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Viruses and phages
Several types of independent genetic units in bacteria
integrated
Plasmid vs. episome = lytic vs. lysogenic phage
an autonomous units as extrachromosomal genomes, self-replicating circular molecules
in a stable and characteristic number of copies
Plasmid:
free form
free form
integrated
integrated
die for transfer
conjugation
lysis
Two pathways of a phage: lytic and lysogeny
lysis
Prophage: latent form; as part of bacterial genome
Integration (repressor on an operator)gene expressionIn a cascade
immunity1. Excision2. Susceptible host3. Conditions of infection
infection
relief the repression
Conditions that favor lysogeny include: • Poor nutritional state of the host population (bacteria) • High multiplicity of infection (MOI). High ratio of infecting phage to host bacteria.
Lytic cycle of a phage
Goal: replicate a large number of progenyMethod: hijack host Tx/Tl machineries/apparatusResult: phage mRNAs are preferentially transcribedProcedure: 1. Infection
2. early event 3. late event
4. lysis
Early infection
Late infection
Make a populatio
n of
phage genome
Assembly
A cascade regulation in lytic phase: accompanied with the similar organization of the genetic map
small number of regulatory switches with cluster organization in a sequential expression order to Maximize economy
Gen
e ex
pre
ssio
n c
asca
de
in a
po
siti
ve
co
ntr
ol
man
ner
positive
(delayed early)
Immediate early
Begin at the phage genome starts to replicate
(i.e. assembly proteins)
Each set of genes are necessary for the expression of next genes
Cascade:
See next
Two types of regulatory event in lytic cascade (switch from early stage to next stage related to gene expression)
by new σ factor is made or RNA polymerase
by making antitermination factor (using the same promoter)
1. 2.
Previous gene expression continues at next stage
T4 phage
Phage genomes show functional clustering
(165kb)
Numbered: essential genesThree-letter: non-essential genes (selective advantage)
Early: host RNA polymeraseMiddle: host RNA polymerase+MotA/AsiA (as activators to compensate for the deficiency of middle gene promoter at -30)
Late: host RNA polymerase + new synthetic sigma factor/modifier from middle phase
(initiation control)
Lambda (λ) phage: two lifestyles(anti-termination control
; positive control)
pN as an anti-terminator
pQ as an anti-terminator
host RNA polymerase
(Repressor control;Negative control)
Morphology 1. Double-stranded DNA genome, adopts both a linear and a circular form. 2. The protein coat, which protects the genome, is composed of a head and a tail. 15 different proteins, all encoded by the viral genome, form the protein coat.
How does anti-termination work in λ phage?
Gene clustering by functionalityImmediate early gene, N, encodes for
an anti-terminator
pN, an anti-terminator, allows the transcript to continue into the delayed early phase
(immediate early phase)
anti-termination(binds to nut site)
3 promoters
How does anti-termination work in λ phage? (delayed early phase)
λDNA join to form a circle after infection
PR
PR’
PL
+Q
-pQ: the transcript will stop at tR3 and is 194 bases long, known as 6S RNA
Anti-termination by pQ
Lysogeny: maintained by cI repressor
By denying RNA polymerase access to these promoters, a repressor protein (encoded by cI) prevents the phage genome from entering the lytic cycle.
cI repressor: 1. maintains the lysogenic state 2. provides immunity
an inefficient promoter for cI (lacking ribosome binding site). RM= repressor maintenancePRM
cI repressor
An autogenous circuit of cI repressorRepressor binding to the operators simultaneously blocks entry to the lytic cycle and promotes its own synthesis
Block lytic cycle Maintain lysogenic cycle
PRM
X X
repressorrepressor
*activate
To ensure the maintenance of lysogenic phase and immunity by cI autogenous circuit
2° infected phage only can enter lysogenic phase but not lytic phase
λvir:mutated OR or OL prevents repressor binding
cI expression is sensitive to its own existenceAbsence of cI repressor will stop the autogenous circuit of its expression
Immunity region
Mechanism
at aa. 111th/113th
UV
Structure of cI repressorDimeric structure of the repressor is crucial in maintaining lysogeny
Inducer binding siteChange conformation
~ ~
dimer simultaneously binds to DNA withhigh affinity
17bp palindromic sequence
Interaction of cI repressor and operator
Major groove34A
Allow binding to a successive major grooves of DNA
Contact with DNA bases
Cross over DNA
(protrusion from helix1)
contact with another face of DNA
Not contacted bases may be twisted to allow optimal contact between operator and repressore.g. phase 434 repressor contact 5 outmost of the half-site (G-C rich)The 3 inner bases (A-T rich) can easier result inwidening angle of two half-sites
DNA binding specificityAAs of helix make directly contact with bases of the operator
Chimera approach to prove the specificity of binding (protein-DNA).
Recognition helix:
Helix 3 forms several H-bonds with DNA basescontribute to binding specificity
Binding helix:
Helix2 forms H-bonds with phosphate backboneNot for specificity Also the ionic bonds contribute for interactions
Hydrophobic
H-bonds H-bonds
Hydrophobic
specificity
Keep relationship between helicesAffinity
K
contact with G in major groove and phosphate backboneMutation makes repressor affinity less than ~1000X
Cooperative DNA binding of repressor dimer
3 repressor-binding sites/operatorNo identical sequenceSeparated by 3-7 bp and AT-rich
Where to start binding O1 has strongest affinityO1 increases the affinity of O2
Usually O3 is not filled with repressor (no enough conc.)
Increases the effective affinity of repressor for the operator at
physiological conc. (or at lower conc)
OL1 and OR1 lie overlapping with RNA polymerase binding sites of PL and PR, respectively.Occupancy of OL1-OL2 and OR1-OR2 physically blocks access of RNA polymerase to the corresponding promoters.
Repressor interacts with RNA polymerase: an autogenous regulation of cI repressor
O2
turn/loopbetween helix 2 and 3
containing acidic patch to interact w/ basic region of RNA polymerase via electrostatic interaction
Stabilize the RNA polymerase binding to PRM promoter site to facilitate cI transcription(transition from closed complex to open complex)
O1
PRM
Protein-protein interaction can release energy that is used to help to initiate transcription
O3
establishmentstabilization-ve feedback
Both O1 and O2 mutations lead to virulence
How is the synthesis of repressor established in the first place?
cI: establishment and maintenance cII: maintenancecIII: maintenance positive regulators for initiation step of cI expression
(repression)pN
(+) via PRE promoter
(cIII protects cII degraded by HflA protease)
PRE promoter needs cII protein to facilitate RNA polymerase initiating transcription
cI
initiation
anti-cro
Inhibits translation of cro mRNA
cl protein expression
RNA transcribed to cI
Three related species of lysogenic bacteriophages have been studied, lambda, 434, and P22. A relatively small region of the phage genome contains all the genetic components of the on-off switch. In each of the three species of phages this region comprises two structural genes coding for the two regulator protein, cro and repressor, that operate the switch and the operator region (OR) on which they act. The operator region (OR) contains three protein binding sites – OR1, OR2 and OR3.
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The two genes are transcribed in opposite directions from their two promoters, which occupy opposite ends of the operator region. When RNA polymerase is bound to the right-hand promoter, cro is switched on, along with the early lytic genes that lie to the right of cro, and lysis results. When the polymerase is bound to the left-hand promoter, repressor is switched on, and cro and the lytic genes are repressed, and the cell survives as a lysogenic strain.
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The structures of their dimers are also known.
Lambda cro molecule Lambda repressor
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Lysogenic phase. Repressor (red) and RNA polymerase (yellow) bound to the switch region in a lysogenic strain of E. coli. The repressor binds to OR1 and OR2, thereby turning off synthesis of cro. The repressor also works as an activator for its own synthesis by facilitating RNA-polymerase binding to the repressor promoter through its binding to OR2
Lytic phase. Synthesis of the cro protein turns off synthesis of the repressor, since cro binds to OR3 and blocks RNA-polymerase bindding to the repressor promoter. Transcription of the phage genes to the right can now occur
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Why does RNA polymerase need cII protein to take place transcription?
PRE promoter has a lack of -35 consensus seq.
-45 -25 -12 +13
cII regulator binds to a region extending from -25 to -45Only when it is added, RNA polymerase can binds to PRE promoter
Cis-acting mutation affects the establishment of lysogenySimilar phenotype as cII or cIII mutant
(trans-acting mutants)
Lysogenic phase of λ phage
All immediate early and delayed early genes are
turn off
Integration into genome
The role of cII in lysogenic phase(establishment)
Acts at PRE to transcribe cIActs at PI to transcribe int (for integration)Acts at Panti-Q to transcribe antisense RNA of Q mRNA, ensures degradation of Q
The role of cI in lysogenic phase(maintenance)
Autogenous circus to turn off all genesvia PRM and OR , OL
Block PR and PL gene expression
The entering lytic cycle:N: anti-termination; cro: repressor of lysogeny
Acts at OR3 to prevent RNA poly binding to PRM,
blocking maintenance of cI repressor. Acts at OR/OL to prevent RNA poly from expressingimmediate early genes, blocking repressor establishment.
The role of cro repressor: dimer/9kD each
Mechanism is similar to cI repressor
A tug of war between cI and croThe occupancy of repressor (cI) and cro at operators
cII is the judge
Mechanism of Lysogen Induction by UV Light UV irradiation damages DNA and thus activates E. coli’s DNA repair systems. One of the DNA repair proteins, RecA, normally functions to repair double-stranded DNA breaks. However, RecA also acts as a coprotease to facilitate the cleavage of CI (lambda repressor). CI itself has latent autoproteolytic activity, but requires the RecA coprotease to stimulate this activity. Lysogenic induction would proceed as follows:
• UV light damages DNA and activates RecA • RecA binds to CI • The RecA-CI complex cleaves CI (autoproteolysis) • In the absence of CI, transcription from the right and left promoters (PR and PL) resumes. • CRO is produced, which inhibits the synthesis of CI and stimulates expression of lytic genes. • Phage particles are formed and the cell is lysed.
The x-ray structures of DNA-binding domain of the lambda cro and repressor are known.
The N-terminal domain of lambda repressor, which binds DNA, contains 92 amino acid residues folded into fives helices. Two of these, 2 (blue) and 3 (red) form a helix-turn-helix motif with a very similar structure to that of lambda cro. The complete repressor monomer contains in addition a larger C-terminal domain.
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In the case of identical domains, the DNA recognition site comprises two half sites that are either direct or inverse (palindromic) repeats of each other.The base pair separation between the two half sites is specific to each protein; it depends upon the linker region between the motifs when these belong to the same sequence, and on the protein-protein dimerization interface otherwise.
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Oct1 pou domainPDBcode: 1octR = 3.0 Å R factor = 0.237
Association of two different DNA-binding motifs (here, homodomain and classic HTH type in oct1 pou protein). The target site encompasses 2 major grooves and one minor.
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Fos (mauve) – Jun (red) –Nfat (blue)PDBcode: 1a02R = 2.7 Å R factor = 0.246
Association between two different DNA-binding domain, one leucine zipper and a more complicated one. The target site becomes 3 major grooves and one minor.
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