Transcript Section L Regulation of Transcription in Prokaryotes

Section L Regulation of
Transcription in Prokaryotes
L1 The lac operon
L2 The trp operon
L3 Transcriptional regulation
by alternative s factor
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
L1 The lac operon
• The operon
• The lactose operon
• The lac repressor
• Induction
• cAMP receptor
protein
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
Nobelists in Biology in 1965
Francois Jacob (44y)
Jacques Monod (55y)
(French)
Lac. Operon Theory
Concept of mRNA
Francois Jacob
Jacques Monod
The operon
Definition: The operon is a unit of gene expression and regulation
which typically includes:
Regulator genes
PlacI
lacI
Operator sequence
Structural genes
Olac
lacY
Plac
regulation
lacZ
lacA
information
• Regulator genes: whose products recognize the control-elements,
for example a repressor which binds to and regulates an operator
sequence.
• Operator sequence: Control elements such as an operator
sequence, which is a DNA sequence that regulates transcription
of the structural genes.
• Structural genes: The structural genes for encoding proteins.
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
The lactose operon
Structural genes: The lactose operon consists of 3 structural genes:
lacZ, lacY, lacA.
Polycistronic mRNA: The three structural genes are encoded in a
single transcription unit lacZYA, which has a single promoter
Plac, and transcribes a single polycistronic mRNA, but more
proteins expressed.
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
The lac repressor
Definition:  The lac I gene encodes the
repressor,  which is active as a
tetramer of identical subunits.  It has
a very strong affinity for the lac
operator-binding site, Olac, and  also
has a generally high affinity for DNA.
Structure:  The lac operator-binding site
consists of 28 bp  which is
palindromic.  This inverted
symmetry of the operator matches the
inherent symmetry of the lac repressor.
DNA
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
The lac repressor
Binding:  In the absence of lactose, the repressor occupies the
operator-binding site.  The lac repressor increases the
binding of the RNA polymerase to the lac promoter by two
order of magnitude.  This means that when lac repressor is
bound to the Olac, the RNA plo is also likely to be bound to
adjacent Plac promoter sequence without move.
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
Induction process
1. No lactose: In the absence of lactose, the lac repressor blocks all
but a very low level of transcription of lacZYA.
2. Uptake: When lactose is added to cell, the low level of permease
allows its uptake, and b-galactosidases catalyzes some lactose to
allo-lactose.
3. Induction: Allo-lactose acts as an inducer and binds to the lac
represser. This causes a change in the conformation of the
repressor tetramer, reducing its affinity for lac operator.
PlacI
lacI
RNAp
RNAp
RNApRNApRNAp
RNAp
Plac Olac
lacZ
lacY
lacA
Low
Lac.
5. Re-Inhibition:
mRNA
Section L: Regulation of transcrip. in Prok.
4. Transcription:Yang Xu, College of Life Sciences
cAMP receptor protein-I
Function:
• The Plac promoter is not a strong
promoter. Plac and related promoters
do not have strong -35 sequences
and some even have weak -10
consensus sequences.
• For high level transcription, they
require the activity of a specific
protein called cAMP receptor
protein (CRP).
• CRP exists as a dimer which cannot
bind to DNA on its own, nor
regulate transcription, but which
can form CRP-cAMP
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
CRP-cAMP (cAMP receptor protein) binding site
IR
IR
-70
-50
site I
● site I:
-40
site II
IR is CRP-cAMP strong binding site（-70 ~ -50）
● site II: is CRP-cAMP weak bingding site（-50 ~ -40）
● CRP-cAMP binds site I
cooperative effect
Promote site II binding
● CRP-cAMP + site II promotion
RNApol. into -35 sequence
into -10 sequence
starting transcription
RNApol
SiteI
siteII GC Island
-35
-10
cAMP receptor protein-II
• Normal Condition: The glucose is present in E. coli.
1. Inactivated operon: The E. coli does not require alternative
carbon sources such as lactose. Therefore lactose operon is
normally inactivated.
2. Inactivated CRP: The glucose can reduce the level of cAMP,
therefor the cAMP is not enough for binding CRP.
• Special Condition: When glucose is absent in E. coli culture.
1. Activated CRP: The levels of cAMP increased, and CRP
binds to cAMP to form the CRP-cAMP complex which is the
activated CRP.
2. Activated operon: The CRPcAMP complex binds to the Plac
just upstream from the site for
RNA pol. CRP binding induces a
90º bend in DNA, and to enhance
RNA pol binding to the promoter,
enhancing transcription by 50-fold.
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
L2 The trp operon
• The tryptophan operon
trp
• The try repressor
• The attenuator
• Leader RNA structure
• Attenuation
• Importance of attenuation
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
The tryptophan operon
Structural genes: The trp operon encodes five structural genes
whose activity is required for tryptophan synthesis.
Transcript: The operon encodes a single transcription unit which
produces a 7 kb transcript which is synthesized downstream
from the trp promoter Ptry and trp operator sites Otrp.
Expression: Like many of the operons involved in amino acid
biosynthesis, the trp operon has evolved systems for coordinated expression of these genes when tryptophan is in
short supply in the cell.
Regulation speed: As with the lac operon, the RNA product of
this transcription unit is very unstable, enabling bacteria to
respond rapidly to changing needs for tryptophan.
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
trpR
P O
E D C B A
Mechanism
Repressor (inactive )
operon on
can not bind
on the O site
(trp absent)
trpR
P O
E D C B A
operon off
tryptophan
repressor + trp
active repressor
Operator
The trp repressor
Trp repressor: A gene product of the trpR operon. It
is a dimer of two subunits.
Operator structure: Ptrp is between -21 and +3. The
core binding site is a palindrome of 18bp.
Mechanism:  The trp repressor can only bind to the
operator when it is complexed with tryptophan.
 The repressor dimer has a structure with a
central core and two DNA-reading heads.
 When tryptophan is bound to the repressor the
reading heads are the correct distance apart, and
the side chains in the correct conformation, to
interact with major grooves of the DNA at Ptrp.
Tryptophan: is the end-product of the enzymes
encoded by the trp operon, it acts as a corepressor and inhibits its own synthesis by endproduct inhibition. The repressor reduces
transcription initiation by around 70-fold.
Section L: Regulation of transcrip. in Prok.
trp
Yang Xu, College of Life Sciences
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
The attenuator
Background: At first, it was thought the repressor was responsible
for all of the transcriptional regulation of the trp operon. And
then, it was observed that the deletion of a sequence between the
operator and the trpE resulted in an increase in both the basal and
the activated (de-repressed) levels of transcription.
Attenuator: This site is termed the attenuator and it lies towards the
end of the transcribed leader sequence of 162 nt that precedes the
trpE initiator codon.
Structure: The attenuator is a -independent terminator site which
has a short GC-rich palindrome followed by eight successive U
residues in RNA sequence.
Function: If this sequence is able to form a 3－4 hairpin structure in
the RNA transcript, then it acts as a highly efficient transcription
terminator and only a 140 nt transcript is synthesized.
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
S3
S4
A
A•U
U
C
G
A
C•G
G•C
C•G
C•G
C•G
G•C
A•U
……NNNN
UUUUUUU-OH
Section L: Regulation of transcrip. in Prok.
Rho-independent T.
Yang Xu, College of Life Sciences
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
Attenuation
Reason of attenuation: Attenuation effect depends on the fact that
transcription and translation are tightly coupled in E. coli;
translation can occur as an mRNA is being transcribed.
Sequence 1:  The 3’OH-end of the trp leader peptide coding
sequence overlaps complementary sequence 1;  the two trp
codons are within sequence 1.
Stop codon: The stop codon in the leader sequence is between
sequence 1 and sequence 2.
3:4 hairpin: It is a conditional terminator (attenuator). When the
tryptophan is non-starved, the 3:4 hairpin forms in the mRNA.
Coordination: As transcription of the trp operon proceeds, the
RNA polymerase pauses at the end of sequence 2 until a
ribosome begins to translate the leader peptide.
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
Importance of attenuation
700-fold regulatory effect:
• Attenuation: The presence of tryptophan gives rise to a 10-fold
repression of trp operon transcription through the process of
attenuation alone.
• Trp repressor: Combined with control by the trp repressor (70fold), this means that tryptophan levels exert a 700-fold
regulatory effect on expression from the trp operon.
His operon: For example
• Histidine codons: The His operon has a leader sequence which
encodes a peptide with seven successive histidine codons.
• Only mechanism: The His operon has no repressor-operator
regulation, and attenuation forms the only mechanism of
feedback control.
Other operons: Attenuation occurs in at least six operons that
encode enzymes concerned with amino acid bio-synthesis.
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
L3 Trans. regulation by alternative s factor
• Sigma factor
• Heat shock
• Bateriophage s factor
groel
hsp47
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
Regulation patterns and s factors
Transcription regulation patterns:
• By transcriptional repressors: such as the lac repressor;
• By transcriptional activators: such as the CRP;
• By different s to direct RNApol binding different promoter:
Functions of s factors :  The abb’w core enzyme of RNA
polymerase is unable to start transcription at promoter sites.
 In order to specifically recognize the consensus -35 and -10
elements of the promoters, it requires the s factor subunit.
 This subunit is only required for transcription initiation,
 being released from the core enzyme after initiation and before
RNA elongation takes place.
Features: Many bacteria, including E.coli, produce a set of s
factors that recognize different sets of promoters.
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences
Heat shock gene
Transcriptional regulation by alternative s factor in E. coli
for stress condition (an example for the use of different s)
70
----- When 37℃ ; genes expressed in E.coli by RNApol with s
----- When ＞ 37℃; (42 ℃: very soon; 50 ℃: the only products)
More then 17 heat-shock proteins are expressed in E.coli
through transcription by RNApol using an alternative s32 ,
which have own specific promoter consensus sequence
Responsive Promoter
-35
-10
Standard s70 ----TTGACA----16-18---------TATAAT-----Heat shock s32
----TTGAA-----13-15---CCCCAT-T----------
Bateriophage s factor
Some phages provide new s subunits to the host RNA polymerase
with a different promoter specificity and hence to selectively
express their own phage genes.
This strategy is an effective alternative to the need for the phage to
encode its own complete polymerase. This pattern allows its own
genes to be transcribed at specific stages during virus infection.
Early gene
middle genes
late genes
host s70
phage s28
phage s-late
T4 in E.coli
proteins
That’s all for Section L
Section L: Regulation of transcrip. in Prok.
Yang Xu, College of Life Sciences