Bacterial Transcription

!1
Bacterial Transcription
(converting DNA Tent

!1
 1. RNA Polymerase Structure
5
~

RNA polymerase from E. coli had shown several

subunits largest
~

– 2 very large subunits are β (150 kD) and β’ (160

kD)
– Sigma (σ) at 70 kD
– Alpha (α) at 40 kD – 2 copies present in
holoenzyme
– Omega (w) at 10 kD
• Appears to play a role in enzyme assembly

!2
 w
very small
=

dispensable
= enz .
Still fu.

top aftertakin o
gotto

!3
Structure of the Core Polymerase
• X-ray crystallography on the Thermus
aquaticus RNA polymerase core reveals an
enzyme shaped like a crab claw
• It appears designed to grasp the DNA
• A channel through the enzyme includes the
catalytic center
– Mg2+ ion coordinated by 3 Asp residues
– Rifampicin-binding site

!4
Fig. 6.35 B +B
pinktolue =

+ yellow
green =
& 0 : athe
back

white = w
Fig. 6.36 active site use metalions (Ng
 Structure of holoenzyme

• Crystal structure of T. aquaticus RNA

polymerase holoenzyme shows an extensive
interface between σ and β- and β’-subunits of
the core

!7
 2. Promoters
(DNA recognized by &NA
sequence ->
polymeme

• Presence of the σ-subunit permitted

recognition of authentic RNA polymerase
binding sites
• Polymerase binding sites are called
promoters
• Transcription that begins at promoters is
specific, directed by the σ-subunit
• sigma recognise -10 and -35 regions in the
promoter region
!8
 Core Promoter Elements
• There is a region common to bacterial promoters described as
6-7 bp centered about 10 bp upstream of the start of transcription
= -10 box
↓ nucleotide upstream
10 of + 1
• Another short sequence centered 35 bp upstream is known as the
-35 box
• Comparison of thousands of promoters has produced a
consensus sequence for each of these boxes

Ou +

first nucleotides on
meNA

Capital letter = most conserved

-
conserve
TATA dox !9
region
 indure gene expression >
-
strength &
ability gene expression
=
to

Promoter Strength
• Consensus sequences:
– -10 box sequence approximates TAtAaT
– -35 box sequence approximates TTGACa
• Mutations that weaken promoter binding:
– Down mutations
– Increase deviation from the consensus
sequence
• Mutations that strengthen promoter binding:
– Up mutations
– Decrease deviation from the consensus
sequence

!10
 UP Element
esp-weak primer

• UP element is a promoter, stimulating

transcription by a factor of 30
• UP is associated with 3 “Fis” sites which
are binding sites for transcription-activator
protein Fis, not for the polymerase itself
• Transcription from the rrn promoters
respond
– Positively to increased concentration of iNTP
– Negatively to the alarmone ppGpp

!11
 The rrnB P1 Promoter

C remove =
gene expression
went down

!12
 3. Binding of RNA Polymerase to
Promoters is facilitated by σ-subunit
• How tightly does core
enzyme v. holoenzyme
bind DNA? ↑
represent B + B all

• Experiment measures
+ counts

binding of DNA to enzyme

using nitrocellulose filters
– Holoenzyme binds filters M+ Sigma
~ only
m

tightly removed

– Core enzyme binding is more

transient

& to dis top

the one that
utility af RNA polymence !13
Sigma
=

:
 Temperature and RNA Polymerase
Binding

highest +
very stade

• As temperature is
lowered, the binding of
RNA polymerase to
DNA decreases
dramatically

!14
 RNA Polymerase Binding
Hinkle and Chamberlin proposed:
• RNA polymerase holoenzyme binds DNA loosely
at first
– Binds at promoter initially
– Scans along the DNA until it finds the promoter
• Complex with holoenzyme loosely bound at the
promoter is a closed promoter complex as DNA
is in a closed ds form
• Holoenzyme can then melt a short DNA region at
the promoter to form an open promoter complex
with polymerase bound tightly to DNA
!15
 oreview of initiation & Most important

Polymerase/Promoter Binding
• Holoenzyme binds DNA
loosely at first
• Complex loosely bound at
promoter = closed
promoter complex, dsDNA
in closed form
• Holoenzyme melts DNA at
promoter forming open
promoter complex -
polymerase tightly bound
one strand of DNA = become a
template

!16
Stages of Transcription Initiation
• Formation of a closed
promoter complex
• Conversion of the closed
promoter complex to an
open promoter complex
• Polymerizing the early
nucleotides – polymerase
at the promoter
• Promoter clearance –

(
transcript becomes long
enough to form a stable
hybrid with template
!17
anything not neaded
duringelongation get rid
=
Local DNA Melting at the Promoter
• From the number of RNA polymerase
holoenzymes bound to DNA, it was
calculated that each polymerase caused a
separation of about 10 bp
• In another experiment, the length of the
melted region was found to be 12 bp
• Later, size of the DNA transcription bubble
in complexes where transcription was
active was found to be 17-18 bp

!18
 4. The Functions of σ
• Gene selection for transcription by σ
causes tight binding between RNA
polymerase and promoters
• Tight binding depends on local melting of
DNA that permits open promoter complex
• Dissociation of σ from core after
sponsoring polymerase-promoter binding

!19
 ~ & Phosphate
j ⑳ =
rediunctive

Sigma Stimulates Transcription

⑨

· >
-

:
C -

every
hu
14

noose
Initiation
radioactive [
& = needed for stable

defect
dinding

T
we nucleotide
,
elonyaction
my radioativity
• Stimulation by σ appears &
on
C-14

to cause both initiation IMRNA

tag on
= 1 U-phosphate
gamma-phosphate

and elongation ↑
• Or stimulating initiation
provides more initiated
chains for core
Iinitiation start

polymerase to elongate ~
/ATP -

higher than
initiation start

4 b+P

214 +
mone
relioactivity incoronate detectCongr!2is
0
V = detect initiation
 Reuse of σ

min
to
at
mon
-add
-

M M-
+

• During initiation σ can be recycled for additional

use in a process called the σ cycle
• Core enzyme can release σ which then
associates with another core enzyme
!21
 Sigma May Not Dissociate from Core
During Elongation

• The s-factor changes its relationship to the

core polymerase during elongation
• It may not dissociate from the core
• May actually shift position and become
more loosely bound to core

!22
 similar to RT-PCR

Fluorescence Resonance Energy

2 -& Transfer
=
no floralence,

↳/ ⑳ & =
see fluorescence
emillion

• Fluorescence resonance energy transfer

(FRET) relies on the fact that two
fluorescent molecules close together will
engage in transfer of resonance energy
• FRET allows the position of σ relative to a
site on the DNA to be measured with using
separation techniques that might displace
σ from the core enzyme

!23
FRET Assay for σ Movement Relative to
DNA

!24
 Structure and Function of σ
• Genes encoding a variety of σ-factors have
been cloned and sequenced
• There are striking similarities in amino acid
sequence clustered in 4 regions
• Conservation of sequence in these regions
suggests important function
• All of the 4 sequences are involved in
binding to core and DNA
!25
Homologous Regions in Bacterial σ
conserved
Factors
most
2 +H =

!26
 E. coli σ70
• Four regions of high sequence similarity are
indicated
• Specific areas that recognize the core promoter
elements, -10 box and –35 box are notes

!27
 Region 1

• Role of region 1 appears to be in

preventing σ from binding to DNA by itself
• This is important as σ binding to
promoters could inhibit holoenzyme
binding and thereby inhibit transcription

!28
 Region 2
• This region is the most highly conserved of
the four
• There are four subregions – 2.1 to 2.4
• 2.4 recognizes the promoter’s -10 box
• The 2.4 region appears to be α-helix

!29
 Regions 3 and 4
• Region 3 is involved in both core and DNA
binding
• Region 4 is divided into 2 subregions
– This region seems to have a key role in
promoter recognition
– Subregion 4.2 contains a helix-turn-helix
DNA-binding domain and appears to govern
binding to the -35 box of the promoter

!30
 Summary
• Comparison of different σ gene sequences reveals
4 regions of similarity among a wide variety of
sources
• Subregions 2.4 and 4.2 are involved in promoter
-10 box and -35 box recognition
• The σ-factor by itself cannot bind to DNA, but DNA
interaction with core unmasks a DNA-binding
region of σ
• Region between amino acids 262 and 309 of β’
stimulates σ binding to the nontemplate strand in
the -10 region of the promoter
!31
 5. Role of α-Subunit in UP Element
Recognition
/TATA Bux

• While σ-factor recognizes the core

promoter elements, what recognizes the
UP element?
• It appears to be the α-subunit of the core
polymerase 22subunits

!32
 Modeling the Function of the C-
Terminal Domain
• RNA polymerase binds to a UP element =
can de
any length
core promoter via its σ-factor, dumbbell arm
grad on to roelement
~
-

no help from C-terminal

domain of α-subunit
• Binds to a promoter with an
UP element using σ plus the
α-subunit C-terminal domains
• Results in very strong
interaction between
polymerase and promoter
• This produces a high level of
transcription -
esp when
.
promoter
core =
weak (down mutation
!33
 Structure of the Holoenzyme-DNA
Complex
Crystal structure of T. aquaticus holoenzyme-DNA
complex as an open promoter complex reveals:
– DNA is bound mainly to s-subunit
– Interactions between amino acids in region 2.4 of
s and -10 box of promoter are possible
– 3 highly conserved aromatic amino acids are able
to participate in promoter melting
– 2 invariant basic amino acids in s predicted to
function in DNA binding
– A form of the polymerase has 2 Mg2+ ions

!34
 & subunits =

guy

2 subunit that plays important

role in initiation

= 6 + a

& pen promote complet

 Elongation >
-
B + 3
dond
involve in phosphodiester

• After transcription initiation is

accomplished, core polymerase continues
to elongate the RNA
• Nucleotides are added sequentially, one
after another in the process of elongation
• Core polymerase contains the RNA
synthesizing machinery as well as binds
to DNA template

!36
 6. Role of β in Phosphodiester Bond
Formation

• Core subunit β lies near the active site of

the RNA polymerase
• This active site is where the
phosphodiester bonds are formed linking
the nucleotides

!37
Role of β’ and β in DNA Binding
• In 1996, Evgeny Nudler and colleagues
showed that both the β- and β’-subunits
are involved in DNA binding
• They also showed that 2 DNA binding sites
are present
– A relatively weak upstream site
• DNA melting occurs where fre nucleotides come in use DNA template
>
- as

staple interaction
• Electrostatic forces are predominant thi
may disrupt

– Strong, downstream binding site where

hydrophobic forces bind DNA and protein
together 4 need to
destony stabilize
= reaction
!38
 RNA-DNA Hybrid
where DNA Linds to RNATDNAmelt important tr stubilize PNA-protein

• The area of RNA-DNA hybridization was

determined in T7 DNA.
• The region of hybrid appears to be 8 bp
long

!39
 Topology of Elongation
• Elongation of transcription involves
polymerization of nucleotides as the RNA
polymerase travels along the template DNA
• Polymerase maintains a short melted region of
template DNA
• DNA must unwind ahead of the advancing
polymerase and close up behind it
• Strain introduced into the template DNA is
relaxed by topoisomerases

!40
Fig. 6.44
2 proposal

RNA
polymense

① more
follow direction of
DNA double helix
-
newly RNA = intertwine

* most accepted
kind
must be some
of constrain

require to nick

one stand

of DNA

& more in straight line

but rotate DNA template

-new &NA : not intertwine
 7. Termination of Transcription
• When the polymerase reaches a terminator
at the end of a gene it falls off the template
and releases the RNA
• There are 2 main types of terminators
– Intrinsic terminators function with the RNA
polymerase by itself without help from other
proteins
– Other type depends on auxiliary factor called
ρ, these are ρ-dependent terminators

!42
7.1 Rho-Independent Termination
• Intrinsic or r-independent termination
depends on terminators of 2 elements:
– Inverted repeat followed immediately by
– T-rich region in nontemplate strand of the gene
• An inverted repeat predisposes a transcript
to form a hairpin structure

!43
 Inverted Repeats and Hairpins
Fold in half complement
sequence when :

• The repeat at right is

symmetrical around its
center shown with a
dot
hairpin structure
• A transcript of this
sequence is self-
complementary
– Bases can pair up to
form a hairpin as seen
in the lower panel

!44
 Model of Intrinsic Termination
Bacterial terminators act by:
• Base-pairing of something
to the transcript to
destabilize RNA-DNA
hybrid
– Causes hairpin to form /T-rich region & H-bond T= A =
very weak
RNA fell off
easily
• Causing the transcription
to pause Stop hairpin
I/ Pause

=
unitude
separate
– Causes a string of U’s to be ↑ encounter = RNA polymerase pause

incorporated just
downstream of hairpin
!45
7.2 Rho-Dependent Termination
• Compare the sedimentation of transcripts
made in presence and absence of ρ
– Without ρ, transcripts cosedimented with the
DNA template – they hadn’t been released
– With ρ present in the incubation, transcripts
sedimented more slowly – they were not
associated with the DNA template
• It appears that ρ serves to release the RNA
transcripts from the DNA template
!46
 multiple subunits

Mechanism of Rho -
>
- donut-like
structure

• No string of T’s in the ρ-

dependent terminator, just
inverted repeat to hairpin
• Binding to the growing
transcript, ρ follows the of
/start process
RNA polymerase disassembly

• It catches the polymerase

as it pauses at the hairpin
• Releases transcript from
the DNA-polymerase
complex by unwinding the
RNA-DNA hybrid

!47
 sets ofthesame
gene promote
e me pathway
that share metriolic

~
Operons: Control of bacteria transcription

!48
 goal- down lactose - glucose
Sireak
produce enz that
.
galactuse
1. The lac Operon
• The lac operon was the first operon
discovered
• Contains 3 genes coding for E. coli
proteins that permit the bacteria to use the
sugar lactose
– Galactoside permease which transports
lactose into the cells
− β-galactosidase cuts the lactose into galactose
and glucose
– Galactoside transacetylase whose function is
unclear
!49
Fig. 7.2
 Genes of the lac Operon
• Genes are grouped:
– lacZ = β-galactosidase
– lacY = galactoside permease
– lacA = galactoside transacetylase
• All 3 genes are transcribed together
producing 1 mRNA, a polycistronic message
that starts from a single promoter
– Each cistron, or gene, has its own ribosome
binding site
– Each cistron can be transcribed by separate
ribosomes that bind independently of each
other
!51
 Control of the lac Operon
• The lac operon is tightly controlled, using 2
types of control needed
inhibit when cell do
not

– Negative control, like the brake of a car, must

remove the repressor from the operator
– An activator, additional positive factor,
responds to low glucose by stimulating
transcription of the lac operon

!52
Negative Control of the lac Operon

• The off-regulation is done by the lac

repressor
– Product of the lacI gene
– Tetramer of 4 identical polypeptides
– Binds the operator just right of promoter

!53
 lac Repressor
• When repressor binds the operator,
operon is repressed
– Operator and promoter are contiguous
– Repressor bound to operator prevents
RNA polymerase from binding to the
promoter
• As long as no lactose is available, lac
operon is repressed

!54
Negative Control of the lac Operon
at all time
transcribed
lact-get

~ I = inhibitor

recognize operator sequence

ory
very

!55
in conformation
change = release from
operator-
can find
 Inducer of the lac Operon
•The repressor is an allosteric protein
– Binding of one molecule to the protein changes shape of
a remote site on that protein
– Altering its interaction with a second molecule
•Inducer (one molecule) of lac operon binds the
repressor
– Causing the repressor to change conformation that
favors release from the operator (the second molecule)
•The inducer is allolactose, an alternative form of
lactose

!56
 Inducer of the lac Operon =
leaky operon

• Inducer (one molecule) of lac operon binds the

repressor
• The inducer is allolactose, an alternative form of
lactose

always have little

exist inside call

can actus inducer

!57
find to the represser
 lac Operators
• There are three lac operators
– The major lac operator lies adjacent to
promoter
– Two auxiliary lac operators - one upstream
and the other downstream
• All three operators are required for
optimum repression
• The major operator produces only a
modest amount of repression I

!58
Fig. 7.11

inhiditor = find to all = able to block the finding

Of RNA Polymense
within coding region
Fig. 7.12

all 3 = repress gene act .

&
Fig. 7.13
 Catabolite Repression of the lac
Operon
al catabolite
ract
• When glucose is present, lac operon is in a
relatively inactive state
• Selection in favor of glucose attributed to
role of a breakdown product, catabolite
• Process known as catabolite repression
uses a breakdown product to repression
the operon
B ga)
-

lactuse > galactose +

glucose
-
↓
do not need to breakdown ↑↑ glucose
!62
anymore : amount of
glucose =
play important role in regulating las
operon
 complex with CAP catabolite activated protein
campe form =

glucose & -
+
↓ ↑ #Ame -
&
require to drive franscription

Positive Control of lac Operon

of lac operon

• Positive control of lac

operon by a substance
sensing lack of
glucose that responds
by activating lac
promoter
– The concentration of
nucleotide, cyclic-AMP,
rises as the
concentration of
glucose drops

!63
 Catabolite Activator Protein
• cAMP added to E. coli can overcome
catabolite repression of lac operon
• Addition of cAMP lead to activation of the lac
gene even in the presence of glucose
• Positive controller of lac operon has 2 parts:
– cAMP
– Protein factor is known as:
• Catabolite activator protein or CAP
• Cyclic-AMP receptor protein or CRP

!64
 Stimulation of lac Operon
CAP-cAMP complex
positively controls the high= gene
expression
activity of β-galactosidase
– CAP binds cAMP tightly
– Mutant CAP not bind
cAMP tightly
– Compare activity and
production of β-
galactosidase using both
complexes
– Low activity with mutant
CAP-cAMP

!65
 The Mechanism of CAP Action
• CAP-cAMP complex binds to the lac
promoter
– Mutants whose lac gene is not stimulated by
complex had the mutation in the lac promoter
– Mapping the DNA has shown that the
activator-binding site lies just upstream of the
promoter
• Binding of CAP and cAMP to the activator
site helps RNA polymerase form an open
promoter complex
!66
 stimulate open promotor complex

CAP Plus cAMP Action

state interaction
~ require very &
CAP-cAMPcoPX =
Strengthen interaction
• The open promoter complex does not form even
is RNA polymerase has bound the DNA unless
the CAP-cAMP complex is also bound

!67
 CAP
• Binding sites for CAP in lac operons contains the
sequence TGTGA. This sequence is also found
in other operons, such as ara and gal.
– Sequence conservation suggests an important role in
CAP binding
– Binding of CAP-cAMP complex to DNA is tight
• CAP-cAMP activated operons have very weak
promoters
– Their -35 boxes are quite unlike the consensus
sequence
– If these promoters were strong they could be activated
even when glucose is present

!68
Fig. 7.17
 Recruitment
• CAP-cAMP recruits polymerase to the
promoter in two steps
– Formation of the closed promoter complex
– Conversion of the closed promoter complex
into the open promoter complex

R + P ←⎯→
KB
RPc ⎯⎯→
k2
RPo
• CAP-cAMP bends its target DNA by about
100° when it binds

!70
Proposed CAP-cAMP Activation of lac
Transcription
• The CAP-cAMP dimer
binds to its target site on
the DNA
• The αCTD (α-carboxy
terminal domain) of
polymerase interacts
with a specific site on
CAP
• Binding is strengthened
between promoter and
polymerase

!71
T
-
E
S
S
·
= -
E = - = =
:
- E =
↑
E E
- 5
-
·
E ii X
a
 2. The ara Operon
• The ara operon of E. coli codes for
enzymes required to metabolize the
sugar arabinose multiple
- sugar
subunit

• It is another catabolite-repressible
operon

!72
 The araCBAD Operon
The ara operon is also called the araCBAD
operon for its 4 genes
– Three genes, araB, A, and D, encode the
arabinose metabolizing enzymes
– These are transcribed rightward from the
promoter araPBAD
– Other gene, araC
• Encodes the control protein AraC
• Transcribed leftward from the araPc promoter

!73
 Features of the ara Operon
• Two ara operators exist:
– araO1 regulates transcription of araC
– araO2 is located far upstream of the promoter
araPBAD between -265 and -294
• CAP-binding site is 200 bp upstream of the
ara promoter, yet CAP stimulates transcription
• This operon has another system of negative
regulation mediated by the AraC protein

!74
The ara Operon Repression Loop
• The araO2 operator controls transcription from a
promoter 250 downstream
• The DNA must loop out to allow interaction

!75
 The ara Control Protein
•The AraC, ara control protein, acts as both a
positive and negative regulator
•There are 3 binding sites
• Far upstream site, araO2
• araO1 located between -106 and -144
• araI is really 2 half-sites
– araI1 between -56 and -78
– araI2 -35 to -51
– Each half-site can bind one monomer of AraC

!76
 AraC Control of the ara Operon
• In absence of arabinose, no araBAD products
needed, AraC exerts negative control
– Binds to araO2 and araI1
– Loops out the DNA in between
– Represses the operon
• Presence of arabinose, AraC changes
conformation
– It can no longer bind to araO2
– Occupies araI1 and araI2 instead
– Repression loop broken
– Operon is derepressed

!77
 Control of the ara Operon
prin
control apart of promoter

overlo

made at
all time
promoter of ara
- from limer

of dimen
loopover of acc
region
demoter

conformation
change
& act as
inducer
CAMP-CAP
can find

release loop = can

transcrip !78
Positive Control of the ara Operon

• Positive control is also mediated by CAP

and cAMP
• The CAP-cAMP complex attaches to its
binding site upstream of the araBAD
promoter
• DNA looping would allow CAP to contact
the polymerase and thereby stimulate its
binding to the promoter

!79
 ara Operon Summary
• The ara operon is controlled by the AraC protein
– Represses by looping out the DNA between 2 sites,
araO2 and araI1 that are 210 bp apart
• Arabinose can derepress the operon causing
AraC to loosen its attachment to araO2 and bind
to araI2
– Break the loop and allow transcription of operon
• CAP and cAMP stimulate transcription by binding
to a site upstream of araI
– AraC controls its own synthesis by binding to araO1
and prevents leftward transcription of the araC gene

!80
 & Posunthesis aperon

3. The trp Operon make tryptophan

• The E. coli trp operon contains the genes for the
enzymes the bacterium needs to make the amino
acid tryptophan
• The trp operon codes for anabolic enzymes,
those that build up a substance
• Anabolic enzymes are typically turned off by a
high level of the substance produced
• This operon is subject to negative control by a
repressor when tryptophan levels are elevated
• trp operon also exhibits attenuation
!81
Tryptophan’s Role in Negative Control
of the trp Operon
• Five genes code for the polypeptides in the
enzymes that convert chorismic acid to
tryptophan
• The trp operator lies wholly within the trp
promoter
• High tryptophan concentration is the signal
to turn off the operon
• Presence of tryptophan helps the trp
repressor bind to its operator
!82
 Negative Control of the trp Operon
measure amount
of
tryptophan

• Without tryptophan no trp

repressor exists, just the
inactive protein,
aporepressor
find
~ not
anywhere
• If aporepressor binds & by itself - not repress operon

tryptophan, changes
conformation with high
affinity for trp operator
dind block

• Tryptophan is a corepressor
=

>
-
not effective

actas 10-repullor
!83
 Attenuation of the trp operon
• Attenuation imposes an extra level of
control on an operon, more than just the
repressor-operator system
• Operates by causing premature
termination of the operon’s transcript when
product is abundant
• Why?
– Repression of the trp operon is weak

!84
Attenuation in the trp Operon
T-rich region start/stap codon
contain inverted repeat + -

!85
 Mechanism of Attenuation
• The transcript of the
leader-attenuator
(t-rich)
region contains a string
&
of U’s and an inverted
repeat, so it can form a
hairpin structure.
• When RNA pol pauses
at the string of U’s, the
hairpin forms. Thus
transcription is
aborted.

!86
 Defeating Attenuation
• Attenuation operates in the E. coli trp operon as
long as tryptophan is plentiful
• If amino acid supply low, ribosomes stall at the
tandem tryptophan codons in the trp leader
• Stalled ribosome will influence the way RNA folds
– Prevents formation of a hairpin
– This is part of the transcription termination signal
which causes attenuation

!87
 Overriding Attenuation
• 2 possible hairpins can form
• The terminator codon is only exposed in
only one hairpin structure.
• Two Trp codons in tandem

!88
Fig. 7.31
translation
transcription
- same time
&acteria =

low Tip

Stop
codon
encodes for Trp
ridosome
follows
=
also full off ridosome stalli

Waiting for Top RNA &

prevent
terminate both from dinding
hairpin :
to
&
translation
-
transcription
cause different happin not fermination
hairpin
=

>
- transcription proceeds
Fig. 7.32
!91
• The attenuation mechanism involves a
coupling of transcription and translation,
where the latter affects the former.
• Thus, this mechanism would not work in
eukaryotes.

!92
 dislynthetic

4. The ribD operon

• Of Bacillus subtilis
• Controls the synthesis and transport of the
vitamin riboflavin and flavin mononucleotide
(FMN) un translated region

• Contain a conserved element in their 5’UTR latthe

known as the RFN element beginning uf
ment

• FMN binds directly to the RFN element and

cause the mRNA to change its
conformation.
use wit switch mechanism
!93
 product
that regulate

/ed ind-changes are

Proman
product low reng die
= no stop hairpin hairpin
= termination
hairpin
 Riboswitches
• Small molecules can act directly on the 5’-
UTRs of mRNAs to control their expression
• Regions of 5’-UTRs capable of altering
their structures to control gene expression
in response to ligand binding are called
riboswitches

!95
 Riboswitch Action
• Region that binds to the ligand is an
aptamer
• An expression platform is another module
in the riboswitch which can be:
– Terminator
– Ribosome-binding site
– Another RNA element that affects gene
expression
• Operates by depressing gene expression
– Some work at the transcriptional level
– Others can function at the translational level

!96
 Model of Riboswitch Action
• FMN binds to aptamer (the
RFN element) in 5’-UTR of
the ribD mRNA
• Binding FMN, base pairing in whereend products change initre when
ad product find

riboswitch changes to create

a terminator
• Transcription is terminated
• Saves cell energy as FMN is
a product of the ribD operon

!97
Shifts in bacterial transcription
time
at the sure

cage depression
hundreds gene
a

Sigma-factor switching
and
Shift in RNA polymerase

!98
 1. Sigma Factor Switching
6 6-17
house keeping
=

• σ is the key factor in determining specificity

of DNA transcription
• To shift the transcription process σ is a
likely candidate
• Study of the process done in B. subtilis and
its phage, SPO1

!99
 Temporal Control of Transcription
virus dacterial produce viral
use transcription to
particle

• SPO1 has a large i

E
genome 3 sets of
gene
(
• Temporal transcription first
+
set to

runscribed
get
express as difftime

program: recognized by host

5 factor

– First 5 minutes: 7
phage 6 factor

expression of early genes

– During 5 – 10 minutes: S recognize
expression of middle
genes
– After 10 minutes to end:
late genes expressed
another of 6 factor
type

& recognize
!100
: G-switching
 Transcription Switching
• This switching is directed by a set of
phage-encoded σ factors that associate
with the host core RNA polymerase
• These σ factors change the host
polymerase specificity of promoter
recognition from early to middle to late
– The host σ factor is specific for the phage early
genes
– Phage gp28 protein switches the specificity to
the middle genes
– Phage gp33 and gp34 proteins switch to late
specificity !101
 Sporulation
• Same type of mechanism applies to
changes in gene expression during
sporulation
• Bacteria can exist indefinitely in vegetative
state if nutrients are available
• Under starvation conditions, B. subtilis
forms endospores, tough dormant bodies
metabolism
+energy + protein prod
+

&
.

transcription of translation
diff
require . set of
genes
slow down

!102
 Sporulation Switching
• During sporulation, a whole new set of
genes is turned on, and vegetative genes
are turned off
• Switch occurs largely at the level of
transcription
• Several new σ-factors displace the
vegetative σ-factor from the polymerase
core
• Each σ-factor has its own preferred
promoter sequence
!103
 -45 prof
=
denature
high
>
-
temp
.

Bacterial Heat Shock

• The heat shock response is a defense by
cells to minimize damage
by denaturation
to comme

• Molecular chaperones are proteins:

– Bind proteins partially unfolded by heating
– Help these proteins refold properly
• Genes encoding proteins that help cells
survive heat are called heat shock genes

!104
 Other σ-Switches
• Heat shock response is governed by an
alternative σ-factor, σ32 or σH >
- more heat resistant

– Directs RNA polymerase to the heat shock

gene promoters
– Accumulation of σH with high temperature is
due to:
• Stabilization of σH
• Enhanced translation of the mRNA encoding σH
• Responses to low nitrogen and starvation
stress also depend on genes recognized
by other σ-factors
!105
 genes motorepussed
at all time
many
have mulple promoter
house keeping ->

Genes With Multiple Promoters

• Some sporulation genes must be expressed
during 2 or more phases of sporulation
when different σ-factors predominate
• Genes transcribed under different conditions
are equipped with two different promoters
– Each promoter is recognized by one of two
different σ-factors
– This ensures their expression no matter which
factor is present
– Allows for differential control under different
conditions
!106
2. The RNA Polymerase switching
• Phage like T7, T3, and φ11 have small genomes
and many fewer genes
• These phage have 3 phases of transcription:
classes I, II, and III
• Of 5 class I genes, gene 1 is necessary for class
II and class III gene expression
– If gene 1 is mutated, only class 1 genes are
transcribed
– Gene 1 codes for a phage-specific RNA polymerase of
just one polypeptide

!107
 Gene 1 RNA Polymerase
• Gene 1 RNA polymerase transcribes only
T7 class II and III genes, not class I genes
• RNA polymerase of phage T7 is unusually
specific
• This polymerase will transcribe virtually no
other natural template

!108
Temporal Control of Transcription
• Host polymerase
transcribes the class I
genes -

recognize by host &NA

polymence

• One of class I genes is

the phage polymerase
• The phage
polymerase then
transcribes the class II
and III genes
S

!109
670 house-keeping
:
G

amount
normally produced high
in
I
interaction O 2 enters
for core RNA polymence ->
depends on
O have
high affinity

* final *

Transcriptional switching in Escherichia coli

during stress and starvation by modulation
of σ70 activity

FEMS Microbiol Rev 34 (2010):

646-657

!110
• What are the factors that determine gene
expression?

!111
 Sigma factors compete for limited
amounts of core RNAP

• A large no. of sigma factors in bacteria

helps in switching on transcription in
response to a variety of environmental
signals.

• They are structurally different, but the core

RNAP-binding surface is conserved.

!112
The concentrations of different sigma factors,
except the Housekeeping σ70, are varied at
different stages of cell growth.
– σ38 levels are below the detection limits at
exponential phase, but go up to 30% of the σ70
levels during stationary phase.

The number of core RNAP molecules inside

the cell is limited and remains almost
constant under various physiological states.
• In addition to the intracellular
concentration, another factor that
determines the probability of a sigma
factor’s association with the core RNAP is
the “affinity” with which it binds to core
RNAP.
• σ70 has the highest affinity for RNAP
• σ38 has the lowest

!114
Mechanisms to subdue to dominant σ70

• (p)ppGpp and DksA alteringfinding

competiveness

– Direct or indirectly altering the core binding

competitiveness of sigma
– In general, they enhance the transcriptional
activity from promoters driven by alternative
sigma factors.

• Rsd and 6S RNA

– Inhibiting σ70– driven transcription
!115
 enhancelinding
:
a
when dind to core Propol & alter coredinding ability

(p)ppGpp and DksA

• E. coli σ38,σ32 and σ24 and Pseudomonas
putida σ54 regulons are transcribed poorly
in the absence of (p)ppGpp.

• The RNAP binding ability of σ38,σ32

compared to σ70 was diminished in
(p)ppGpp mutant strain.

!116
 Rsd
• Is an anti- σ70
• Binds to σ70 and interferes with the core
binding and recognition of -35 of E. coli
promoters, preventing the association
between σ70 and RNAP.

!117
 6S RNA
• Binds to RNAP- σ70 complex and inhibits
transcription from certain promoters by
preventing the access of RNAP to the
promoter regions on DNA.

• That DNA region is believed to be -35

region

!118