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6.2.4 Termination of Transcription
1. Intrinsic (factor-independent) termination: abouthalf of the transcription units in E.coli
2. Factor-dependent termination (Rho factor): the
other half
3. Damage-dependent termination (Mfd factor)
Three processes destabilize the elongation
complex and release the transcript:
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The factor-independent (intrinsic)
has two essential components:
1. A GC-rich hairpin that forms in the emergingtranscript and is closed about 9 nucleotides
upstream of the RNA release site
2. An adjacent U-rich segment: the weakness
of the rU/dA hybrid is thought to facilitate its
unwinding and dissociation
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Factor-Independent Terminator
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Model of Rho-Independent
Termination
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Forms hexamers carrying six RNA-binding sites
and six ATP-binding sites
Binds to a loading site (rut = rho utilization):~ 70 nucleotides long contains a significant
number of cytosine residues and no secondarystructure
Formation of mRNA-Rho complex leads to
activation of Rho ATPase facilitating
translocation in a 5' 3' direction
Rho-Dependent Termination
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Rho can terminate transcription frequently in
operons
Rho can attenuate transcription conditionally
at the beginning of operons
Rho can attenuate transcription even within
open reading frames when mRNA is
uncovered due to a nonsense mutation Rho is responsible for silencing horizontally
transferred DNA elements, some of which aredetrimental to the host
Rho-Dependent Termination Sites
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Transcription Termination by Rho
E Nudler (2002) Genes Cells 7: 755
1. Rho monomers attach to unstructured nascenttranscripts
2. Using ATP, RNA wraps around the internal surface of
the Rho hexamer (or the outer rim)
3. Wrapping might pull the RNA-3 end from the RNAP
active centre or Rho may invade the active centre
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Damage-Dependent Termination
Mfd = Mutation frequency decline; 130 kDa
monomer
Mfd consists of three functional domains:
-DNA-binding domain
- ATPase domain
- RNAP-binding domain
Mfd recruits the DNA excision repair to the site
of DNA damage
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J. Roberts (2004) Curr. Opin. Microbiol. 7: 120
Effect of the DNA Translocase Activity
of Mfd on the RNA Polymerase
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V Pags (2003) DNA Repair 2: 273
Readthrough Transcription
Definition:A downstream gene will be transcribed at a
reduced rate due to a transcriptional terminator
5-10%
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6.2.5 Antitermination and
Attenuation of Transcription
AntiterminationThe default pathway results in premature
termination, and a regulatory moleculepromotes transcription readthrough
AttenuationThe default pathway is readthrough, and a
regulatory molecule induces transcription
termination
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1. Attenuation of the E. coli trp operon2. Attenuation of the B. subtilis trp operon
3. Antiterminator protein N of phage4. Antiterminator protein Q of phage
5. Antitermination of the bgl operon of E. coli
6. tRNA-mediated antitermination of Gram-
positive bacteria
Examples:
St t f th O d f
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Structure of the t r pOperon and of
Trp and Other Leader Peptides
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Features of the Regulatory Leader
Transcript of theE. c o l i t r p
Operon
Leader transcript: 141 nucleotides long
Can form three alternative RNA secondary
structures 1:2 = pause or antiterminator structure
2:3 = antiterminator structure
3:4 = terminator structure (intrinsic terminator)
14 amino acids long
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Transcription Attenuation of the E. co l i
t r pOperonPause allows a ribosome to
bind which disrupts thepausing RNAP complex
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Attenuation of the t r pOperon
Principle:
Formation of alternative secondary structures
Regulator:
Tryptophanyl-tRNA
M d l f T i ti Att ti f th
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Model of Transcription Attenuation of the
B. subtilis trp Operon
P. Gollnick (2002) BBA 1577: 240
TRAP: 11-mer
Binds 11 Trp
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Organization of the Phage Genome
Early transcription starts from two divergent
promoters PL and PR
Most of the two early transcripts are
terminated at strong terminators located after
the genes N and cro
St ct e of the Phage Antite
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Structure of the Phage Antiter-
mination Complex
Nus = N utilization substanceN binds to nut (N utilization): boxA and boxB
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The N Protein of Phage
N protein modifies RNAP into a termination-resistant from that allows readthrough of
downstream termination signals
N antiterminates at both Rho-dependent and
intrinsic termination sites
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Mechanisms of Antitermination
At Rho sites:
Antipausing speeds RNAP through the critical
release sites
By blocking some step of termination
At intrinsic terminators:
By directly preventing the formation of thehairpin
Structure of Phage Q Anti
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Structure of Phage Q Anti-
Termination Region
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Paused Complex With Q
Structure of Phage Q Antitermination
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Structure of Phage Q Antitermination
RegionTTGACTTATTGAATAAAATTGGGTAAATTTG
+1
ACTCAACGATGGGTTAATTCGCTCGTTGTG
pause site
GTAGTGAG ATG
1. Elongating RNAP pauses
2. Q binds to qut (in blue)
3. Q shifts RNAP into a new -35 region
(underlined) immediately upstream of the start
codon
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Model for Antitermination Control
by the BglG Antiterminator Protein
P. Gollnick (2002) BBA 1577: 240
Ribonucleic AntiTermination
BglG stabilizes an alternativestructure
M d l f A tit i ti C t l f
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Model for Antitermination Control of
b g lOperon Expression
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Conclusion
The antiterminator protein occurs in two forms
An active form (dimer)
An inactive form (phosphorylated monomer)
Active BglG stabilizes an alternative
secondary structure
Model of the Interaction of Uncharged
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Model of the Interaction of Uncharged
tRNAThr with the B . s u b t i l i s t h r S Leader
RNA
Regulation of Aminoacyl tRNA
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Regulation of Aminoacyl tRNA
Synthetase Operons in Gram-positive
Bacteria by Antitermination
Intrinsic terminatorstructures in the mRNA
leader sequences
Folding in alternative
structures
T B T i ti A tit i ti
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T Box Transcription Antitermination
~250 genes identified in Gram-positive
bacteria contain an about 300 n leader region
able to form a conserved secondary structure Most of these genes are involved in amino
acid biosynthesis Uncharged cognate tRNA interacts with the
leader region in at least two places to
establish an antiterminator structure
Specificity is achieved by binding of the
anticodon of the tRNA to a specifier codonbulged out of the secondary structure
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6.2.6 Regulators of Transcription
Three classes of transcriptional regulators:
1. Sigma factors
2. Transcriptional repressors
3. Transcriptional activators
T i ti l R l t Bi d t
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Transcriptional Regulators Bind to
DNA Using an Helix-Turn-Helix Motif
Gly
Most DNA-Binding Proteins Are Dimers
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Most DNA Binding Proteins Are Dimers
Classification of Known E co l i
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Classification of Known E. co l i
Transcription Factors
Of 71 transcription factors are
1. 18 activators
2. 20 repressors
3. 33 dual regulators
What determines whether a
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at dete es et e a
transcription factor acts as an
activator or a repressor ?
The position of the binding site relative to thetranscription start site
Activator binding sites:
Located in most cases upstream of the promoter
Repressor binding sites:
Located within or downstream of the promoter or
upstream in conjunction with a downstream site
E l i t i ti f t
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E. co l i transcription factors:
More than 300 transcription factors
Seven transcription factors (ArcA, CRP, Fis,
FNR, IHF, Lrp, NarL) control 50% of all
regulated genes
~60 transcription factors control only a single
promoter
Mechanisms of Repression
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Mechanisms of Repression
1. By steric hindrance
2. By a post-RNAP binding step:
- repression at the open complex
formation step
- repression at the promoter clearance
step3. By DNA looping
4. By modulation of an activator
Repression by Steric Hindrance
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Repression by Steric Hindrance
DF Browning (2004) Nature Rev. Microbiol. 2:1
Steric hindrance = Inhibiting RNAP binding tothe promoter
Repressing by DNA Looping
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DF Browning (2004) Nature Rev. Microbiol. 2:1
Looping = Binding to distal sites and
interacting by looping
Repression by Modulation of an
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DF Browning (2004) Nature Rev. Microbiol. 2:1
Activator
Modulation of an activator= Repressor binds to
an activator and prevents the activator from
functioning
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How is the activity of the repressor
modulated ?
i i l
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Transcriptional Repressors
1. The LacI repressor
2. The ArgR repressor
3. The RheA repressor
4. The HspR repressor5. The OmpR repressor
6. The LexA repressor7. The GalR repressor
The E. co l i l a c I Repressor
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p
LacI repressor = Tetramer
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Principle:
The inducer inactivates the
repressor
Inducers of the LacI Repressor
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p
Location of the Three l a cOperators
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Repression factor by binding to O1 only: 20-fold
Repression factor by binding to O1 and either O2
or O3: 50-fold
NA Becker (2005) J.
Mol.Biol. 349: 716
Absence of Lactose
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Absence of Lactose
CJ Wilson (2007) Cell. Mol. Life Sci. 64: 3
Presence of Lactose and Glucose
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Presence of Lactose and Glucose
CJ Wilson (2007) Cell. Mol. Life Sci. 64: 3
Presence of Lactose
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Presence of Lactose
CJ Wilson (2007) Cell. Mol. Life Sci. 64: 3
The E. co l i a r g R Repressor
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Principle:
The end product activates therepressor
The RheA Repressor
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At low temperature: The repressor prevents
expression of hsp18 and autoregulates its own
expression
At high temperature: The repressor is inactive
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Principle:
The repressor acts as thermosensor
The HspR Repressor
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The HspR Repressor
DnaK is Needed as a Corepressor
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P i i l
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Principle:
The corepressor is titrated bydenatured proteins
The OmpR
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The OmpR
Repressor
Target genes:
ompF
micF
Principle:
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p
The repressor becomes active after
phosphorylation
The LexA Repressor
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Principle:
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Principle:
The repressor cleaves itself after
interaction with activated RecA (RecA*)
protein = proteolytic cleavage
Pathway of Repressosome Formation
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Pathway of Repressosome Formation
S Roy (2005) Biochem. 44: 5373
Principle:
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Principle:
The repressor is inactivated by the
inducer galactose = conformational
change
6.2.6.2 Activation of Transcription
by Positive Regulators
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by Positive Regulators
(Transcriptional Activators)
Three different mechanisms:
1. Recruitment of RNAP
- activation at simple promoters
- activation at complex promoters2. Pre-recruitment of RNAP
3. Promoting isomerization of RNAP
Transcription Activation by
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Recruitment of the RNAP
Principle:
Promoter is weak causing no or insufficient
binding of RNAP
Activator binds first and then recruits the RNAP
Activation of Simple Promoters
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1. Class I activation
2. Class II activation
3. Activation by a conformational change
Example: CAP
Class I Activation
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DF Browning (2004) Nature Rev. Microbiol. 2:1
The activator is bound to an upstream site and
contacts the CTD of the RNAP
Example: CAP
The CAP Activator
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Catabolite Activator Protein (CAP) = Cyclic AMP
Receptor Protein (CRP)
Functions as a homodimer in the presence of
the allosteric effector cAMP
CAP-binding site: 22 bp inverted repeat; each
half recognized by one monomer
Induces a bend in the DNA of about 90
Class II Activation
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DF Browning (2004) Nature Rev. Microbiol. 2:1
The activator binds to a target site adjacent to thepromoter 35 hexamer interacting with domain 4
of 70Example: CI of phage
Activation by Conformation Change
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Activator binds at or very near the promoter
MerR-type activators twist the DNA to
reoriente the 35 and 10 elements
Activation at Complex Promoters
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1. Repositioning of an activator2. Co-dependence on independent contacts by two
activators3. Co-dependence due to co-operative binding of
activators
4. Co-dependence due to bacterial nucleoid proteins
5. Modulation by an epigenetic mechanism
6. Anti-activators
7. Subcellular relocalization of transcription factors
Repositioning of a Primary by a
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Secondary Activator
Activator 1 binds to a site where it is unable
to activate transcription
Activator 2 repositions activator 2
Example: MalT is repositioned by CAP
Co-dependence on Independent
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Contacts by Two Activators
Activator 1 is unable to contact the RNAP
Activator 2 bends the DNA
Co-dependence on Independent
Contacts by Two Activators
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Architecture at the E. coli proP P2 promoter
SM McLeod (2002) J. Mol. Biol. 316: 517
Co-Dependence Due to Co-
Operative Binding of Activators
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Activation of the E. coli melAB operon is
dependent on two different activators
TA Belyaeva (20002) Mol. Microbiol. 36: 211
- melibiose + melibiose
Co-Dependence Due to Bacterial
Nucleoid Proteins
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Activation of the E.
coli nir promoter
DF Browning (2000)
Mol. Microbiol. 37:
1258
A
B
odulation by an Epigenetic Mechanism
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A Barnard (2004) Curr. Opin. Microbiol. 7: 102
Anti-Activators
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Definition:
Protein which forms a complex with the
activator thereby preventing it from interacting
with the DNA
Examples:
NifA (activator) NifL (anti-activator)
PspF (activator) PspA (anti-activator)
The NifA - NifL Pair
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NifA: N-dependent activator of nitrogen fixationgenes
NifL: anti-activator; prevents transcription under
detrimental environmental conditions
J Barrett (2001) Mol. Microbiol. 39: 480
The PspF PspA Pair
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AJ Darwin (2005) Mol. Microbiol. 57: 621
PspB or/and PspC
may sense aninducing signal
Subcellular Relocalization of the
Activator
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A Bhm (2004) Curr. Opin. Microbiol. 7: 151
The Maltose System
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Consists of ten genes involved in uptake and
utilization of maltose and maltodextrins LamB acts as a specific diffusion pore
Uptake is mediated by a periplasmic-binding
protein-dependent ABC transporter
MalSQPZ: degrade maltose to glucose and
glucose -1-phosphate
The Regulatory Network Controlling
the Activity of MalT
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red MalT = inactive
green MalT = active
Transcription Activation by Pre-
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Recruitment
Definition:
1. RNAP holoenzyme and the transcriptional
activator form a complex in solution (in thecytoplasm)
2. This complex screens for the appropriatepromoter
The SoxS Activator of E. co l i
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Expression of soxS activated by oxidative stress
SoxS binds to a DNA sequence called Soxbox The Soxbox occurs more than 600 times on the
chromosomal DNA of E. coli Question: How does SoxS recognize the
appropriate Soxboxes ? Answer: By pre-recruitment
The Pre-Recruitment Mechanism
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Transcription Activation by
Isomerization of the RNA
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Polymerase
Components:
1. EN (54)2. Transcriptional activator
- with DNA-binding domain (binds to an
enhancer)
- without DNA-binding domain
The Isomerization Step Needs an
Activator
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S Wigneshweraraj (2008) Mol. Microbiol. 68: 538
E54 is unable to isomerize spontaneously
Most Activators Consist of Three
Functional Domains
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1. An N-terminal regulatory domain
2. A central ATPase (AAA+) which binds ATP
and a transcriptional activation domain
3. A C-terminal DNA-binding domain (HTH)
Some activators lack a DNA-binding domain
Enhancer-
D d
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DependentActivation
LL Beck (2007) Trends
Microbiol. 15: 530
Enhancer-
I d d t
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IndependentActivation
LL Beck (2007) Trends
Microbiol. 15: 530
Activator does not
contain a DNA-binding
domain
Function of the ATPase Domain
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The activator ATPase functions to remove a set
of repressive nucleoprotein interactions
Mechanisms controlling the
i t ti f th l t d i
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interaction of the regulatory domainwith the ATP-binding domain:
1. The regulator domain is phosphorylated by
a kinase (part of a two-component signal
transduction system)
Example one: NtrC (Nitrogen assimilation
regulator C)Phosphorylation causes oligomerization
Example two: DctD (Dicarboxylic acid
transport regulation D)
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2. The regulator binds a small ligandExample one: XylR of Pseudomonas putida
XylR binds toluene
Example two: NifA
NifA binds 2-oxoglutarate
NifA forms a repressive complex with NifL
transport regulation D)Phosphorylation relieves the inhibitory
effect of the N-terminal domain on the 54
interaction module
Interactions
Between NifL
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and NifA in
Response to
Environmental
Cues
Review Articles
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M Rappas (2007) Curr. Opin. Struct. Biol. 17: 110
LL Beck (2007) Trends Microbiol. 16: 530
S Wigneshweraraj (2008) Mol. Microbiol. 68: 538
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