1' Introduction to DNA and RNA structure

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1. Introduction to DNA and RNA structure
BIOCHEMISTRY 441, WINTER 2008
View the The DNA Story movie at one of the
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DNA is a polymer of2-deoxyribonucleotides
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RNA is a polymer of ribonucleotides
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RNA is easily hydrolyzed under alkaline conditions
H2O
mixture of 2- and 3- monophosphate derivatives
RNA
shortened RNA
The reaction proceeds through a 2,3-cyclic
monophosphate intermediate. Enzymatic hydrolysis
of RNA by RNase proceeds through a similar
intermediate. Because DNA lacks the 2-OH group,
it is stable under alkaline conditions.
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Why does DNA contain T rather than U?
Cytosine deaminates non-enzymatically to form
uracil. If this happens in DNA, it constitutes a
mutation. Cells have a proof-reading system that
recognizes the error and replaces the U by C.
Deamination of cytosine is of less consequence in
RNA, which is not the permanent repository of
genetic information.
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The phosphate groups of DNA and RNA are
negatively charged
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A phosphodiester group has a pKa of about 1, and
so will always be ionized and negatively charged
under physiological conditions (pH 7). Nucleic
acids require counterions such as Mg2,
polyamines, histones or other proteins to balance
this charge.
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The sugars are always in the b-furanose (cyclic)
form
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The nucleotide base can rotate with respect to
the sugar
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ns/coll/pauling/dna/pictures/franklin-typeBphoto.h
tml
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H-bonds between Watson-Crick base pairs stabilize
a double helix
Base-pairing explains Chargaffs rules for the
base composition of DNA A T G C
It has not escaped our notice that the specific
pairing we have postulated immediately suggests a
possible copying mechanism for the genetic
material. -- J.D. Watson F.H.C. Crick, Nature
171 737 (1953)
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The B-form DNA helix has a diameter of about 20 Å
A base pair is more exposed to the solvent on one
side (the major groove, at the top in these
views) than the other (the minor groove,
bottom).
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B-form DNA consists of a right-handed double
helix with antiparallel strands
3.4 Å per base-pair
34 Å (10 bp) per turn
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These dimensions are for DNA fibers. In
solution, there are 10.5 base-pairs per turn.
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The antiparallel orientation of the two strands
was demonstrated by synthesizing DNA
enzymatically from labeled nucleoside
triphosphates
Kornberg synthesized labeled double-stranded DNA
enzymatically from a-32P-labeled 5-nucleoside
triphosphates and hydrolyzed the DNA with a DNase
that released the 3-mononucleotides. For
example, with labeled ATP and unlabeled TTP, GTP
and CTP, the sequence shown below gives
H2O
DNase
In this example, 2/4 of the Tp and 1/5 of the Gp
are labeled. What would you expect if the two
strands were parallel? (Answer 1/4 of the Tp and
2/5 of the Gp would be labeled.)
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The two strands of the double helix separate
reversibly at high temperatures
The temperature at which this denaturation or
melting occurs depends on the pH and salt
concentration, and increases with the GC content
of the DNA. (The curves drawn here are
schematic.)
If the temperature is lowered, the strands
recombine. The rate of recombination is
inversely proportional to the complexity of the
DNA.
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Double-stranded and single-stranded DNA differ in
their optical absorption at 260 nm
The conjugated p-electron systems of the purine
pyrimidine bases absorb strongly in the UV.
(Thats why UV light is mutagenic and
carcinogenic.)
The absorbance of double-stranded DNA (dsDNA) at
260 nm is less than that of either
single-stranded DNA (ssDNA) or the free bases.
This is called hypochromism.
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Hypochromism results from dipole-dipole
interactions between neighboring bases
The excited states of an interacting pair of
molecules can be described as linear combinations
of the excited states of the individual
molecules. In certain geometries, some of the
absorption strength in the near-UV moves to bands
at higher energies.
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Summary of the main structural features of B-form
DNA

• Right-handed helix
• Two antiparallel strands held together by
  Watson-Crick hydrogen bonds
• Pitch (repeat length) 34 Å (3.4 nm)
• 36o rotation between residues
• Helix diameter of 20 Å (2.0 nm)
• Wide major groove, narrow minor groove
• Chargaffs Rules A T G C
• Charged phosphates
• Bases in anti configuration
• The strands separate at high temperatures
• The solution structure is dynamic

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Hoogsteen pairs can stabilize triplex DNA
structures
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Tautomeric forms of G T can cause mutations due
to mispairing during DNA replication
The ring NH atoms of G and T have pKa values of
about 9. At physiological pH, about 99 of the
base is in the keto form and 1 in the enol form.
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Palindromic sequences (inverted repeats) in DNA
or RNA can form hairpin or cruciform structures
Mirror repeats cannot form these structures.
A palindrome reads the same in either direction
(Radar, Madam, Im Adam).
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Cells contain a variety of types of RNA with
different functions
Eucaryotic cells contain an additional type,
small nuclear RNA (snRNA).
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Most RNA molecules consist of a single strand
that folds back on itself to form double-helical
regions
The loops and hairpins have few or no base-pairs
In RNA, G pairs with C and A pairs with U.
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DNA base sequences can be determined by using DNA
polymerase and dideoxynucleotides
Synthesize DNA enzymatically in the presence of a
small amount of a dideoxynucleotide (e.g., ddTTP)
and larger amounts of ordinary ATP, GTP, CTP and
TTP, using a primer and one strand of the DNA to
be sequenced as a template. DNA polymerase
synthesizes the new strand in the 5 ? 3
direction. The dideoxynucleotide interrupts DNA
synthesis wherever it is incorporated. This
happens at random points where the corresponding
base occurs in the sequence. In this
illustration, each DNA fragment ends in ddT,
which is labeled with a red dye.
synthesis
(continued )
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DNA sequencing can be automated
Repeat the procedure with ddC, ddA and ddG, using
a different fluorescent dye to tag the
dideoxynucleotide in each case. Then mix all the
DNA fragments and separate them according to size
by electrophoresis.
Separating the DNA fragments gives a ladder of
bands, each ending in a particular
dideoxynucleotide at its 3 end and tagged with
the corresponding dye. This procedure has been
automated in machines that can sequence more than
3x106 bases in a month.