Download pyrimidine

Nucleic Acids:
Chemistry & Structure
Andy Howard
Introductory Biochemistry
8 October 2009
Biochemistry:Nucleic Acids I
10/08/2009
What we’ll discuss

Nucleic acid
chemistry



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Pyrimidines: C, U, T
Purines: A, G
Nucleosides &
nucleotides
Oligo- and
polynucleotides

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DNA duplexes and
helicity
DNA sequencing
DNA secondary
structure: A, B, Z
Folding kinetics
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Chemistry Nobel Prize 2009
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Structural studies of the ribosome
Venki Ramakrishnan, LMB Cambridge
Thomas Steitz, HHMI Yale University
Ada Yonath, Weizmann Institute
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10/08/2009 Biochemistry:Nucleic Acids I
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p. 3 of 57
6
5
Pyrimidines
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N
4
2
N
3
Single-ring nucleic acid bases
pyrimidine
6-atom ring; always two nitrogens in the ring,
meta to one another
Based on pyrimidine, although pyrimidine itself
is not a biologically important molecule
Variations depend on oxygens and nitrogens
attached to ring carbons
Tautomerization possible
Note line of symmetry in pyrimidine structure
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1
p. 4 of 57
H
N
O
Uracil and thymine
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Uracil is a simple dioxo
derivative of pyrimidine:
2,4-dioxopyrimidine
Thymine is 5-methyluracil
Uracil is found in RNA;
Thymine is found in DNA
We can draw other
tautomers where we move
the protons to the oxygens
10/08/2009 Biochemistry:Nucleic Acids I
O
HN
uracil
HN
O
N
H
thymine
p. 5 of 57
O
H
N
O
NH
O
Tautomers


Lactam and
Lactim forms
Getting these right
was essential to
Watson & Crick’s
development of
the DNA double
helical model
HN
O
uracil - lactam
H
HN
O
N
O
uracil - lactim
HN
N
H
thymine - lactam
10/08/2009 Biochemistry:Nucleic Acids I
O
O
N
thymine - lactim
p. 6 of 57
OH
H
N
O
Cytosine
NH2
N
cytosine

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
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This is 2-oxo,4-aminopyrimidine
It’s the other pyrimidine base found in
DNA & RNA
Spontaneous deamination (CU)
Again, other tautomers can be drawn
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Cytosine:
amino and imino forms

Again, this tautomerization needs to be
kept in mind
H
N
O
NH2
H
N
O
N
cytosine -amino form
NH
N
cytosine -imino form
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7
6
5
1N
Purines


8
4
2
N
N
3

H
N
9
Derivatives of purine; again, the purine
root molecule isn’t biologically
important
Six-membered ring looks a lot like
pyrimidine
Numbering works somewhat
differently: note that the glycosidic
bonds will be to N9, whereas it’s to
N1 in pyrimidines
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Adenine
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This is 6-aminopurine
Found in RNA and DNA
We’ve seen how important adenosine
and its derivatives are in metabolism
Tautomerization happens here too
NH
NH2
H
N
N
N
N
adenine - amino form
H
N
HN
N
N
adenine - imino form
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Guanine
This is 2-amino-6-oxopurine
 Found in RNA, DNA
 Lactam, lactim forms
OH

O
H
N
H
N
N
HN
H2N
N
guanine - lactam
N
H2N
N
N
guanine - lactim
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Other natural purines
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Hypoxanthine and xanthine
are biosynthetic precursors
of A & G
Urate is important in
nitrogen excretion
pathways
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Tautomerization and H-bonds
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Lactam forms predominate at neutral pH
This influences which bases are H-bond
donors or acceptors
Amino groups in C, A, G make H-bonds
So do ring nitrogens at 3 in pyrimidines
and 1 in purines
… and oxygens at 4 in U,T, 2 in C, 6 in G
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O
HO
Nucleosides
NR1R2
OH
HO
N-glycoside of ribofuranose

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As mentioned in ch. 8, these are
glycosides of the nucleic acid bases
Sugar is always ribose or deoxyribose
Connected nitrogen is:
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
N1 for pyrimidines (on 6-membered ring)
N9 for purines (on 5-membered ring)
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Pyrimidine nucleosides

Drawn here in amino and lactam forms
OH
OH
HO
HO
OH
O
N
H2N
N
OH
O
O
N
O
N
H
cytidine
10/08/2009 Biochemistry:Nucleic Acids I
O
uridine
p. 15 of 57
OH
Pyrimidine
deoxynucleosides
OH
H
H
OH
O
N
O
N
H
OH
O
OH
2'-deoxyuridine
O
N
H
OH
N
O
2'-deoxythymidine
O
N
H2N
O
N
O
deoxycytidine
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A tricky nomenclature issue


Remember that thymidine and its
phosphorylated derivatives ordinarily
occur associated with deoxyribose, not
ribose
Therefore many people leave off the
deoxy- prefix in names of thymidine and
its derivatives: it’s usually assumed.
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p. 17 of 57
Purine nucleosides

Drawn in amino and lactam forms
NH2
O
N
N
N
HN
N
N
H2N
N
N
O
O
HO
HO
OH
OH
HO
adenosine
10/08/2009 Biochemistry:Nucleic Acids I
HO
guanosine
p. 18 of 57
Purine deoxynucleosides
O
NH2
N
N
HN
N
N
H2N
N
N
N
O
O
OH
OH
HO
deoxyadenosine
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HO
deoxyguanosine
p. 19 of 57
Conformations around the
glycosidic bond
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Rotation of the base around the glycosidic bond
is sterically hindered
In the syn conformation there would be some
interference between the base and the 2’hydroxyl of the sugar
Therefore pyrimidines are always anti, and
purines are usually anti
Furanose and base rings are roughly
perpendicular
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Glycosidic bonds

This illustrates the
roughly perpendicular
positionings of the
base and sugar rings
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Solubility of nucleosides and
lability of glycosidic linkages
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The sugar makes nucleosides more
soluble than the free bases
Nucleosides are generally stable to basic
hydrolysis at the glycosidic bond
Acid hydrolysis:

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Purines: glycosidic bond fairly readily
hydrolyzed
Pyrimidines: resistant to acid hydrolysis
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Chirality in nucleic acids
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Bases themselves are achiral
Four asymmetric centers in
ribofuranose, counting the glycosidic
bond.
Three in deoxyribofuranose
Glycosidic bond is one of those 4 or 3.
Same for nucleotides:
phosphates don’t add asymmetries
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Monophosphorylated
nucleosides
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NH2
N
N
N
We have specialized names for HO
the 5’-phospho derivatives of the
nucleosides, i.e. the nucleoside
monophosphates:
They are nucleotides
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N
O
OO
P
HO
O
adenylate
Adenosine 5’-monophosphate =
AMP = adenylate
GMP = guanylate
CMP = cytidylate
UMP = uridylate
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O-
p. 24 of 57
pKa’s for base N’s and PO4’s
Nucleotide pKa base-N pK1 of PO4 pK2 of PO4
5’-AMP
3.8(N-1)
0.9
6.1
5’-GMP
9.4 (N-1)
0.7
6.1
2.4 (N-7)
5’-CMP
4.5 (N-3)
0.8
6.3
5’-UMP
9.5 (N-3)
1.0
6.4
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UV absorbance

These aromatic rings absorb around
260
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Deoxynucleotides

O
Similar nomenclature
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dAMP =
deoxyadenylate
dGMP =
deoxyguanylate
dCMP =
deoxycytidylate
dTTP (= TTP) =
deoxythymidylate =
thymidylate
N
HN
H2N
N
10/08/2009 Biochemistry:Nucleic Acids I
N
O
OO
OP
HO
O
deoxyguanylate
p. 27 of 57
Di and triphosphates

Phosphoanhydride bonds link second and
perhaps third phosphates to the 5’-OH on
the ribose moiety
O
N
O
H2N
O
O
O
P
P
P
O
N
O
O-
O
O-
OH
O-
Mg2+
OH
HO
cytidine triphosphate
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Cyclic
phosphodiesters
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3’ and 5’ hydroxyls are both involved
in -O-P-O bonds
cAMP and cGMP are the important ones
(see earlier in the course!)
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Oligomers and Polymers
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Monomers are nucleotides or
deoxynucleotides
Linkages are phosphodiester linkages
between 3’ of one ribose and 5’ of the next
ribose
It’s logical to start from the 5’ end for
synthetic reasons
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Typical DNA dinucleotide
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Various notations: this is pdApdCp
Leave out the p’s if there’s a lot of them!
-O
OP
O
O
O
-O
N
O-
N
P
O
O
O
O
N
-O
P
O
HN
O
NH2
O
N
O
10/08/2009 Biochemistry:Nucleic Acids I
N
NH2
p. 31 of 57
DNA structure
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Many years of careful
experimental work enabled
fabrication of double-helical
model of double-stranded
DNA
Explained [A]=[T], [C]=[G]
Specific H-bonds stabilize
double-helical structure:
see fig. 10.20
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What does double-stranded
DNA really look like?


Picture on previous slide emphasizes
only the H-bond interactions; it ignores
the orientation of the sugars, which are
actually tilted relative to the helix axis
Planes of the bases are almost
perpendicular to the helical axes on both
sides of the double helix
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Sizes (cf fig. 10.20, 11.7)
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Diameter of the double helix: 2.37nm
Length along one full turn:
10.4 base pairs = pitch = 3.40nm
Distance between stacked base pairs =
rise = 0.33 nm
Major groove is wider and shallower;
minor groove is narrower and deeper
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What stabilizes this?

Variety of stabilizing
interactions
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Stacking of base pairs
Hydrogen bonding between
base pairs
Hydrophobic effects (burying
bases, which are less polar)
Charge-charge interactions:
phosphates with Mg2+ and
cationic proteins
10/08/2009 Biochemistry:Nucleic Acids I
Courtesy
dnareplication.info
p. 35 of 57
How close to instability is it?
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Pretty close.
Heating DNA makes it melt: fig. 11.14
pH > 10 separates strands too
The more GC pairs, the harder it is to
melt DNA thermally

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Weaker stacking interactions in A-T
One more H-bond per GC than per AT
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iClicker quiz, 1st question

1. What positions of a pair of aromatic
rings leads to stabilizing interactions?
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(a) Parallel to one another
(b) Perpendicular to one another
(c) At a 45º angle to one another
(d) Both (a) and (b)
(e) All three: (a), (b), and ( c)
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iClicker question 2

2. Which has the highest molecular mass
among the compounds listed?
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(a) cytidylate
(b) thymidylate
(c) adenylate
(d) adenosine triphosphate
(e) they’re all the same MW
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p. 38 of 57
Base composition for DNA

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As noted, [A]=[T], [C]=[G] because of
base pairing
[A]/[C] etc. not governed by base pairing
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Can vary considerably (table 10.3)
E.coli : [A], [C] about equal
Mycobacterium tuberculosis: [C] > 2*[A]
Mammals: [C] < 0.74*[A]
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Supercoiling

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Refers to levels of organization of DNA
beyond the immediate double-helix
We describe circular DNA as relaxed if
the closed double helix could lie flat
It’s underwound or overwound if the ends
are broken, twisted, and rejoined.
Supercoils restore 10.4 bp/turn relation
upon rejoining
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Supercoiling
and flat DNA
Diagram courtesy SIU Carbondale
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Sanger dideoxy method

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Incorporates DNA replication as an analytical
tool for determining sequence
Uses short primer that attaches to the 3’ end of
the ssDNA, after which a specially engineered
DNA polymerase
Each vial includes one dideoxyXTP and 3
ordinary dXTPs; the dideoxyXTP will be
incorporated but will halt synthesis because the
3’ position is blocked.
See figs. 11.3 & 11.4 for how these are read out
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Automating dideoxy
sequencing


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Laser fluorescence detection allows for
primer identification in real time
An automated sequencing machine can
handle 4500 bases/hour
That’s one of the technologies that has
made large-scale sequencing projects
like the human genome project possible
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p. 43 of 57
DNA secondary structures

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:


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 perpendicular
to helix axis
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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
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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)


Rotation about the
backbone axis
Successive base-pairs
rotated with respect to
each other by ~ 32º
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Propeller
twist


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 and DNA-RNA hybrids because
the 2’-OH gets in the way of B-RNA
Broader



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)



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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p. 54 of 57
Getting from B to Z


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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p. 55 of 57
DNA is dynamic



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?

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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