Figure 2.6 Part 1
Joining DNA after a single enzyme has cut it
Vector
DNA
Donor
DNA
Vector
plus one
or more donor
fragments
EcoRI
AATTC
G
AATTC
G
G
CTTAA
G
CTTAA
Reclosed
vector
Circularized donor
Copyright 2006 by E.A. Birge
Figure 2.6 Part 2
Joining DNA after two enzymes have cut it
Vector
DNA
Donor
DNA
Vector
plus one
or more donor
fragments
Keep large fragment
EcoRI
PstI
CTGCA
G
Discard
G
CTTAA small
fragment
G
G
CTTAA
ACGTC
AATTC
G
G
ACGTC
Only one possible
structure can form
and be joined by
DNA ligase
Copyright 2006 by E.A. Birge
Figure 4.7
RNA Polymerase
Binds all the time
lacZ lacY lacA
lacI
PI
Transcripts
Binds only when
CRP and cAMP
are present
P3 P2 O3 P1 O1 O2
Low efficiency
promoters
Translation gives
repressor monomer
O1
tL
High efficiency
promoter
Both of these mRNA
moleculestetramer
are
Repressor
may
translatable
to give
bind to operator
sites,
b-galactosidase,
forming
a loop in the DNA
galactoside
permease,
that prevents
RNA
O2 and thiogalactoside
polymerase
transacetylase binding
lacY
Repressor monomer may lacI P1
bind to allolactose, the
true inducer
Copyright 2006 by E.A. Birge
Figure 4.8
RNA polymerase
binds here in the
absence of cAMP
and CRP
RNA polymerase binds
here when repressor absent and cAMP
and CRP bound
galM
galK
galE galE
galT galT
galK galK
galU galUgalS galS
galR galR
PR PRtR OtRE P2OP
E 1PO
2 IP1 OI
Translation
tL PUtL PtUU PtSU PtSS
tS
Arrows indicate
mRNA transcripts
Translation
Repressor monomer
Alternate repressor
Inside of loop
works best at PS but
also binds OI and OE
Repressor binds
to galactose and
leaves promoter open
P2
OE
P1
OI
galE
Repressor dimer bridges OE and
OI, blocking transcription from P1 but not P2
Copyright 2006 by E.A. Birge
Fig.
4.10
Regions
DNA duplex
that can
in form
the attenuator
stems and
region
loops
1
2
3
4
RNA polymerase pause site.
Ribosome near the end of
Approach of ribosome starts
the leader peptide
Polymerase moving again.
No translation
Ribosome
paused at
tryptophan
codons
2
2
1
3
2
4
3
1
4
1
Attenuator loop
forms, transcription
stops
Antiterminator loop
forms, transcription
continues
Copyright 2006 by E.A. Birge
3
4
Fig.
trpE trpD trpC trpF trpB trpA hisH tyrA argE
p
p
4.11
Leader region
1 50
100
Excess
tryptophan
150
Antiterminator
loop
Limiting
tryptophan
1
50
Trap wheel
binds to DNA
150
100
100
150
Potential
terminator
loop
Terminator
loop
50
1
trp operon has two
promoters, only 1st is
attenuated
mtrB encodes
trap protein mtrA mtrB
p
Tryptophan
Copyright 2006
by E.A.2002
Birge
Copyright
by E. A. Birge
Uncharged tRNA
stabilizes antitermination loop
I
UA
GC
G
C
C
G
GU
GCCA
Tryptophan in short supply,
tRNA uncharged
Fig.
4.12
Antiacceptor loop
Potential attenuator stem
Codon
equivalent
Tryptophan prevents
binding to antiacceptor
loop
II
III
UA
G C
C
G
Tryptophan in excess,
tRNA is charged
Copyright 2002 by E. A. Birge Copyright 2006 by E.A. Birge
GCCATrp
UGGC
Terminator
loop forms
Figure 5.4
Gap
Nick
Replicate
Cut strand
D-loop
forms
Replicate
Branch migration to the right
Copyright 2006 by E.A. Birge
Isomerization (can
branch migrate in
either direction)
Figure 5.10
Chi site
+
As this strand emerges,
other strand binds to it
+
3’
or
Eventually
Exo
V
is site
RecBCD
Now have a
Properly
oriented
Chi
marching
along DNA single
triggers
endonucleolytic
cut strand
Final Products
that can bind
RecA and
Nucleoprotein
filament
form a D-loop
D-loop
Holiday
Structure
synapses with new DNA
Copyright 2006 by E.A. Birge
Figure 6.6
Wild type
phage
Mutant
phage
with
same phenotype
Mutant
phage
E. coli
host cell
Trans test
(same cistron)
Trans test
(different cistrons)
Cis
test
a
B
A
b
No progeny can result
because a and b affect
the same enzyme
a
b
A
B
Both enzyme
A & B are
functional,
lysis occurs
Copyright 2006 by E.A. Birge
a
B
A
b
Complementation
DNA protrudes
through
membrane Primer
Figure 7.9
RF IV
Infecting
virus
Coat protein
accumulates
in membrane
Complementary strand
synthesis
Gene V
New virus protein
replaces SSB
Displaced strand
Unpackaged
stabilized by SSB
DNA
Copyright 2006 by E.A. Birge
RF II (nicked by
protein A)
Rolling Circle
Displaced DNA
is coated with SSB
Protein A can
rebind and nick
DNA duplex
Figure 8.2
Box B loop
RNA
Nutpolymerase
has two
binds
and starts
subsites,
A and
transcription
B
nut
P
(Early
promoter)
NusA protein
required for
termination
NusB binds to
Box A and
S10
Box A binding site
Delayed early
Genes located
here
l DNA
T
(Transcription
terminator)
Ribosomal protein
Lambda N
S10 binds to RNA
protein binds
pol and NusB
to NusA and Box B
NusG normally
causes termination.
Displaced by NusB
Copyright 2006 by E.A. Birge
Figure 8.4, lytic response
Possible transcripts
PRM
PL
PRE
PR
att int xis red cIII N OL rex cI OR cro cII O P Q
CR
OL1
CR
OL2
PL
Repressor binds to
1, then 2, then 3
CR
OL3
CR
OR3
PRM
CR
OR2
CR
OR1
PR
Repressor
Left and right
Cro
binds
to
3,
All transcripts
maintenance
transcripts
then
2,
then
1
turned off
promoter
turned off
turned off
on
Copyright 2006 by E.A. Birge
Figure 8.4a, Temperate response
PL
PRM (L1M transcript)
PRE (L1E transcript)
att int xis red cIII N OL rex cI OR cro cII O P Q
R
OL1
R
OL2
R
OL3
R
OR3
Cro
PRM
binds
Left and right
Repressor binds to All
transcripts to 3, then 2,
transcripts
1, then 2, then 3
except repressor then 1
turned off
turned off
PL
Copyright 2006 by E.A. Birge
R
OR2
R
OR1
PR
Repressor
maintenance
promoter
turned on
Figure 8.4b l Repressor Binding
Adjacent dimers form tetramers
OL1 OL2 OL3
Repressor dimers
Binding of
final tetramer
blocks last
active promoter
OR3 OR2 OR1
Tetramers then form an octamer,
looping the DNA
OL3 OL2 OL1
PRM
OR3 OR2 OR1
The issue that remains
is how PRE turns on
Copyright 2006 by E.A. Birge
Turning on PRE
PInt
PRE
att int xis red cIII N OL rex cI OR cro cII O P Q
cIII protein
antagonizes
ftsH protein
Critical Genes are
cII and cIII
cII protein binds
to –35 region of
two promoters, has
same effect as CRP
on lac promoter
Host protein ftsH
cleaves cII, keeps
PRE turned off
Therefore, if cII or cIII proteins are mutated, very difficult to turn
on these promoters. If they do turn on, get a normal lysogen.
Copyright 2006 by E.A. Birge
Figure 10.1, DNA Uptake
Input DNA
Pseudopilin
subunits
Outer membrane
Peptidoglycan
NucA
Pilin
subunits
N
N
Cell
Membrane
ATPase
Single-strand fragment
Copyright 2006 by E.A. Birge
Fig. 10.2 DNA Entry into
Hemophilus
Donor
is cleaved
DonorDonor
DNA
DNADNA
Donor
DNA
Donor DNA is internalized
DNA Receptor Protein
Outer membrane
Cytoplasmic
membrane
One strand is degraded,
the other is translocated
to cytoplasm
Copyright 2006 by E.A. Birge
Fig. 11.2 Interrupted Mating
If you extrapolate from
late value, intercept
doesn’t make sense
Need to
extrapolate
from earliest
time points
Copyright 2006 by E.A. Birge
Fig. 11.8 Transfer DNA
Replication
Transfer to
light, radioactive
medium
Transfer to light,
nonradioactive
medium
Replicating Hfr
Note that all
DNA labeled with
By chance, someDNA
labelis either
13 and 15N (heavy
the
or
If C
new round of will be at oriT,heavy:light
isotopes)
of
Next round of
replication
begins plasmid originheavy:heavy
replication
replication begins
at plasmid oriT,
get radioactive
light:light DNA early
At this point, the
At this point, all
radioactive DNA
DNA is heavy:
becomes light:light
light
Copyright 2006 by E.A. Birge
Fig. 12.6 Conjugal Plasmid Interactions
R100 Plasmid
finO
traJ finP
Naturally defective F Plasmid
protein in F,
therefore
finO
transfer
always is
traJ finP
efficient
FinO works in
trans so R100
reduces transfer
by F as well as
and FinP levels build up, and conjugal itself
ability decreases. Transfer is very
efficient right after a previous transfer.
Required for
The combination of FinO and
conjugal functions
FinP proteins inhibits traJ function
to be expressed
When R100 arrives in a cell, FinO
Copyright 2006 by E.A. Birge
Fig. 13.1 Plasmid R1Copy Number
Origin of
Promoter 2
Copy number control region
replication
CopA RNA
Prevents translation of tap
Promoter 1
tap
copB
CopT RNA
Translation of tap allows
translation of repA
CopB inhibits
promoter 2
activity
RepA
stimulates
replication
initiation
Copyright 2006 by E.A. Birge
CopB RNA
Translation gives
CopB protein
repA
Fig. 13.1 pIP501Copy Number
Origin of
When bound,
2
causes replication
RNA IIIPromoter
attenuation of RepR mRNA
Promoter 1
copR
repR
CopR RNA
Translation gives
CopR protein
RepR RNA
CopB inhibits
promoter 2
activity and
stimulates
Promoter III
Copyright 2006 by E.A. Birge
RepR stimulates
replication
initiation
F or P1 Plasmid Partitioning
ADP plus
sopA protein
inhibits promoter
(autoregulation)
ADP
ATP plus proteins
causes partitioning of
plasmid at ¼ and ¾
ATP of distance to pole of cell
F
sopA
sopB
sopC
P1
parA
parB
parS
Copyright 2006 by E.A. Birge
Fig. 14.2 Nitrogen Regulation
(Global Regulatory Network)
NtrC (inactive)
NtrB (kinase phosphatase)
GlnD + glutamine
GlnB-UMP
NtrC-P
GlnD + 2-oxoglutarate
NtrC-P
NtrC-P
NtrC-P
Need two
glnA ntrBC
nifL nifA
glnK amtB
Now focus only
factors to
on
regulatory
s54
s54
s54
activate
proteins
NifA
GlnK
transcription,
Activates
NifL
(interferes
activator must
Promoter
promoter
Inhibits
nifHDK
with NifL)
bind to upstream
NifA
54
enhancer
s
function
GlnB
Nitrogenfixation
fixation
Nitrogen
turned
occursoff
occurs
Copyright 2006 by E.A. Birge
Fig. 14.4 Sigma Factor Production
Sigma A and
phosphorylated
Spo0A trigger this
promoter
Transcription
and translation
sigE
Prosigma E
Sigma E activates
this promoter
spoIIID Transcription
and translation
Sigma E
Sigma E
Activates newly
spliced gene
SpoIIID protein
Activation
spoIIIC spoIVCA spoIVCB
spoIIIC spoIVCB
Prosigma
K
DNAActivated
sequencespoIVCA
analysis shows
is a that the DNA
coding
for
Sigma K
Two components of
sigma
recombinase
K is actually
thatsplit
catalyzes
by a gene called
spoIVCA
spoIVCA
turns
on
late genes in
Sigma K
excision of its own gene
is discarded
mother
cell, turns off sigE
Copyright 2006 by E.A. Birge
Fig. 15.2 Gin-catalyzed Inversion
–1
This is a ribbon diagram with blue on
one side of
the ribbon
and brown on the
+1/2
+1/2
other. The circular molecule is folded so
that the enhancer (red) passes between
the two vertical strands. The large arrows
are the gix +1
sites.
+1
–1
Before recombination
After recombination
Copyright 2006 by E.A. Birge
Copyright 2002 by E. A. Birge
Fig. 15.4 Tn10 Transposition
IS10L
Bent target DNA
TetR
IS10R
Nearly precise excision
or
Precise excision
Copyright 2006 by E.A. Birge
Fig 15. 4A Phage Mu Transposition
Bacterial DNA
L1 L2 L3 E R3 R2 R1
attL
attR
Mu Prophage
B B
Gene A product
Transposome assembles
is a transposase
A
A
A
A
IHF
IHF
IHF bends Mu DNA
Target DNA
Copyright 2006 by E.A. Birge
ATP
ADP
PhageAMu
B
Protein
causes
protein binds
hydrolysis
of
to followed
target
ATP
bysequence
release of
B protein
Fig 15.5B Molecular Rearrangements
This is strand transfer complex
Single
Mu prophage
strand
nicks
in in
host
prophage
DNA
Replication begins,
target sequence
duplicated at each end
Offset
cuts
Target
DNA
Target
DNA
separates
5 bp apart
Copyright 2006 by E.A. Birge
Fig. 15.4B After Replication Finishes
ButThis
the regions
molecule
containing
is lined upthefortwo
recombination
prophages are
Whenreally
you straighten
Aout
crossover
circular
occurs
DNA,
you get
part of
within
one
big
thethe
cointegrate
prophages
molecule
within the prophages
Copyright 2006 by E.A. Birge
After Recombination Finishes
These regions are also duplicated, but that event
Noteoccurred
that this during
was essentially
a resolvase
reaction
the previous
transposition
Original DNA molecule restored, although
prophage is actually a recombinant
Prophage is flanked by short duplication
Target DNA molecule is more complex
Copyright 2006 by E.A. Birge
Copyright 2002 by E. A. Birge
Ch 17 Operon Organization
bgl operon
Phospho-bglucosidase
bglB
Pts
Enzyme
II
bglF
antiterminator
bglG
transcript
Degrades
sugar
Transports sugar
Copyright 2006 by E.A. Birge
or phosphorylates
(inactivates)
antiterminator
protein
Defective
promoter
can be
activated by
bglO or leuO
Ch 17 Tree Building
• The sequence differences must be
informative
• Simple example
Progenitor Sequence
Descendant 1
Descendant 2
Descendant 3
AAGGCCTT
AAGGCCTT
AAGGCCTT
ATGGGCTT
ATGGGCTT
ATCGGCTT
ATCGGCTT
ATCGGCTT
ATCGCCTT
ATCGCCTT
ATCGCCTT
Overall, descendant 2 has two differences
The
indicated
are
different
from
from
the progenitor
and therefore
most
Descendant
1descendant
matches
the
progenitor
2 is
and
3 do
not,
This time
3 bases
matches
thebut
progenitor
but
distant
it. but
1 closely
and
2 are
equally
distant.
1 is
more
closely
to not
progenitor
than
2 or
3, which
1so
and
2 do
not,
so 3from
isrelated
more
related
to the
progenitor
progenitor
from
each
other
There is
enough
information
arenot
related
to each
other to sort
so theyout
are
uninformative
1 and
3
Copyright 2006 by E.A. Birge