1. Viruses and phages

2. Several types of independent genetic units in bacteria
lysis
die for transfer
free form
integrated
integrated
free form
integrated
an autonomous units as extrachromosomal genomes,
self-replicating circular molecules
in a stable and characteristic number of copies
conjugation
Plasmid:
Plasmid vs. episome = lytic vs. lysogenic phage

3. Two pathways of a phage: lytic and lysogeny
infection
gene expression
In a cascade
Integration (repressor on an operator)
Prophage: latent form;
as part of bacterial genome
lysis
Excision
Susceptible host
Conditions of infection
immunity
relief the repression

4. 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.

5. Lytic cycle of a phage Early infection Late infection
Goal: replicate a large number of progeny
Method: hijack host Tx/Tl machineries/apparatus
Result: phage mRNAs are preferentially transcribed
Procedure: 1. Infection
2. early event
3. late event
4. lysis
Make a population of
phage genome
Early infection
Late infection
Assembly

6. A cascade regulation in lytic phase
Immediate
early
A cascade regulation in lytic phase
: accompanied with the similar organization of the genetic map
Cascade:
positive
Each set of genes are necessary for the expression of next genes
small number of regulatory switches with
cluster organization in a
sequential expression order to
Maximize economy
See next
(delayed early)
Gene expression cascade in a positive control manner
Begin at the phage genome starts to replicate
(i.e. assembly proteins)

7. Two types of regulatory event in lytic cascade
(switch from early stage to next stage related to gene expression) 1.
by new σ factor is made
or RNA polymerase
by making antitermination factor
(using the same promoter) 2.
Previous gene expression continues at next stage

8. Phage genomes show functional clustering
T7 phage
Immediate
early
early
late

9. T4 phage Phage genomes show functional clustering (initiation control)
(165kb)
Numbered: essential genes
Three-letter: non-essential genes (selective advantage)
Early: host RNA polymerase
Middle: 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

11. (anti-termination control
Lambda (λ) phage: two lifestyles
(anti-termination control
; positive control)
(Repressor control;
Negative control)
host RNA polymerase
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.
pN as an anti-terminator
pQ as an anti-terminator

14. Immediate early gene, N, encodes for
How does anti-termination work in λ phage?
(immediate early phase)
Immediate early gene, N, encodes for
an anti-terminator
Gene clustering by functionality
3 promoters
anti-termination
(binds to nut site)
pN, an anti-terminator, allows the transcript to
continue into the delayed early phase

16. How does anti-termination work in λ phage?
(delayed early phase)
λDNA join to form a circle after infection
Anti-termination by pQ PL PR
-pQ: the transcript will stop at tR3 and
is 194 bases long, known as 6S RNA
PR’ +Q

20. prevents the phage genome from entering the lytic cycle.
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
cI repressor
PRM
an inefficient promoter for cI (lacking ribosome binding site). RM= repressor maintenance

21. An autogenous circuit of cI repressor
Repressor binding to the operators simultaneously blocks entry to the lytic cycle and
promotes its own synthesis
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
Maintain lysogenic cycle
Block lytic cycle
λvir:
mutated OR or OL prevents
repressor binding
*activate
repressor
repressor
PRM
Immunity region X X
cI expression is sensitive to its own existence
Absence of cI repressor will stop the
autogenous circuit of its expression

22. Mechanism Structure of cI repressor ~ UV
Dimeric structure of the repressor is
crucial in maintaining lysogeny
Structure of cI repressor
at aa. 111th/113th ~
Inducer binding site
Change conformation UV
dimer simultaneously
binds to DNA with
high affinity

23. contact with another face of DNA
Interaction of cI repressor and operator
Not contacted bases may be twisted to allow
optimal contact between operator and repressor
e.g. phase 434 repressor contact 5 outmost of
the half-site (G-C rich)
The 3 inner bases (A-T rich) can easier result in
widening angle of two half-sites
Allow binding to a successive
major grooves of DNA
Major groove
Cross over DNA
34A
contact with another face of DNA
(protrusion from helix1)
Contact with DNA bases
17bp palindromic sequence

24. DNA binding specificity
AAs 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 bases
contribute to binding specificity
Binding helix:
Helix2 forms H-bonds with phosphate backbone
Not for specificity
Also the ionic bonds contribute for interactions
Hydrophobic
Hydrophobic
Affinity
H-bonds
H-bonds
Keep relationship between helices
specificity K
contact with G in major groove and phosphate backbone
Mutation makes repressor affinity less than ~1000X

25. Cooperative DNA binding of repressor dimer
3 repressor-binding sites/operator
No identical sequence
Separated by 3-7 bp and AT-rich
O1 has strongest affinity
O1 increases the affinity of O2
Usually O3 is not filled with repressor
(no enough conc.)
Where to start binding
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.

26. Repressor interacts with RNA polymerase
: an autogenous regulation of cI repressor
PRM
turn/loop
between helix 2 and 3
Both O1 and O2 mutations
lead to virulence
containing acidic patch to
interact w/ basic region of
RNA polymerase via
electrostatic interaction
-ve feedback
stabilization
establishment O3 O2 O1
Stabilize the RNA polymerase binding to PRM promoter site to facilitate cI transcription
(transition from closed complex to open complex)
Protein-protein interaction can release energy that is used to help to initiate transcription

27. How is the synthesis of repressor established in the first place?
cI: establishment and maintenance
cII: maintenance
cIII: maintenance
(+) via PRE promoter pN
positive regulators for initiation step of cI expression
(repression)
initiation
(cIII protects cII degraded by HflA protease)
cl protein expression
Inhibits translation of cro mRNA cI
anti-cro
RNA transcribed to cI
PRE promoter needs cII protein to facilitate RNA polymerase initiating transcription

28. 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.

29. 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.

30. The structures of their dimers are also known.
Lambda repressor
Lambda cro molecule

31. Lambda repressor/operator complex

32. Cro-DNA interactions

33. 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

36. Why does RNA polymerase need cII protein to take place transcription?
PRE promoter has a lack of -35 consensus seq.
cII regulator binds to a region extending from -25 to -45
Only when it is added, RNA polymerase can binds to
PRE promoter
-45
-25
-12
+13
Cis-acting mutation affects the establishment
of lysogeny
Similar phenotype as cII or cIII mutant
(trans-acting mutants)

37. Lysogenic phase of λ phage
The role of cII in lysogenic phase
(establishment)
Acts at PRE to transcribe cI
Acts 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 genes
via PRM and OR ， OL
Block PR and PL gene expression
Integration into genome
All immediate early and
delayed early genes are
turn off

38. The entering lytic cycle:
N: anti-termination; cro: repressor of lysogeny
The role of cro repressor: dimer/9kD each
Acts at OR3 to prevent RNA poly binding to PRM,
blocking maintenance of cI repressor.
Acts at OR/OL to prevent RNA poly from expressing
immediate early genes, blocking repressor establishment.
Mechanism is similar to cI repressor

39. A tug of war between cI and cro
The occupancy of repressor (cI) and cro at operators
cII is the judge

42. Role of cII

43. 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.

45. 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 a helices. Two of these, a2 (blue) and a3 (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.

46. 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.

47. 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.
Oct1 pou domain
PDBcode: 1oct
R = 3.0 Å
R factor = 0.237

48. Fos (mauve) – Jun (red) –
Nfat (blue)
PDBcode: 1a02
R = 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.