
each other. This structure places all four DNA
binding domains of LacR on the same side of the
tetramer, and results in a deep, V-shaped cleft
between the two dimers. Each monomer contributes
a carboxyl-terminal helix to an antiparallel
four-helix bundle that functions as a
tetramerization domain.
- structure is consistent with it binding a
palindromic sequence (as with restriction enzymes)
25
Figure 26.18 of Mathews
26
The lac operon is also subject to POSITIVE
regulation, by CRP in the presence of cAMP

• The lac operon is induced by the presence of
  lactose in the medium, but E. coli prefers to use
  glucose (better energy source)
• lac operon is also regulated by glucose levels
• glucose high ? Low transcription of lac
  operon
• glucose low ? High transcription of lac
  operon
• This correlates to the activity of CRP which
  activates lac operon in the presence of cAMP
  (cyclic AMP)
• glucose low ? cAMP high
• glucose high ? cAMP low

27
Figure 26.21
28
- the lac operator is 35 bp long - it is
downstream overlapping with the promoter - it
has a two-fold sequence symmetry (imperfect)
i.e., a palindromic sequence 22 bp out of 35 are
protected from nucleases during footprinting
experiments - the affinity of the repressor for
the operator sequence is 4x106 higher than for
other DNA sequences
Operator Sequence (spans region shown)
Figure 26.19 (Biochemistry)
29
Levels of Control of Lac Operon Expression 3
Scenarios

• No Lactose around
• Operon switched off, essentially no mRNA
  regardless of glucose
• Lactose present glucose also present
• The presence of lactose inactivates the repressor
  
• ? low level of transcription occurs
• Glucose present ? cAMP is low ? CRP does not
  help transcription and thus it remains at low
  level
• Lactose present no glucose
• The presence of lactose inactivates the repressor
• ? Transcription occurs
• NO Glucose ? cAMP is high ? cAMP binds CRP
  (becomes activated) ? CRP binds Helps
  Transcription
• High Level of transcription