Transcript Nucleic Acids: RNA and chemistry

Nucleic Acids:
DNA, RNA and chemistry
Andy Howard
Introductory Biochemistry
7 October 2010
Biochemistry:Nucleic Acids II
10/07/2010
DNA & RNA
structure & function
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DNA and RNA are dynamic molecules,
but understanding their structural realities
helps us understand how they work
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What we’ll discuss
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DNA structure
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Characterizations
B, A, and Z-DNA
Dynamics
Function
RNA:
structure & types
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mRNA
tRNA
rRNA
Small RNAs
DNA & RNA
Hydrolysis
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alkaline
RNA, DNA
nucleases
Restriction
enzymes
DNA & RNA
dynamics and
density
measurements
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DNA secondary structures
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If double-stranded DNA were simply a straightlegged ladder:
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Base pairs would be 0.6 nm apart
Watson-Crick base-pairs have very uniform
dimensions because the H-bonds are fixed lengths
But water could get to the apolar bases
So, in fact, the ladder gets twisted into a helix.
The most common helix is B-DNA, but there are
others. B-DNA’s properties include:
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Sugar-sugar distance is still 0.6 nm
Helix repeats itself every 3.4 nm, i.e. 10 bp
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Properties of B-DNA
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Spacing between base-pairs
along helix axis = 0.34 nm
10 base-pairs per full turn
So: 3.4 nm per full turn is pitch
length
Major and minor grooves, as
discussed earlier
Base-pair plane is almost
From Molecular
perpendicular to helix axis
Biology web-book
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Major groove in B-DNA
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H-bond between adenine
NH2 and thymine ring
C=O
H-bond between cytosine
amine and guanine ring
C=O
Wide, not very deep
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Minor groove in
B-DNA
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H-bond between
adenine ring N and
thymine ring NH
H-bond between
guanine amine and
cytosine ring C=O
Narrow but deep
From Berg et al.,
Biochemistry
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Cartoon of
AT pair in
B-DNA
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Cartoon
of CG pair
in B-DNA
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What holds duplex
B-DNA together?
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H-bonds (but just barely)
Electrostatics: Mg2+  –PO4-2
van der Waals interactions
 - interactions in bases
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Solvent exclusion
Recognize role of grooves in defining
DNA-protein interactions
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Helical twist
(fig. 11.9a)
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Rotation about the
backbone axis
Successive basepairs rotated with
respect to each
other by ~ 32º
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Propeller
twist
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Improves overlap of
hydrophobic
surfaces
Makes it harder for
water to contact the
less hydrophilic
parts of the
molecule
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A-DNA (figs. 11.10)
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In low humidity this forms naturally
Not likely in cellular duplex DNA,
but it does form in duplex RNA &
DNA-RNA hybrids because the
2’-OH gets in the way of B-RNA
Broader
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2.46 nm per full turn
11 bp to complete a turn
Base-pairs are not
perpendicular to helix axis:
tilted 19º from perpendicular
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Z-DNA (figs.11.10)
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Forms in alternating Py-Pu
sequences and
occasionally in
PyPuPuPyPyPu, especially
if C’s are methylated
Left-handed helix rather
than right
Bases zigzag across the
groove
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Getting from B to Z
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Can be accomplished without
breaking bonds
… even though purines have their
glycosidic bonds flipped (anti ->
syn) and the pyrimidines are
flipped altogether!
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Summaries of A, B, Z DNA
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DNA is dynamic
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Don’t think of these diagrams as static
The H-bonds stretch and the torsions
allow some rotations, so the ropes can
form roughly spherical shapes when not
constrained by histones
Shape is sequence-dependent, which
influences protein-DNA interactions
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What does DNA do?
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Serve as the storehouse and the propagator of
genetic information:
That means that it’s made up of genes
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Some code for mRNAs that code for protein
Others code for other types of RNA
Genes contain non-coding segments (introns)
But it also contains stretches that are not parts of
genes at all and are serving controlling or
structural roles
Avoid the term junk DNA!
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Ribonucleic acid
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We’re done with DNA for the moment.
Let’s discuss RNA.
RNA is generally, but not always, singlestranded
The regions where localized base-pairing
occurs (local double-stranded regions)
often are of functional significance
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RNA physics & chemistry
RNA molecules vary widely in size, from a few bases in
length up to 10000s of bases
 There are several types of RNA found in cells
Type
% %turnSize, Partly Role
RNA
over
bases DS?
mRNA
3
25
50-104 no
protein template
tRNA
15
21
55-90 yes
aa activation
rRNA
80
50
102-104 no
transl. catalysis &
scaffolding
sRNA
2
4
15-103 ?
various
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Messenger RNA
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mRNA: transcription vehicle
DNA 5’-dAdCdCdGdTdAdTdG-3’
RNA 3’- U G G C A U A C-5’
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typical protein is ~500 amino acids;
3 mRNA bases/aa: 1500 bases (after splicing)
Additional noncoding regions (see later) brings it
up to ~4000 bases =
4000*300Da/base=1,200,000 Da
Only about 3% of cellular RNA but instable!
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Relative quantities
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Note that we said there wasn’t much
mRNA around at any given moment
The amount synthesized is much
greater because it has a much shorter
lifetime than the others
Ribonucleases act more avidly on it
We need a mechanism for eliminating it
because the cell wants to control
concentrations of specific proteins
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mRNA processing in Eukaryotes
Genomic DNA
Unmodified mRNA produced therefrom
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# bases (unmodified mRNA) =
# base-pairs of DNA in the gene…
because that’s how transcription works
BUT the number of bases in the unmodified
mRNA > # bases in the final mRNA that actually
codes for a protein
SO there needs to be a process for getting rid of
the unwanted bases in the mRNA: that’s what
splicing is!
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Splicing: quick summary
Genomic DNA
transcription
Unmodified mRNA produced therefrom
exon
intron
exon
intron
exon
intron
splicing
exon
exon
(Mature transcript)
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exon
translation
Typically the initial eukaryotic message
contains roughly twice as many bases as the
final processed message
Spliceosome is the nuclear machine
(snRNAs + protein) in which the introns are
removed and the exons are spliced together
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Heterogeneity via
spliceosomal flexibility
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Specific RNA sequences in the initial
mRNA signal where to start and stop
each intron, but with some flexibility
That flexibility enables a single gene to
code for multiple mature RNAs and
therefore multiple proteins
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Transfer RNA
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tRNA: tool for engineering protein
synthesis at the ribosome
Each type of amino acid has its
own tRNA, responsible for
positioning the correct aa into the
growing protein
Roughly T-shaped or Y-shaped
molecules; generally 55-90 bases
long
15% of cellular RNA
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Phe tRNA
PDB 1EVV
76 bases
yeast
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Secondary and Tertiary
Structure of tRNA
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Extensive H-bonding creates four double
helical domains, three capped by loops, one
by a stem
Only one tRNA structure (alone) is known
Phenylalanine tRNA is "L-shaped"
Many non-canonical bases found in tRNA
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tRNA
structure:
overview
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Amino acid
linkage to
acceptor stem
Amino acids are linked to the 3'-OH
end of tRNA molecules by an
ester bond formed between the
carboxyl group of the amino acid
and the 3'-OH of the terminal
ribose of the tRNA.
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Yeast phetRNA
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Note
nonstandard
bases and
cloverleaf
structure
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Ribosomal RNA
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rRNA: catalyic and scaffolding
functions within the ribosome
Responsible for ligation of new
amino acid (carried by tRNA)
onto growing protein chain
Can be large: mostly 500-3000
bases
a few are smaller (150 bases)
Very abundant: 80% of cellular
RNA
Relatively slow turnover
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23S rRNA
PDB 1FFZ
602 bases
Haloarcula
marismortui
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Small RNA
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sRNA: few bases / molecule
often found in nucleus; thus it’s often
called small nuclear RNA, snRNA
Involved in various functions, including
processing of mRNA in the spliceosome
Protein Prp31
Some are catalytic
complexed to U4
Typically 20-1000 bases
snRNA
Not terribly plentiful: ~2 % of total RNA
PDB 2OZB
33 bases +
85kDa
heterotetramer
Human
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iClicker quiz
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1. Shown is the lactim
form of which nucleic
acid base?
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Uracil
Guanine
Adenine
Thymine
None of the above
HN
O
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N
OH
lactim
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iClicker quiz #2
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Suppose someone reports that he has
characterized the genomic DNA of an
organism as having 29% A and 22% T. How
would you respond?
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(a) That’s a reasonable result
(b) This result is unlikely because [A] ~ [T] in
duplex DNA
(c) That’s plausible if it’s a bacterium, but not if
it’s a eukaryote
(d) none of the above
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Unusual bases in RNA
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mRNA, sRNA mostly ACGU
rRNA, tRNA have some odd ones
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Other small RNAs
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21-28 nucleotides
Target RNA or DNA through
complementary base-pairing
Several types, based on function:
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Small interfering RNAs (q.v.)
microRNA: control developmental timing
Small nucleolar RNA: catalysts that (among
other things) create the oddball bases
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
snoRNA77
courtesy Wikipedia
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siRNAs and gene
silencing
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Small interfering RNAs block specific
protein production by base-pairing to
complementary seqs of mRNA to form
dsRNA
DS regions get degraded & removed
This is a form of gene silencing or RNA
interference
RNAi also changes chromatin structure
and has long-range influences on
expression
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Viral p19
protein
complexed to
human 19-base
siRNA
PDB 1R9F
1.95Å
17kDa protein
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Do the differences between
RNA and DNA matter? Yes!
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DNA has deoxythymidine, RNA has uridine:
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cytidine spontaneously degrades to uridine
dC spontaneously degrades to dU
The only dU found in DNA is there because
of degradation: dT goes with dA
So when a cell finds dU in its DNA, it knows
it should replace it with dC or else
synthesize dG opposite the dU instead of dA
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Ribose vs. deoxyribose
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Presence of -OH on 2’ position makes
the 3’ position in RNA more
susceptible to nonenzymatic cleavage
than the 3’ in DNA
The ribose vs. deoxyribose distinction
also influences enzymatic degradation
of nucleic acids
I can carry DNA in my shirt pocket, but
not RNA
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Backbone hydrolysis of
nucleic acids in base
(fig. 10.29)
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Nonenzymatic hydrolysis in base occurs
with RNA but not DNA, as just mentioned
Reason: in base, RNA can form a specific
5-membered cyclic structure involving
both 3’ and 2’ oxygens
When this reopens, the backbone is
cleaved and you’re left with a mixture of
2’- and 3’-NMPs
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Why alkaline hydrolysis works
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Cyclic phosphate intermediate stabilizes
cleavage product
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The cyclic intermediate
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Hydroxyl or water
can attack fivemembered Pcontaining ring on
either side and
leave the –OP on
2’ or on 3’.
O
H
N
O
O
O-
O
P
N
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O
OO
O
P
O-
O
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Consequences
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So RNA is considerably less stable
compared to DNA, owing to the formation
of this cyclic phosphate intermediate
DNA can’t form this because it doesn’t
have a 2’ hydroxyl
In fact, deoxyribose has no free
hydroxyls!
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Enzymatic cleavage of oligoand polynucleotides
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Enzymes are phosphodiesterases
Could happen on either side of the P
3’ cleavage is a-site; 5’ is b-site.
Endonucleases cleave somewhere on
the interior of an oligo- or polynucleotide
Exonucleases cleave off the terminal
nucleotide
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An a-specific
exonuclease
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A b-specific
exonuclease
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Specificity in nucleases
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Some cleave only RNA, others only DNA,
some both
Often a preference for a specific base or
even a particular 4-8 nucleotide
sequence (restriction endonucleases)
These can be used as lab tools, but they
evolved for internal reasons
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Enzymatic RNA
hydrolysis
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Ribonucleases operate through
a similar 5-membered ring
intermediate: see fig. 19.29 for
bovine RNAse A:
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His-119 donates proton to 3’-OP
His-12 accepts proton from 2’-OH
Cyclic intermediate forms with
cleavage below the phosphate
Ring collapses, His-12 returns
proton to 2’-OH, bases restored
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PDB
1KF8
13.6 kDa
monomer
bovine
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Variety of nucleases
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Restriction endonucleases
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Evolve in bacteria as antiviral tools
“Restriction” because they restrict the
incorporation of foreign DNA into the bacterial
chromosome
Recognize and bind to specific palindromic DNA
sequences and cleave them
Self-cleavage avoided by methylation
Types I, II, III: II is most important
I and III have inherent methylase activity; II has
methylase activity in an attendant enzyme
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What do we mean by
palindromic?
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In ordinary language, it means a phrase that
reads the same forward and back:
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Madam, I’m Adam. (Genesis 3:20)
Eve, man, am Eve.
Sex at noon taxes.
Able was I ere I saw Elba. (Napoleon)
A man, a plan, a canal: Panama!
(T. Roosevelt)
With DNA it means the double-stranded
sequence is identical on both strands
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Palindromic DNA
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G-A-A-T-T-C
Single strand isn’t symmetric: but the
combination with the complementary
strand is:
G-A-A-T-T-C
C-T-T-A-A-G
These kinds of sequences are the
recognition sites for restriction
endonucleases. This particular
hexanucleotide is the recognition
sequence for EcoRI.
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Cleavage by restriction
endonucleases
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Breaks can be
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cohesive (if they’re off-center within the sequence) or
non-cohesive (blunt) (if they’re at the center)
EcoRI leaves staggered 5’-termini: cleaves
between initial G and A
PstI cleaves CTGCAG between A and G, so it
leaves staggered 3’-termini
BalI cleaves TGGCCA in the middle: blunt!
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iClicker question 3:
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3. Which of the following is a potential
restriction site?
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(a) ACTTCA
(b) AGCGCT
(c) TGGCCT
(d) AACCGG
(e) none of the above.
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Example for EcoRI
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5’-N-N-N-N-G-A-A-T-T-C-N-N-N-N-3’
3’-N-N-N-N-C-T-T-A-A-G-N-N-N-N-5’
Cleaves G-A on top, A-G on bottom:
5’-N-N-N-N-GA-A-T-T-C-N-N-N-N-3’
3’-N-N-N-N-C-T-T-A-AG-N-N-N-N-5’
Protruding 5’ ends:
5’-N-N-N-N-G
A-A-T-T-C-N-N-N-N-3’
3’-N-N-N-N-C-T-T-A-A
G-N-N-N-N-5’
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How often?
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4 types of bases
So a recognition site that is 4 bases long
will occur once every 44 = 256 bases on
either strand, on average
6-base site: every 46= 4096 bases, which
is roughly one gene’s worth
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EcoRI
structure
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Dimeric structure
enables recognition of
palindromic sequence
 sandwich in each
monomer
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EcoRI pre-recognition
complex
PDB 1CL8
57 kDa dimer + DNA
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Methylases
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
A typical bacterium protects
its own DNA against
HhaI methyltransferase
cleavage by its restriction
PDB 1SVU
endonucleases by
2.66Å; 72 kDa dimer
methylating a base in the
restriction site
Methylating agent is
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
generally Sadenosylmethionine
Structure
courtesy
steve.gb.com
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The biology problem
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How does the bacterium mark its own DNA
so that it does replicate its own DNA but not
the foreign DNA?
Answer: by methylating specific bases in its
DNA prior to replication
Unmethylated DNA from foreign source
gets cleaved by restriction endonuclease
Only the methylated DNA survives to be
replicated
Most methylations are of A & G,
but sometimes C gets it too
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How this works
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When an unmethylated specific
sequence appears in the DNA, the
enzyme cleaves it
When the corresponding methylated
sequence appears, it doesn’t get cleaved
and remains available for replication
The restriction endonucleases only bind
to palindromic sequences
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Use of restriction enzymes
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Nature made these to protect bacteria; we
use them to cleave DNA in analyzable ways
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Similar to proteolytic digestion of proteins
Having a variety of nucleases means we can get
fragments in multiple ways
We can amplify our DNA first
Can also be used in synthesis of inserts that
we can incorporate into plasmids that enable
us to make appropriate DNA molecules in
bacteria
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Intercalating agents
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Generally: aromatic compounds that can
form -stack interactions with bases
Bases must be forced apart to fit them in
Results in an almost ladderlike structure
for the sugar-phosphate backbone locally
Conclusion: it must be easy to do local
unwinding to get those in!
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Instances
of intercalators
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Denaturing and Renaturing DNA
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See Figure 11.17
When DNA is heated to 80+ degrees
Celsius, its UV absorbance increases by
30-40%
This hyperchromic shift reflects the
unwinding of the DNA double helix
Stacked base pairs in native DNA absorb
less light
When T is lowered, the absorbance drops,
reflecting the re-establishment of stacking
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Heat denaturation
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Figure 11.14
Heat denaturation of DNA from various sources, so-called
melting curves. The midpoint of the melting curve is
defined as the melting temperature, Tm.
(From Marmur, J., 1959. Nature 183:1427–1429.)
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GC content
vs. melting
temp
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High salt and
no chelators
raises the
melting
temperature
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How else can we melt DNA?
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High pH deprotonates the bases so the Hbonds disappear
Low pH hyper-protonates the bases so the
H-bonds disappear
Alkalai is better: it doesn’t break the
glycosidic linkages
Urea, formamide make better H-bonds
than the DNA itself so they denature DNA
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What happens if we
separate the strands?
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We can renature the DNA into a double
helix
Requires re-association of 2 strands:
reannealing
The realignment can go wrong
Association is 2nd-order, zippering is first
order and therefore faster
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Steps in denaturation
and renaturation
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Rate depends on complexity


The more complex DNA is, the longer it
takes for nucleation of renaturation to
occur
“Complex” can mean “large”, but
complexity is influenced by sequence
randomness: poly(AT) is faster than a
random sequence
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Second-order kinetics





Rate of association: -dc/dt = k2c2
Boundary condition is fully denatured
concentration c0 at time t=0:
c / c0 = (1+k2c0t)-1
Half time is t1/2 = (k2c0)-1
Routine depiction: plot c0t vs. fraction
reassociated (c /c0) and find the halfway
point.
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Typical c0t curves
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Hybrid
duplexes

We can associate DNA
from 2 species



Closer relatives hybridize
better
Can be probed one gene
at a time
DNA-RNA hybrids can
be used to fish out
appropriate RNA
molecules
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GC-rich DNA is denser




DNA is denser than RNA or protein, period,
because it can coil up so compactly
Therefore density-gradient centrifugation
separates DNA from other cellular
macromolecules
GC-rich DNA is 3% denser than AT-rich
Can be used as a quick measure of GC
content
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Density
as
function
of GC
content
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