Download Phage Strategies

Exodus 15:26
26 … If thou wilt diligently hearken to
the voce of the LORD thy God, and
wilt do that which is right in his sight,
and wilt give ear to his
commandments, and keep all his
statutes, I will put none of these
diseases upon thee, which I have
brought upon the Egyptians: for I am
the LORD that healeth thee.
©2001 Timothy G. Standish
Phage
Strategies
Timothy G. Standish, Ph. D.
©2001 Timothy G. Standish
Phage Reproduction:
The Lytic Cycle
Infection
Destruction of
the bacteria’s
DNA
Production of
viral parts
Lysis
Packaging
Replication of
the viral
genome
©2001 Timothy G. Standish
Phage Reproduction:
The Lysogenic Cycle
Infection
Circularization
of phage DNA
Many generations
of bacteria
Exit of phage
On to the
lytic cycle
Temperate
phage
Integration of
phage DNA into
the bacterial
genome
©2001 Timothy G. Standish
PRE
nut
t L
L N
CIII
g
Recombination
CI
Replication
t
nut RI
cro
R
CII
Regulation
O
P
Q
b
a
xis
The l Genome
int
att
J
I K
LM
Z
U
H
V
TG
Tail Genes
FII
PR
tR3
Lysis
S
cos
R
A
W
B
C
Nu3
Head Genes
D
E
FI
©2001 Timothy G. Standish
Order of Gene Immediate
Expression
Early
N
CI
cro
CIII
g
Recombination
CII
Regulation
O
Replication
P
Q
Delayed
Early
b
a
xis
int
Late
att
J
I K
LM
Z
U
H
V
TG
Tail Genes
FII
Lysis
S
cos
R
A
W
B
C
Nu3
Head Genes
D
E
FI
©2001 Timothy G. Standish
The l Regulatory Region
Terminator N utilization
left
left
tL
N utilization Terminator
right
right
nutR tR1
nutL
cIII
N
cI
Positive
regulator
Antiterminator
Repressor
cro
Antirepressor
cII
Positive
regulator
“c” = clear referring to the clear plaques resulting from functional c genes
©2001 Timothy G. Standish
The l Regulatory Region
Promoter
Promoter/
Promoter
Promoter/
Represser
Operator left Represser Operator right EstablishMaintainence
ment
tL
PRM OR/PR
PL/OL
PRE
nutR tR1
nutL
cIII
N
cI
Positive
regulator
Antiterminator
Repressor
cro
Antirepressor
cII
Positive
regulator
“c” = clear referring to the clear plaques resulting from functional c genes
©2001 Timothy G. Standish
The l Regulatory Region:
Infection
Host RNA Polymerase recognizes PL and PR and
begins transcription of the N and cro proteins
tL
cIII
PRM OR/PR
PL/OL
nutR tR1
nutL
N
PRE
cI
cro
STOP
cII
STOP
Transcription
Transcription
©2001 Timothy G. Standish
The l Regulatory Region:
Infection - Establishment of Lysogeny
Host RNA Polymerase recognizes PL and PR and
begins transcription of the N and cro proteins
tL
cIII
PRM OR/PR
PL/OL
nutR tR1
nutL
N
pN
PRE
cI
cro
cII
As they are the very
first genes to be
expressed, N and cro
are called “immediate cro
early” genes.
©2001 Timothy G. Standish
The l Regulatory Region:
Infection - Establishment of Lysogeny
pN binds to nut sites allowing transcription
through tR1 and tL
tL
cIII
PRM OR/PR
PL/OL
nutR tR1
nutL
N
pN
PRE
cI
cro
cII
cro
©2001 Timothy G. Standish
The l Regulatory Region:
Infection - Establishment of Lysogeny
pN binds to nut sites allowing transcription
through tR1 and tL
tL
cIII
PRm OR/PR
PL/OL
nutR tR1
nutL
N
pN
Transcription
PRE
cI
cro
pN
cII
Transcription
cro
©2001 Timothy G. Standish
The l Regulatory Region:
Infection - Establishment of Lysogeny
The cII and cIII proteins are produced. In vivo a
protein called HflA rapidly breaks down cII unless it is
combined with cIII. cII up regulates transcription from PRE.
tL
cIII
cIII
PRm OR/PR
PL/OL
nutR tR1
nutL
N
pN
PRE
cI
cro
cro
cII
pN cIIcIII
cII
©2001 Timothy G. Standish
The l Regulatory Region:
Infection - Establishment of Lysogeny
Transcription from PRE makes mRNA
complimentary to the cro mRNA thus inhibiting
cro translation
tL
cIII
PRm OR/PR
PL/OL
nutR tR1
nutL
N
pN
PRE
cI
cro
cII
pN cIIcIII
Transcription
cro
©2001 Timothy G. Standish
The l Regulatory Region:
Infection - Establishment of Lysogeny
The transcript from PRE provides a 3’ untranslated region
with an efficient ribosome binding site before the start of
the cI gene, thus cI repressor is efficiently produced.
tL
cIII
PRm OR/PR
PL/OL
nutR tR1
nutL
N
pN
PRE
cI
cro
cII
pN cIIcIII
Transcription
©2001 Timothy G. Standish
The l Regulatory Region:
Infection - Establishment of Lysogeny
The repressor gene product of cI binds to OR and OL
preventing transcription from PR and PL.
tL
cIII
PRm OR/PR
PL/OL
nutR tR1
nutL
N
pN cI
PRE
cI
cI
cro
cII
pN cIIcIII
cI
©2001 Timothy G. Standish
The l Regulatory Region:
Infection - Establishment of Lysogeny
The repressor up-regulates expression from PRM
maintaining repressor production and preventing
expression of any other proteins and establishing lysogeny.
tL
cIII
PRM OR/PR
PL/OL
nutR tR1
nutL
N
cI
PRE
cI
cI
cro
cII
Transcription
©2001 Timothy G. Standish
The l Repressor Protein
Repressor protein has two functional domains:
1 An amino acid (residue) 1-92 N-terminal DNA
binding domain which binds to OR and OL
2 A C-terminal protein binding domain essential for
dimerization running from amino acids 132-236
A 40-residue connector runs between the two
domains
Repressor
Repressor dimer
Repressor only
C
CC
binds DNA
efficiently when it is
a dimer
N
N N
©2001 Timothy G. Standish
The l Regulatory Region:
Infection - Establishment of The Lytic Cycle
Host RNA Polymerase recognizes PL and PR and
begins transcription of the N and cro proteins
tL
cIII
PRM OR/PR
PL/OL
nutR tR1
nutL
N
pN
PRE
cI
cro
cII
As they are the very
first genes to be
expressed, N and cro
are called “immediate cro
early” genes.
©2001 Timothy G. Standish
The l Regulatory Region:
Infection - Establishment of The Lytic Cycle
Host RNA Polymerase recognizes PL and PR and
begins transcription of the N and cro proteins
cI
PRM
OR/PR
cro
©2001 Timothy G. Standish
The l Regulatory Region:
Infection - Establishment of The Lytic Cycle
Host RNA Polymerase recognizes PL and PR and
begins transcription of the N and cro proteins
OR/PR
5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’
3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’
cI
OR3
PRM
OR2
OR1
cro
©2001 Timothy G. Standish
C
CC
N
N N
The l Regulatory Region:
Infection - Establishment of The Lytic Cycle
5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’
3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’
cI
OR3
OR2
OR1
cro
The N terminus of the repressor binds to OR1 and 2 with high affinity
The N terminus of the repressor binds to OR1 and 2 with high affinity
The N terminus of the repressor binds to OR1 and 2 with high affinity
©2001 Timothy G. Standish
The l Regulatory Region:
Infection - Establishment of The Lytic Cycle
Host RNA Polymerase recognizes PL and PR and
begins transcription of the N and cro proteins
RNA
Polymerase
5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’
3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’
cI
RNAOR3
Polymerase
OR2
OR1
cro
©2001 Timothy G. Standish
The l Regulatory Region:
Infection - Establishment of The Lytic Cycle
Right and left
operator sites
share a
common
consensus
sequence.
OR3 CTATCACCGCAAGGGATAA
OR2 CTAACACCGTGCGTGTTGA
OR1 TTACCTCTGGCGGTGATAA
OL3 TAACCATCTGCGGTGATAA
OL2 TTATCTCTGGCGGTGTTGA
OL1 ATACCACTGGCGGTGATAC
CON -TAYCWCYGGCGGTGWTR©2001 Timothy G. Standish
©2001 Timothy G. Standish