Transcription regulation in
prokaryotes
Background: major and minor grooves of DNA
Available interactions in minor
and major grooves
Schematic view of available chemical
groups in minor and major grooves
• In major groove all 4 nucleotides can be differentiated by a
binding protein
• In minor groove only AT or GC base pairs can be
differentiated
• The minor groove is also much narrower, which makes it
less accessible for binding proteins
• As a consequence, most sequence specific DNA binding
proteins bind to major groove
Helix-turn-helix DNA binding motif
•
Helix-turn-helix motif is the most common
DNA-binding motif in prokaryotes, present
in many transcription repressors and
activators
• One of the helices, DNA recognition helix,
gets inserted in the major groove of DNA
• Helix-turn-helix proteins are often dimeric,
with two recognition helices recognizing
two adjacent DNA sequences
• Why dimeric?
1) Dimer binds to DNA stronger than
monomer
2) By changing the relative positions of
monomers, the dimer activity can be easily
turned on and off
A common principle to activate or inactivate
dimeric helix-turn-helix proteins
(ligand)
• Ligand changes the position of DNA binding a
helices, so they do not bind DNA any more
• Or the opposite – ligand changes the position of
helices, so they do bind to DNA
The main features of interactions between DNA and
the helix-turn-helix motif in DNA-binding proteins
• 1. Sequence unspecific
interactions contribute to
overall stability of complex,
but do not differentiate the
ound DNA sequence
• 2. Sequence specific
interactions determine, with
which regions of DNA the
interaction will occur
Bacterial promoters
Transcription start
UP element
-35 element
+1
-10 element (Pribnow box)
pre –10 element
+1
• Most bacterial promoters have –35 and –10
elements
• Some have UP element
• Some lack –35 element, but have extended –10
region
The s factors
• s factors are required for promoter recognition
and transcription initiation in prokaryotes
• s factors have analogous function as general
transcription factors in eukaryotes
• A variety of s factors exist in E.coli
• For expression from most promoters s70 is
required
• For expression from some bacterial promoters one
of other s subunits is needed instead
• s70 is essential for cell growth in all conditions,
while other sigmas are required for special events,
like nitrogen regulation (s54), response to heat
shock (s32), sporulation, etc
The overview of s
factor function
RNA pol
s
Holoenzyme
-35
-10
Promoter region
Closed complex
Open complex
Promoter escape
Elongation
s release
mRNA
The promoter specificity of some
s factors in E.coli
s70
TTGACA – 17 bp – TATAATN3-6-A
-35
-10
+1
s32
CTTGAAA – 16 bp – CCCCATNTN3-10-T/A
-35
-10
+1
s54 GG – N12 – GC/T – 12bp – A
-24
-12
+1
Some s70 promoters in E.coli
The –35 and –10 sequences of individual s factors are conserved (yellow
boxes)
The spacer sequence between –35 and –10 is not conserved, but the
spacer length is 171 bp
The 3D structure of bacterial RNA
polymerase holoenzyme
s3
s factor domains :
N-term s1
s2
s3
s4
Inhibition
-10 binding
-10 binding
-35 binding
The UP element
RNAP
RNAP
a NTD
a CTD
UP
s4
-35
s
s2-3
-10
+1
• UP element is an AT rich motif present in
some strong (e.g. rRNA) promoters
• UP element interacts directly with Cterminal domain of RNA polymerase a
subunits
Constitutive and inducible promoters
• Certain genes are transcribed at all times and
circumstances
-Examples – tRNAs, rRNAs, ribosomal proteins, RNA
polymerase
-Promoters of those genes are called constitutive
• Most genes, however, need to be transcribed only
under certain circumstances or periods in cell life
cycles
-The promoters of those genes are called inducible and
they are subject to up- and down- regulation
Regulation at promoters
• Promoters can be regulated by repression
and/or activation
• Many s70 promoters are controlled both by
repression and activation, whereas, for
example s54 promoters are controled solely
by activation
Mechanisms of repression
•
•
•
•
•
Repression by steric hindrance
Inhibition of transition to open complex
Inhibition of promoter clearance
Anti-activation
Anti-sigma factors
a) Repression by steric hindrance (most cases)
• Examples: Trp repressor, lac
repressor
b) Inhibition of transition to open complex
• RNA Polymerase – s complex (RNAP-s) can bind to
promoter, but transition to open complex is blocked
c) Inhibition of promoter clearance
• The transcription bubble can be formed, but
further RNAP-s movement is blocked
e) Anti-sigma factors
• An anti-s factor is defined by the ability to prevent its
cognate s factor to compete for core RNA polymerase
• Mostly used for s factors, other than s70, for example in
life cycle regulation (sporulation, etc)
• Some bacteriophages use their own anti-s factors to
prevent expression of cellular proteins
RNAP
RNAP
anti-s
s
s
-10
-35
-10
-35
d) Anti-activation
• Repressor molecule removes the activator
RNA pol - s
Activator
ABS
weak promoter
+1
Activator binding sequence
Activator
Repressor
ABS
RNA pol - s
weak promoter
+1
Two examples of steric hindrance
• Trp repressor
• Lac repressor
The tryptophan repressor
• The trp repressor controls the operon for the
synthesis of L-tryptophan in E.coli by a simple
negative feedback loop
In the absence of tryptophane
the trp repressor (red blob)
shows no affinity to promoter
(black box) and the RNA
polymerase (yellow blob)
transcribes the operon
When enough tryptophane
(blue dots) is made, it binds to
repressor, which now is able to
bind to promoter and block
RNA polymerase binding
The 3-D structure of trp repressor
The conformational change upon binding
tryptophan molecules induces a
conformational change in trp repressor
The lac promoter
Lac promoter is widely used in artifical plasmids, designed
for protein production
For practical purposes it is easier to use non-hydrolyzable
lactose analog – IPTG (isopropyl-b-thiogalactoside)
instead of native lactose
The structure of lac repressor monomer
Hinge helix
DNA binding
domain
Core N subdomain
Inducer binding pocket
Core C subdomain
Tetramerization helix
Functional lac repressor is a
homotetramer
-82
+11
• Each dimer binds to a distinct DNA
sequence at –82 and +11 respective to
transcription start site
• This results in DNA looping,
preventing the DNA polymerase from
binding to –35 and –10 sequences
-35
-10
-82
+11
lac
repressor
The lac repressor binds both to
major and minor grooves of DNA
A cartoon, ilustrating events upon IPTG binding to
lac repressor
(IPTG)
As IPTG binds, the DNA binding domains scissor apart
Mechanisms of activation
• a) Regulated recruitment
• b) Polymerase activation
• c) Promoter activation
a) Regulated recruitment
• Activator “extends” the binding site for
RNA polymerase
strong or
weak affinity
RNA pol - s
Activator
ABS
strong affinity
weak promoter
weak affinity
+1
Catabolite Activator Protein: CAP
• Activates transcription from more than 150
promoters in E.coli
• Upon activation by cAMP (cyclic Adenosine
MonoPhosphate), CAP binds to promoter and
helps RNAP-s to bind as well
• All CAP–dependent promoters have weak –35
sequence, so that RNAP-s is unable to bind the
promoter without CAP assistance
Models for Class I and Class II promoter activation
Class I CAP binding sites
can be from –62 to –103.
CAP interacts with the
carboxy terminal domain of
the RNAP a-subunit (aCTD)
Class II CAP binding sites
usually overlap the –35.
CAP interacts with the
aCTD, aNTD (N-terminal
domain), and the s factor
Busby and Ebright, 2000, J. Mol. Biol. 293:199-213
Model for Class III promoter activation
• Activation of Class III promoters requires binding of at
least two CAP dimers or at least one CAP dimer and one
regulation-specific activator
• Interactions can be similar to those of ClassI and/or
ClassII promoters, except that each aCTD subunit is
making different interactions
3-D structure of CAP-cAMP-DNA complex
80º
Binding of CAP
causes DNA
bending by 80º
C C
Cyclic AMP binds to the N-terminal domain and causes the two
long “C” helices to reorient and move the DNA binding
domains apart, so that CAP can bind to DNA
CAP activates lac operon
AraC – repressor and activator of arabinose promoter
DNA binding domain of AraC
AraC
RNAP-s
promoter
+ arabinose ( )
Transcription
RNAP-s
Structure of AraC dimers in presence (A) and
absence (B) of arabinose
• Without arabinose, the monomers interact
with the b barrel domains. An important
interaction is stacking of Tyr31 of one
subunit on Trp 95 of the other
• Arabinose binds in close proximity to Trp95,
making the stacking interaction impossible
Monomers associate in a different way – 4
helix bundle interactions by the helical
domains
b) Polymerase activation
• This works for s54 promoters
• RNAP-s54 forms a stable complex with DNA,
but needs to be activated to form an open
complex
RNAP-s54 activation
ATP ATP+Pi
s54
s54
• RNAP-s54 open complex formation requires ATP hydrolysis
• Activator protein with ATP-ase activity binds to “enhancer” site
about 160 bp upstream from –24 sequence. DNA then gets looped
and activator interacts with RNAP-s54 resulting in the open bubble
formation upon ATP hydrolysis
c) Example of promoter activation:
MerR activator family
• MerR is an activator that controls genes involved
in the response to mercury poisoning
• Other MerR family activators (CueR, BmrR, etc)
respond to a variety of different toxic compounds
such as other heavy metal atoms or drugs
• In MerR activated promoters, -10 and –35 regions
are separated by 19bp instead of optimal 17bp
The BmrR-DNA-drug complex
• BmrR binds to a variety of toxic compounds, including
tetraphenylphosphine (TPP)
• Both TPP bound and unbound forms of BmrR bind to
promoter, but only TPP bound form induces transcription
• In 19-bp spacer variant, -35 and –10 binding regions not only are too
far from each other, but also on the opposite sides of double helix
• In BmrR-TPP bound vairiant, DNA double helix is underwound, so
that –10 and –35 regions are at the same distance as in regular 17-bp
spacer
Transcription termination
• In prokaryotes two types of transcription
termination occur – rho indepedent termination
and rho dependent termination
• In rho independent case, the termination is
achieved by a secondary structure of mRNA –
RNA stem-loop, followed by an AU rich region
• A rho protein is required for rho-dependent
termination
Rho independent termination
Attenuation
• Regulation of transcription by the behavior
of ribosomes
• Observed in bacteria, where transcription
and translation are tightly coupled
• Translation of a mRNA can occur as the
mRNA is being synthesized
Attenuation in trp operon
Rho dependent termination
As polymerase transcribes
away from the promoter,
rho factor binds to RNA and
follows the polymerase
When polymerase reaches
some sort of pause site, rho
factor catches up with
polymerase and unwinds
the DNA-RNA hybrid,
resulting in release of
polymerase
Anti-termination