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Bases
In
DNA
there
are
four
bases:
adenine
(abbreviated
A),
guanine
(G),
thymine,
(T)
and
cytosine
(C).
Adenine
and
guanine
are
purines;
Thymine
and
cytosine
are
pyrimidines.
Nucleoside
A
nucleoside
is
a
pyrimidine
or
purine
base
covalently
bonded
to
a
sugar.
In
DNA,
the
sugar
is
deoxyribose
and
so
this
is
a
deoxynucleoside.
There
are
four
types
of
deoxynucleoside
in
DNA;
deoxyadenosine,
deoxyguanosine,
deoxythymidine
and
deoxycyGdine
NucleoGde
A
nucleoGde
is
base
+
sugar
+
phosphate
covalently
bonded
together.
In
DNA,
where
the
sugar
is
deoxyribose,
this
unit
is
a
deoxynucleoGde.
Phosphodiester
Bond
In
DNA
the
nucleoGdes
are
covalently
joined
together
by
3-5
phosphodiester
bonds
to
form
a
repeGGve
sugarphosphate
chain
which
is
the
backbone
to
which
the
bases
are
aPached.
DNA
Sequence
The
DNA
sequence
is
the
sequence
of
A,
C,
G
and
T
along
the
DNA
molecule
which
carries
the
geneGc
informaGon.
In
a
DNA
double
helix,
the
two
strands
of
DNA
are
wound
round
each
other
with
the
bases
on
the
inside
and
the
sugarphosphate
backbones
on
the
outside.
ProkaryoGc
Chromosomes
The
DNA
in
a
bacterium
is
a
supercoiled
double-stranded
circular
molecule
that
is
packaged
in
the
nucleoid
region
of
the
cell.
The
DNA
is
negaGvely
supercoiled,
complexed
to
several
histone-like
proteins
and
organized
(c)
15
O
P
O
P
P
O
P
O
10111 O
O
O
O
O
1
O
2
T
A
H2 C
O
T
A
H2C
O
3
H H
H H
4
H H
H H
5
H
HO
H
HO
5'
5'
6
7
Fig. 1. DNA synthesis. In this schematic diagram, the incoming dTTP hydrogen bonds with the adenine on the
8
template DNA strand and a 3!5! phosphodiester bond is formed, releasing pyrophosphate.
9
ReplicaGon Forks
ReplicaGon
starts
at
1111origin,
is
bi-
a
single
2
direcGonal
and
3
semi-conservaGve.
4
Each
r5eplicaGon
6 (or
eye)
bubble
7
consists
8 of
two
replicaGon
forks.
9
10111
1
2
(a)
Replication bubble
Okazaki Fragment
3'
5'
5'
3'
Leading strand
Okazaki fragments
RNA
Primer
160
1111
2
3
4
5
6
7
8
9
10111
1
2
3
4
5
6
7
8
9
20111
1
2
3
4
5
6
7
8
9
(a)
3!
5!
Primase
(b)
5!
3!
RNA primer
(c)
5!
3!
(d)
RNA primer removed
and gap filled with
DNA by DNA polymerase I
(e)
(f)
DNA fragments
joined by
DNA ligase
(g)
Fig. 4. Details of DNA replication. (a) Primase binds to the DNA templa
and (b) synthesizes a short RNA primer (dotted line); (c) DNA polymeras
RNA primer by synthesizing new DNA (thick line); (d) during synthesis of
adjacent Okazaki fragments are separated by the RNA primers; (e) the R
7
8
9
10111
1
2
3
4
5
6
7
8
9
20111
1
2
3
4
5
6
7
8
9
30111
1
2
3
4
5
6
7
8
9
40111
1
2
3
4
5
6
7
Accessory Proteins
Two interlocked
daughter DNA molecules
Binding of topoisomerase II
!
Fig. 5.
Replica(on
3
3
5
3
5
Replica(on
Overall direction
of replication
3
3
5
3
5
3
5
Replica(on
Overall direction
of replication
3
5
3
5
3
5
3
5
Replica(on
Overall direction
of replication
3
3
5
3
5
3
5
Replica(on
Overall direction
of replication
3
3
5
Okazaki fragment
3
5
3 5
3
5
Replica(on
Overall direction
of replication
3
3
5
Okazaki fragment
3
5
35
3
5
Replica(on
3
5
3
5
3
5
3 5
35
3
5
Replica(on
3
5
3
5
3
5
35
35
3
5
Replica(on
3
5
3
5
3
5
35
35
3
5
Replica(on
3
3
5
3
5
35
3
5
TranscripGon
in
Prokaryotes
TranscripGon
by
E.
coli
RNA
polymerase
occurs
in
three
phases;
iniGaGon,
elongaGon
and
terminaGon.
IniGaGon
involves
binding
of
the
enzyme
to
a
promoter
upstream
of
the
gene.
During
elongaGon,
the
anGsense
DNA
strand
is
used
as
the
template
so
that
the
RNA
made
has
the
same
base
sequence
as
the
sense
(coding)
strand,
except
that
U
replaces
T.
A
terminaGon
signal
is
eventually
encountered
that
halts
synthesis
and
causes
release
of
the
completed
RNA.
T TG A C A
! 35 sequence
"1
5 8 bp
TAT A A T
!10 sequence
(Pribnow box)
Transcriptional
start site
ElongaGon
Following
iniGaGon,
the
subunit
dissociates
from
RNA
polymerase
to
leave
the
core
enzyme
(2)
that
conGnues
RNA
synthesis
in
a
5
3
direcGon
using
the
four
ribonucleoside
5
triphosphates
as
precursors.
The
DNA
double
helix
is
unwound
for
transcripGon,
forming
a
transcripGon
bubble,
and
is
then
rewound
aber
the
transcripGon
Section G RNA synthesis and processing
complex
has
passed.
Sense strand
RNA polymerase
Direction of
transcription
Rewinding
3!
5!
Unwinding
5!
3!
3!
5ppp
Transcription
elongation
Newly synthesized
RNA strand
Antisense
strand
but then peels away from the DNA as transcription proceeds. The DNA is
unwound ahead of the transcription bubble and after the transcription
complex has passed, the DNA rewinds.
ElongaGon
5!
5!
3!
O
O
H 2C
H
P
O-
P
O
PPi
DNA
template
strand
OH
H 2C
H
HO
H
O
3!
DNA
template
strand
H
O
OH
O
O
O-
O
O
H2C
H
H
OH
U
H
H
HO
H2 C
5!
H
HO
5!
OH
Fig. 2. Transcription by RNA polymerase. In each step the incoming ribonucleotide selected is that which can basepair with the next base of the DNA template strand. In the diagram, the incoming nucleotide is rUTP to base-pair with
the A residue of the template DNA. A 3!5! phosphodiester bond is formed, extending the RNA chain by one
nucleotide, and pyrophosphate is released. Overall the RNA molecule grows in a 5! to 3! direction.
TerminaGon
A
common
terminaGon
signal
is
a
hairpin
structure
formed
by
a
palindromic
GC-rich
region,
followed
by
an
AT-rich
sequence.
Other
signals
are
also
used
which
require
the
assistance
of
rho
protein
for
eecGve
terminaGon.
G
G
G C
A U
C G
C G
G C
C G
C G
G C
5
A U U U U OH 3
RNA
Processing
Messenger
RNA
(mRNA)
transcripts
of
protein-coding
genes
in
prokaryotes
require
liPle
or
no
modicaGon
before
translaGon.
LAC Operon
P
lacI
mRNA
lac
lac
lacI
lacZ
lacY
lacA
lacI
lacZ
lacY
lacA
-Galactosidase
lac repressor lac repressor
tt
mRNA
Permease Transacetylase
LAC
Operon
The
lac
operon
contains
lacZ,
lacY
and
lacA
genes
encoding
-
galactosidase,
galactose
permease,
and
thiogalactoside
transacetylase,
respecGvely,
preceded
by
an
operator
site
(Olac)
and
a
promoter
(Plac).
The
operon
is
transcribed
by
RNA
polymerase
to
produce
a
single
polycistronic
mRNA
that
is
then
translated
to
produce
all
three
enzymes.
These
enzymes
are
involved
in
lactose
metabolism.
When
lactose
is
absent,
E.
coli
makes
only
small
amounts
of
these
enzymes
but
the
presence
of
lactose
induces
synthesis
of
large
amounts
of
all
three
enzymes.
The
mechanism
of
inducGon
is
that
the
background
level
of
-
galactosidase
converts
some
lactose
to
allolactose
which
then
acts
as
an
inducer
and
turns
on
transcripGon
of
the
lac
operon.
IPTG
can
also
act
as
an
inducer.
TranscripGon
of
the
operon
is
controlled
by
the
lac
repressor
protein
encoded
by
the
lacI
gene.
CRP/CAP
Catabolite
acGvator
protein,
CAP
(also
called
cAMP
receptor
protein,
CRP)
is
an
acGvator
required
for
high
level
transcripGon
of
the
lac
operon.
The
acGve
molecule
is
a
CRP
dimer
that
binds
35
cyclic
AMP
to
form
a
CRPcAMP
complex.
CRPcAMP
binds
to
the
lac
promoter
and
increases
the
binding
of
RNA
polymerase,
sGmulaGng
transcripGon
of
the
lac
operon.
CRP
dimer
without
cAMP
cannot
bind
to
this
DNA.
The
acGon
of
CRP
depends
upon
the
carbon
source
available
to
the
bacterium.
When
glucose
is
present,
the
intracellular
level
of
cAMP
falls,
CRP
cannot
bind
to
the
lac
promoter
and
the
lac
operon
is
only
weakly
transcribed.
When
glucose
is
absent,
the
level
of
intracellular
cAMP
rises,
the
CRPcAMP
complex
sGmulates
transcripGon
of
the
lac
operon
and
allows
lactose
to
be
used
as
an
alternaGve
carbon
source.
tRNA
Each
tRNA
has
a
cloverleaf
secondary
structure
containing
an
anGcodon
arm,
a
D
(or
DHU)
arm,
a
T
or
TC
arm,
and
an
amino
acid
acceptor
stem
to
which
the
relevant
amino
acid
becomes
covalently
bound,
at
the
3
OH
group.
Some
tRNAs
also
have
a
variable
(or
opGonal)
arm.
The
three-dimensional
structure
is
more
complex
because
of
addiGonal
interacGons
between
the
nucleoGdes.
tRNA
10
(b)
3!OH
I
A3!OH
C I
C A
C
C
Amino acid
5!
acceptor stem
Amino acid
5!
acceptor stem
a)
(b)
5!
3!
Acceptor
3!
stem
Acceptor
stem
TC loop
DHU loop
T loop
D loop
5!
TC arm
TC loop
T loop
D loop
TC arm
DHU loop
Optional arm
DHU arm
Optional arm
DHU arm
Anticodon arm
Variable
arm
Anticodon arm
Variable
arm
Anticodon
loop
Anticodon
Anticodon loop
Anticodon loop
Anticodon
Anticodon
Anticodon
loop
Fig. 1. (a) Cloverleaf secondary structure of tRNA; (b) tertiary structure of tRNA (from
Anticodon
Genetics: a Molecular Approach, second edition, T.A. Brown, Kluwer Academic Publishers,
Promoter
tRNA gene
tRNA gene
5!
3! DNA
Transcription
5!
3! Pre-tRNA
RNA folding
RNA processing
(cleavage and trimming by RNases)
tRNA
tRNA
GeneGc
Code
The
geneGc
code
is
the
rules
that
specify
how
the
nucleoGde
sequence
of
an
mRNA
is
translated
into
the
amino
acid
sequence
of
a
polypepGde.
The
nucleoGde
sequence
is
read
as
triplets
called
codons.
The
codons
UAG,
UGA
and
UAA
do
not
specify
amino
acids
and
are
called
terminaGon
codons
or
Stop
codons.
AUG
codes
for
methionine
and
also
acts
as
an
iniGaGon
(Start)
codon.
Codon sequence
1st base
3rd base
2nd base
(5!end)
Fig. 1.
(3!end)
U
Phe
Phe
Leu
Leu
Leu
Leu
Leu
Leu
Ile
Ile
Ile
Met
Val
Val
Val
Val
Ser
Ser
Ser
Ser
Pro
Pro
Pro
Pro
Thr
Thr
Thr
Thr
Ala
Ala
Ala
Ala
A
Tyr
Tyr
Stop
Stop
His
His
Gln
Gln
Asn
Asn
Lys
Lys
Asp
Asp
Glu
Glu
G
Cys
Cys
Stop
Trp
Arg
Arg
Arg
Arg
Ser
Ser
Arg
Arg
Gly
Gly
Gly
Gly
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
TranslaGon
in
Prokaryotes
During
translaGon
the
mRNA
is
read
in
a
5
to
3
direcGon
and
protein
is
made
in
an
N-terminal
to
C-terminal
direcGon.
TranslaGon
relies
upon
aminoacyl-tRNAs
that
carry
specic
amino
acids
and
recognize
the
corresponding
codons
in
mRNA
by
anGcodon
codon
base-pairing.
TranslaGon
takes
place
in
three
phases;
iniGaGon,
elongaGon
and
terminaGon.
1111
Each
tRNA
molecule
has
a
2
3
cloverleaf
secondary
structure
4
5
consisGng
of
three
stem
loops,
6
one
of
which
bears
the
7
anGcodon
at
its
e89 nd.
The
amino
acid
i10111
s
covalently
1
bound
to
the
3
O23 H
group
at
the
4
3
end
by
aminoacyl
synthetase
5
to
form
aminoacyl-tRNA.
6
7
The
reacGon,
called
amino
acid
8
9
acGvaGon,
occurs
in
two
steps
20111
1 to
form
an
and
requires
ATP
2
3
intermediate,
aminoacyl-
4
adenylate.
5
Amino
acid
5"
UCC
6
7
8
9
30111
1
2
3
NH2
I
CH2
I
C!0
I
O
I
A
C
C
Anticodon
Fig. 1.
Structure of an aminoacyl-tRNA.
TerminaGon
The
appearance
of
a
UAA
or
UAG
terminaGon
(stop)
codon
in
the
A
site
causes
release
factor
RF1
to
bind
whereas
RF2
recognizes
UGA.
The
release
factors
trigger
pepGdyl
transferase
to
transfer
the
polypepGde
to
a
water
molecule
instead
of
to
aminoacyl-tRNA.
The
polypepGde,
mRNA,
and
free
tRNA
leave
the
ribosome
and
the
ribosome
dissociates
into
its
subunits
ready
to
begin
a
new
round
of
translaGon.
Promoter
Transcription unit
5
3
3
5
Start point
RNA polymerase
DNA
1
5
3
Unwound
DNA
3
5
Template strand of
RNA DNA
transcript
2
Rewound
RNA
5
3
3
5
3
5
RNA
transcript
3 Termination. Eventually, the RNA
transcript is released, and the
polymerase detaches from the DNA.
5
3
3
5
5
Completed RNA
transcript
Point
mutaGons
Point
mutaGons
involve
alteraGons
in
the
structure
or
locaGon
of
a
single
gene.
Generally,
only
one
or
a
few
base
pairs
are
involved.
Point
mutaGons
can
signcantly
aect
protein
structure
and
funcGon
Point
mutaGons
may
be
caused
by
physical
damage
to
the
DNA
from
radiaGon
or
chemicals,
or
may
occur
spontaneously
Point
mutaGons
are
oben
caused
by
mutagens
Mutagens
Mutagens
are
chemical
or
physical
agents
that
interact
with
DNA
to
cause
mutaGons.
Physical
agents
include
high-energy
radiaGon
like
X-rays
and
ultraviolet
light
Chemical
mutagens
fall
into
several
categories.
Chemicals
that
are
base
analogues
that
may
be
subsGtuted
into
DNA,
but
they
pair
incorrectly
during
DNA
replicaGon.
Interference
with
DNA
replicaGon
by
inserGng
into
DNA
and
distorGng
the
double
helix.
Chemical
changes
in
bases
that
change
their
pairing
properGes.
Viral Mutagens
Point MutaGon
C T
G U A
C A
mRNA
mRNA
G A
Normal hemoglobin
Glu
Sickle-cell hemoglobin
Val
SubsGtuGons
A
base-pair
subsGtuGon
is
the
replacement
of
one
nucleoGde
and
its
partner
with
another
pair
of
nucleoGdes
Silent
-
changes
a
codon
but
codes
for
the
same
amino
acid
Missense
-
subsGtuGons
that
change
a
codon
for
one
amino
acid
into
a
codon
for
a
dierent
amino
acid
Nonsense
-subsGtuGons
that
change
a
codon
for
one
amino
acid
into
a
stop
codon
Wild type
mRNA
Protein
A U G
Met
A A G U U U G G C U A A
Lys
Phe
Gly
3
Stop
Amino end
Carboxyl end
Base-pair substitution
No effect on amino acid sequence
U instead of C
A U G A A G U U U G G U U A A
Met
Lys
Missense
Phe
Gly
Stop
A instead of G
A U G A A G U U U A G U U A A
Met
Nonsense
Lys
Phe
Ser
Stop
U instead of A
A U G U A G U U U G G C U A A
Met
Stop
InserGons
a
nd
D
eleGons
InserGons
and
deleGons
A U GA A GU U U GG C U A A
Met
Lys
Gly
Phe
Stop
Amino end
Carboxyl end
Base-pair insertion or deletion
Frameshift causing immediate nonsense
Extra U
AU GU A AG U U U G GC U A
Met
Stop
Frameshift causing
extensive missense
U Missing
A U G A A GU U G G C U A A
Met
Lys
Leu
Ala
A A G Missing
A U G U U U G G C U A A
Met
Phe
Gly
Stop
5-TTT
TTT
TTT
TTT
TTT
TTT
TTT
TTG
CTG
GTT
CAA
GGG
CTT
TAT
TCC
ATC
TCT
CTC
GGT
GCA
AGA
GGC
GGC
GGG
TGG
GGG
GCT
GCC
TGC
GGG
CTG
CGT
CTA
GTT
GCA
GTA
GTT
CTC
CAG
CTG
GTA
GAG
GGA
GCA
GAT
GCT
GGT
ACA
GCA
TTG
GTT
CCC
AAT
GCC
CAG
CTT
TTT
GAA
GGG
CCC
CTC
CAG
GGC
CCA
GGG
CTT
CAG
GTT
GTC
CTG
CAC
CAA
GGG
CCC
CCC
CCA
ATT
CCC
CCT
GCC
CCA
CCT
GGA-3