Nucleotides and Nucleotides

Chapter 8
Some basics
Nucleic Acid Structure
Nucleic Acid Chemistry
Other functions of Nucleotides

Learning Outcomes
Structure and function of nucleic acids
Draw and ID purine and pyramidine nitrogeneous bases
Draw and understand the mechanism for formation of ring in
pentose ring in nuclotides
Draw the mechanism for formation and cleavage of Nglycosidic bond
ID and draw the mechanism for the formation of
phosphodiester bond which links nucleotides together in DNA
What are the major characteristics of DNA that highlight the
important chemical interactions that impart structure and
functionality

Nucleotides:
S
Building Blocks of Nucleic Acids
DNA = _________________
acid
Deoxyribonucleic
information
Nucleotide sequences in DNA provide _____________
for ______
Amino Acids____ sequence in all proteins in cells
For nucleotide sequence in ______
Section of DNA containing nucleotide information is
Genes
called ____________
Many _________ of genes and DNA molecules in cells
storage
Only known function of DNA is _________
and
______________ of biological information

Nucleotides:
Building Blocks of Nucleic Acids
Ribonucleic acid
RNA = _____________
acid
RNAs have several functions:
____________ RNAs (rRNAs) are part of ribosomes
Ribosomes are where proteins are synthesized
Messenger
_____________
RNAs (mRNAs) carry information
from one or more genes to the ribosome
______________ RNAs (tRNAs) are molecules
which translate mRNA sequence into a specific
amino acid
Other RNAs exist and have other purposes

Definitions:
Nucleotides,
nucleosides and
nucleobases
Nucleotide = ______________,
____________, and ____
Nitrogeneous Base
1
3
Phosphate
to ____
_____________
groups
Pentose (sugar)
Nucleoside = ____________ base and ____________
Nitrogeneous
Nucleobase = ______________
base
Fig 8.1a

Definitions:
D-Ribose and 2deoxy-D-Ribose

D-Ribose has ______

OH group

2 prime

at 2 (and is used in ________)

2-deoxy- D-Ribose has _____ at 2 (and is used in

______)
DNA
Pentose has 1 to 5 numbers to distinguish
_________ the
numbered elements from the ____________ base
Fig 8.1a

b-furanose
(closed 5 membered
ring of pentose)
Know how to draw the sugar

Straight
In solution free forms of __________
_______
(aldehyde) and _______
(b-furanose) exist in _________
Ring
equilibrium

Ribose
RNA contains b-D-_______________
Ribose
DNA contains b-2-________D-____________
Deoxy

Fig 8.3a

Puckered b-furanose
conformations
(endo or exo)

In ribofuranose ring formation 4 of 5 atoms are in

_________ plane
C2
C3
_____
or _____
are either on the same side (______) or
opposite side (_____) the plane relative to C-5
____________
base usually attached to ______
Nitrogenous
C5
__________ group is usually attached to _____
Fig 8.3b

Definitions:
Nitrogenous bases (purine
or pyrimidine)

________
in nitrogeneous bases are not _________
Numbers
primed
Adenine
Purine is parent compound of _________
(A) and
__________
(G)
Guanine
Pyrimidine
Cytosine
_____________
is parent compound of ________(C),
__________
(T) and ________
(U in RNA)
Thymine
Uracil
Bases are __________ compounds (contain N and C)
Fig 8.1b

Purine Nitrogenous bases

Purine has 5 C and 4 N and 4 double bonds

Adenine
___________
has NH2 group at C6
__________ has NH2 group at C2 and ketone (=O) at C6
Bond
All purines are goodHydrogen
________donors
and ____________

Adenine pKa at N1 is 3.8

Guanine pKa at N7 is 2.4

Neutral molecules at pH 7
Fig 8.1b and Fig 8.2

Pyrimidine Nitrogenous bases

Pyrimidine has 4 C and 2 N and 3 double bonds

Cytosine
_________
has NH2 at C4, ketone (=O) at C2 and no db N1 - C2
Methyl Group
C2 & ___
C4 and no db N1 - C2
Thymine has _____
CH3 at C5, (=O) at ___
_______ has ketones (=O) at C2 and C4 and no ____ N1 C2
All pyrimidines are _____ H-bond donors and __________
Acceptors
Cytosine pKa at N3 is 4.5
Neutral molecules at pH 7

Thymine pKa at N3 is 9.5

Fig 8.1b and Fig 8.2

Deoxyribonucleotides
free form at pH 7.0

Draw these out, know which are what, what binds where
Fig 8.4a

Ribonucleotides
free form at pH 7.0

If phosphate group is in a different location, the name

will change
Fig 8.4b

Nomenclature

The type of pentose defines the nucleic acid

(So 2-deoxy-D-ribose is in DNA even if base is uracil)

Nomenclature for Minor Bases

Common names may be used (hypoxanthine for inosine)
Name usually includes the ________
of the ring where the
Number
substitution occurred (but not the ________ where the
substitution occurred)
Number
If the substitution is ________ the ring, then the ________
is indicated along with its connection to the ring (N6)
Indicatin the methyl is attatched to the n on
the 6th carbon

Fig 8.5a

Minor Bases in DNA

Methylation is common change to major bases for DNA
Viral DNA bases can be hydroxymethylated (-CH2OH) or
glucosylated (sugar moiety added to N or C or O group)

Fig 8.5a

Minor Bases in DNA

Changes are often made to the bases after the DNA has
Made / Synthesized
been _______________
Eukaryotes
5methylcytosine is found in ____________
and bacteria
N6-________________ is found in bacteria but not
eukaryotes
markers
Epigenetic ____________
(which are not coded on genes

but which are passed on as the cells replicate) are useful

Foreign
for distinguishing __________
DNA and when to ______

Replication
on gene ___________

Minor Bases in tRNA

Changes are often in the location of ribose attachment
Thiol or methyl groups can also be attached

Fig 8.5b

Minor Bases in RNA

Inosine can be found in the ______
Wobble position of anticodon
for tRNA (wobble position is the third base in the codon
position for an amino acid and allows for _______ than
61 expected tRNA for the number of codons present)
Adenosine
It is made from de-amination of ____________
Provides richer __________ codes
Pseudouridine is ________
in tRNA and rRNA
found
Eukaryotes
Found in _____________
and some eubacteria
Enzymatic
Made from uridine by _______________
isomerization
after RNA ______________
Synthesized
May be used in the _______________
of tRNA structure
Stab ilization
May help in the ____________
of rRNA
Folding

Location of Phosphate group on pentose

Phosphate groups are usually attached on ____
of ribose
C5
Attatched
Phosphate can be ____________
at C2
ribonucleoside 2- monophosphate
Phosphate can be attached at the C3 and is the end
product of ___________
of RNA by some ribonucleases
Hydrolysis
ribonucleoside 3- monophosphate

Fig 8.6

Location of Phosphate group on pentose

Phosphate can be attached at C2 and C3 and is able to
Intermediate
be isolated as an _________________
adenosine 2,3-cyclic monophosphate
Insert Drawing here.

Fig 8.6

Linkages in DNA
b-N-glycosidic
bond
between
_________
ring and the _____________
Sugar
Nitrogeneous Base
N-9 of purine (A or G) or N-1 of
pyrimidine base (C or T) is linked to
the C- 1 of the __________
Bond is formed with the anomeric
carbon (C with ___________ in the b
Axial
configuration (OH is up in _______
and
Equitorial
____________
positions))
Bond is _________
towards hydrolysis
Stable
especially in _____________
Acid
Bond cleavage is catalyzed by _______
Fig 8.3a

Conformation about the b-N-glycosidic bond

When nucleotides are free in solution,
rotation about the b-N-glycosidic bond is
possible
Torsion angle in bond between N of base
and C1 of pentose is called c
O4-C1-N9-C4 for purine
O4-C1-N1-C2 for pyrimidine
Angle near 0 is syn conformation
Angle near 18 0 is anti conformation
Anti conformation is found in B-DNA

Fig. 8.16a

Anti and Syn Conformation about

the b-N-glycosidic bond

Fig. 8.16b

Linkages in DNA
Hydrophilic
Backbone of DNA and RNA are _____________since
the
Bonds
OH groups of pentose formHydrogen
_______
with water in the cell
Phosphate groups have pKa near _____
At neutral pH (pH = 7) phosphate groups are ___________
- ve
charged
Charges are neutralized by ionic interactions with _______
metal
charges on proteins or ________
ions or polyamines
Phosphodiester
____________
linkages always are oriented 5 to 3 with 5
end missing a nucleotide and 3 end missing a nucleotide
Free
5 and 3 ends may have be ______
or have attached
Phosphate
____________
groups

Phosphodiester Linkage
Note 5 end and 3 end which
is the orientation of the
strands
Note
base
differences
between RNA and DNA
Covalent backbone of the
DNA and RNA is subjected to
slow ___________
hydrolysis
Non enztmatic
of phosphodiester bonds
_____
in test tubes undergoes
RNA
rapid hydrolysis due to the
OH
on
C2
under
_______conditions
(basic pH)
Alkaline
Fig. 8.7

Cyclic 2,3-monophosphate nucleotides

Phosphodiester Linkage
Cyclic 2,3-monophosphate nucleotides are first products of
the action of alkali on ______
and _________
further to
Hydrolysis
RNA
form a mix of 2 and 3 nucleoside monophosphates

Fig. 8.8

Short form to writing the DNA sequence

Phosphate groups are in circled P
C1
C5
Deoxyribose is a linear line with ____
at top and _____
at bottom but is in the closed ring __-furanose formation
Phosphate group is linked from ____
of one pentose to
C3
_____
of next pentose
C5
Always written 5 end to 3 end, with 5 end on the _____

Short cut form

Fig pg 286

Short form to writing the DNA sequence

pA-C-G-T-AOH
pApCpGpTpA
pACGTA
Oligonucleotide
Definition of _____________
is 50 or less nucleotides
Polynucleotide
___________
are long chains of nucleotides

Fig pg 286

Properties affecting Nucleotides

Free nucleotides are basic compounds and are called
Nitrogenous
______ bases
Aromatic
Purines and pyrimidines in DNA and RNA are ________
compounds (have rings with double bonds)
Presence of Double
_______
Bonds ______ influences structure,
electron distribution and light absorption
Electron delocalization due to double bonds gives some
bonds _______ double character
Presence of double bonds means purines are ________
planar in nature and pyrimidines are planar in nature
Bases will exist in one of several __________
states
Tautomeric
depending on pH

Tautomeric states of Uracil

Uracil has three states when free in solution

Lactam at pH 7
Lactim and double lactim exist in lower pH solutions
Other bases do have these forms but they are rare in
solution

Fig. 8.9

Absorption of
Common
nucleotides

Abs range is 250 to 270 nm

Absorbance due to p p* electronic transitions
Excited states of common nucleobases decay rapidly via
radiationless transitions
No fluorescence from these nucleic acids
Spectra are nearly identical for all species
Mixture of nucleotides would use Abs at 260 nm for
measurements and analysis
Fig. 8.10

Properties affecting Nucleotides

hydrophobic
Free nucleobases are ___________
and usually
_________ in water at near neutral pH in cells
In acidic or basic environments, the bases become
________
and are more ________ in water
Charged
Bases are able to ________
with their rings parallel (like
Stack
stacking coins) which allows for hydrophobic stacking
interactions
Stacking involves van der Waals and Dipole
______-dipole
interactions between bases
Stacking reduces base interaction with water and helps in
maintaining the _______
structure of DNA
3D

Functional groups in Nucleobases

Presence of ring nitrogens, carbonyl groups, and
exocyclic amino groups
Hydrogen bonding is very important
Involves carbonyl and amino groups
Interactions between purine and pyrimidine base
pairing
Interaction between two (and up to four)
complementary strands of nucleic acids
A bonds to T (or U in RNA) and G bonds to C
This base paring allows for duplication of DNA and
RNA strands

Nucleobases paring in DNA

Fig. 8.11

Polynucleotides
___________
is phosphodiester linkage based so it is
Backbone
negatively
The backbone is fairly stable but it can undergo
__________ in the presence of enzymes (DNAase)
______
backbones are not as stable (RNA can last for
DNA
years in water but only for a few hours inside cells)
The backbone does not contain __________
or branches
Cross Links
You can only read the sequence from 5 to 3
A different ____________
will be created if you read
Sequence
from 3 to 5

Summary of Basics
Nucleotide has purine or pyrimidine nitrogenous base,
pentose sugar and at least one phosphate group
Phosphodiester bonds join the 5 OH of one pentose to
the 3 OH of the next pentose
Two types of nucleic acid: RNA and DNA
RNA has ribose (sugar) and usually Uracil, Cytosine,
Adenine and Guanine (bases)
DNA has deoxyribose (sugar) and usually Thymine,
Cytosine, Adenine and Guanine (Bases)
Recall it is the sugar that determines the type of nucleic
acid not the base

Nucleic Acid Structure

1953 Watson and Crick discovered the structure of DNA
Structure is hierarchial in complexity
__________
covalent structure and nucleotide
Primary
sequence
__________
regular or stable structure obtained by
Secondary
any or all nucleotides
____________ - folding of large chromosomes or
large tRNA or rRNA molecules

DNA double helix that stores genetic information

1868 DNA first isolated by Friedrich Miescher and was
called _____________
nuclein
1940s work by Oswald T Avery, Colin MacLeod, and
MacIyn McCarty proved that DNA was genetic based
material
Avery and collegues took DNA from a virulent strain of
Streptococcus pneumoniae bacterium and injected into a
non-virulent strain of the bacterium (and the nonvirulent
strain became virulent)
DNA _____________
information about the virulence.
Contained

DNA double helix that stores genetic information

1952 - Alfred D. Hershey and Martha Chase studied
infection of bacteria cells by bacteriophage (virus) by
radioactive labeled DNA (showed DNA not proteins
contained genetic information)
Late 1940s Erwin Chargaff and colleagues determined
4
that there were ________
bases in DNA
Ratios
Bases occurred at different _______
between different
organisms

Chargaffs Conclusions
1. Base composition varies from one species to another
2. DNA from different tissues of the same species have
same base composition
3. Base composition does not change with changes to the
organisms age, nutritional state or surrounding
environment
4. A = T in number and G = C in number within a specific
DNA strand
Sum of pyrine bases = sum of pyrimidine bases
** information above useful in determining 3D structure of
DNA, how info is encoded in DNA and how it passes
between generations

Use of X-ray
diffraction to prove
DNA

1950s Rosalind Franklin and Maurice Wilkins used X-ray

diffraction to look at DNA fibers
___________
pattern was found
Reproducable
They deduced that DNA was helical in with two periodicities
along their long axis (___________
was 3.4 and
Primary
^
_________ was 34 )
Angstroms
Fig. 8.12

How do you get a 3D model to fit this data?

Enter Watson and Crick
1953 Watson and Crick proposed a 3D structure DNA
using base pairing and X-ray data
Two helical DNA chains wound around a central axis to
form a right handed double helix
Be able to identify :
Right handed clockwise
left handed counterclockwise

Make 2 fists and point your thumbs away from you

Does the helix curve to the right or the left to wrap around
your thumb?
Is the spiral going around your right thumb in the direction
where there four other fingers curled up (clockwise)? Box. 4.1

How do you get a 3D model to fit this data?

Enter Watson and Crick
Backbone keep deoxyribose and phosphate groups on
the outside of double helix right beside the water in the
cell
Deoxyribose
Each ____________
is in a C2-endo conformation (ring
formation)
Purine and pyrimidine bases are on the inside of the helix
Planar nature allows for close stacking
Perpendicular
Rings are _____________
to the long backbone axes
Since bases are inside the helix they are in a
hydrophobic environment

Major and Minor Grooves in DNA structure

Smaller groove in the double helix is the
minor groove (the backbone is closer
together)
Larger
________
groove in double helix is the
________ groove (backbone is further
apart)
Pairing of bases a purine on one strand
pairs in the same plane with a
pyrimidine on the other strand

Fig. 8.13a

Major and Minor Grooves in DNA structure

A to T has a double H bond
C to G has a triple H bond
Higher the ratio of GC to AT base pairs
the ________
it is to separate the
Harder
paired DNA strands
Other pairing of bases will lead to
_______________
of the double helix
Destabilization

(b) is ball and stick model of double

helix
(c) is space filling model of double helix
Fig. 8.13b and c

Parallel or Antiparallel strands in DNA

Parallel strands would have 3,5 phosphodiester bonds in
the same direction
Antiparallel strands have 3 and 5 running in opposite
directions
Watson and Crick thought the antiparallel orientation was
the better model
DNA polymerase provided experimental results that the
strands were antiparallel
X-ray diffraction results also confirmed antiparallel strands

Spacing of bases and Turns in the double helix

DNA model has the bases ____________ to the backbone
primary
Bases needed to be 3.4 apart (_________
repeating
feature in the x-ray analysis)
____
base pairs per turn of the DNA double helix (a
10
distance of 34 ) which was the second repeating feature
in the x-ray analysis)
If DNA is in ________ and not in the cell, the bases per
10.5
turn is about _____
Base pairs are also not identical in the sequence or
Complimentary
composition but are __________________
A partners (hydrogen bonds) with T
G partners (hydrogen bonds) with C

What forces hold DNA duplex together?

Hydrogen bonds between the base
pairs
2 H-bonds between A and T
3 H-bonds between C and G
Base stacking interactions
Non specific in nature but make
major contributions to the
stability of double helix

Fig. 8.14

Complimentary nature of

the strands
Complementary nature of DNA
allows for easy replication
Watson and Crick were able to
foresee this feature before there
was confirmatory proof
Two strands separate
Each parent strand is the guide
for
making
a
precise
complimentary strand with A
pairing with T and G pairing with
C
Fig. 8.15

A and B and Z forms of DNA

DNA is flexible
Rotation
about
the
covalent
____________________
backbone
and changes in temperature can
cause bending, stretching and
_________
of bases through melting
unpairing
These changes result in different
B
DNA structure (Form ___)
from the
one proposed by Watson and Crick
Reagents used to crystallize DNA
cause it to ________
and so most
Dehydrate
short DNA molecules will appear in
Form A
Fig. 8.17

A, B and Z forms of DNA:

3 changes can occur
Different Structural conformation
of deoxyribose (C2 or C3 endo
or exo)
Rotation about the bonds in the
phosphodeoxyribose backbone
Rotation about the C1-N-glycosyl
bond
purines (A and G) can be
Syn
either _______
or _______
Anti
pyrimidines can be only
Anti Conformation
______ (C and T) due to steric
hinderance of carbonyl oxygen
at C2

Fig. 8.16

A and B forms of DNA

B form DNA is most stable under
physiological conditions
A form and Z form have been studied
under crystal form
A form appears in solutions with little H2O
A form is right handed,
wider (11 bases per turn)
plane of bases tilted by 20 from the
backbone
minor groove is shallower and major
groove is deepened
Fig. 8.17

B and Z forms of DNA

Z form is left handed helix
12 bases per turn (but it looks more
slender and elongated)
Backbone looks zig zag in appearance
Pyrimidine alternating with purine bases fit
better in to a Left handed helix (alternating
C and G or 5-methyl-C and G residues)
Purines are in the ____
Syn conformation and
pyrimidines are in the _____
conformation
Anti
Major groove is hard to find in Z form but
the minor groove is narrow and deep
Z form in __________
and eukaryotes
Bacteria

Fig. 8.17

Comparison of A and B and Z forms of DNA

Fig. 8.17

Structures within DNA due to specific base

sequences
4 or more Adenosine in a row lead to a bend (6 A = bend of
18 )
Bending may assist in binding proteins to DNA
Presence of palindromes (sequences which are the same
forward and backwards like ROTATOR or NURSES RUN) and
their location influence how a strand of DNA may fold upon
itself
Term used when sequences of DNA have inverted repeats of
base sequence over two strands of DNA

Fig. 8.18

Structures within DNA due to specific base

sequences
Palindrome on two complementary strands of DNA to
superimpose, the sequence is rotated by 180 in horizontal
axis and then rotated by another 180 in the vertical axis.

Minor repeat has a repeating sequence on the same strand

of DNA and only needs to rotated by 180 in the vertical axis

Fig. 8.17

Structures within DNA due to specific base

sequences
Since the repeats are within the same strand, they are
able to participate in other structures
Hairpin turns can not formed from complementary
strands of DNA (only single stranded DNA)
If both strands are participating in the hairpin turn it is
called a cruciform (cross shape).

Fig. 8.19

Structures within DNA due to specific base

sequences
These hairpin and cruciform can be found in all large DNA
molecules
Cruciforms have been found in in vivo E. coli
Single stranded DNA and RNA fold into many hairpin
turns when they are in solution
Other unusual structures involve three or four strands of
DNA
Three or four strands can be found in the major groove
when there is additional hydrogen bonding
Protonated Cytosine can pair with G of
Thymidine can pair with A in A/T base pair

Hoogsteen pairing

Fig. 8.20a

Structures within DNA due to specific base

sequences
N-7, O6 and N6 of purines (known as Hoogsteen positions)
can participate in H-bonding of triplex DNA
Unusual (non Watson-Crick base pairing) base pairing
called Hoogsteen pairing
These triplex DNAs are most common in low pH (since
cystosine must be protonated)
pKa of cystosine is greater than 7.5 (normal pKa value is
4.2)

Triplex DNA

Fig. 8.20b

Structures within DNA due to specific base

sequences
Triplexes will form when there are long stretches of only
purine or pyrimidine bases in one strand
These stretches may contain two purine and one
pyrimidine strand or (two pyrimidine and one purine
strand)
If there are high content of
guanosine residues then
tetraplex DNA strands will
form
These strands can be
parallel or antiparallel in
nature
Fig. 8.20e

Guanosine tetraplex

Fig. 8.20c

Four successive tetraplets from a G

tetraplex structure

Fig. 8.20d

Structures within RNA due to specific base

sequences
RNA is the intermediary carrying the information from the
gene in DNA to the _________
where the amino acid
Ribosome
sequence is assembled to make functional proteins
1950s RNA was thought to be the molecule which moved
the information from the nucleus to the ribosome
1961 Franois Jacob and Jacques Monod proposed
messenger RNA (mRNA) to carry the information from
DNA to ribosomes
Process of forming mRNA on DNA template is
Transcription
_____________________

Structures of mRNA
Bacteria and archaea use a single mRNA to encode many
polypeptide chains
One chain is monocistronic
Many chains is polycistronic
Cistron = gene

Fig. 8.21

Structures of mRNA
Minimum length of a mRNA is defined by the length of the
gene
If there is are 100 amino acids, the chain would need to be
minimum 300 nucleotides (1 amino acid = 3 nucleotides =
1 codon)
Noncoding
Chains are always longer because there are ___________
sequences for protein synthesis

Structures of other RNA

Transfer RNA (tRNA) is used to bring the amino acids to
the growing protein chain one ________
______
at a time
Acid
Amino
tRNA is covalently linked to the amino acid and will
partner with mRNA so that the correct sequence is created
for the desired protein (from the DNA gene)
Ribosomal
Ribosomal RNA (rRNA) are part of the ____________
Ribozymes have enzymatic activity
RNA molecules have a ________
diversity in structure than
Richer
DNA

Single stranded RNA

Transcription product is single
stranded (ss) RNA Single stranded are
supposed to be right handed
ss RNA will form a right
handed helix influenced by
base stacking interactions
Purine
________-purine
is stronger
Purine
than _______-pyrimidine
or
Pyrimidine
__________-pyrimidine
Pyrimidine bases can be
Displaced
___________
to offer purinepurine stacking to occur
Self
complementary
sequences in the strand offer
more complex structures

Fig. 8.22

Structure Formation of double stranded RNA

rare
____ to have two strands of RNA be 100% complementary
Strands will bond over shorter regions and form a right
A Form
handed ________
double helix
Weak interactions involving base stacking help to stabilize
the RNA structures
Z-form RNA has been made in the lab (under High
____ salt
concentration or _____
temperature conditions)
High
No B-form RNA has been made
A-form helix is not continuous due to mismatches (_____
Bulge
Internal
in one strand or _______
loop on both strands) or from
hairpin turns from nearby self complementary sequences

Structure Formation of double stranded RNA

Fig. 8.23

Pairing differences in
RNA
In RNA, A pairs with U and C
pairs with G
BUT G can pair with U
Pairing of RNA strands is
antiparallel
Hairpins
are
the
predominant
secondary
structure

Fig. 8.24

Hairpin turns in RNA

Short sequences (such as UUGG) make tight turns and
are located at the end of a hairpin turn
These sequences are often very stable
It is possible that these sequences will act as starting
points for 3D folding of RNA molecules
Other stabilizing mechanisms come from non-WatsonCrick bonding of base pairs
2OH of ribose can bond to other groups
Such bonding is seen in phenylanine tRNA of yeast, and
two RNA enzymes (ribozymes) whose functions depend
on their structure

Summary of Nucleic Acid Structure -1

Avery-MacLeod-McCarty experiment showed DNA
carries inheritable characteristiscs
Hershey Chase experiment proved DNA not proteins
carried the information for replication
Watson and Crick used available data to provide the
double helix model based on B-DNA (A pairs with T and G
pairs with C; bases are perpendicular to the backbone
and 3.4 and 10.5 bases per turn)
DNA can be in A-form, B-form or Z-form

Summary of Nucleic Acid Structure -2

DNA can have hairpin turns or cruciforms depending on
palindrome sequences or be in triplex or tetraplex
strands
mRNA carries the genetic information from DNA to the
ribosome
tRNA and rRNA are involve in protein synthesis
Single RNA can fold into hairpin turns, double stranded
sections or complex loops

Nucleic Acid Chemistry

Genetic information in DNA needs to be _________
Stable
Absence
Chemical transformations of DNA are slow in _________
of enzymatic catalyst activity
Even with the slow changes, DNA alterations can be
Significant
physiologically ________________
Slow accumulation of irreversible alterations to DNA may
be linked to ________
and _______________
Aging
Carcinogenesis (cancer causing)
Non-destructive changes also occur such as Strands
________
separation for DNA replication and ______________
Transcription
Studying DNA has lead to the development of some very
specific and interesting Technology
___________ which helps in
analysis of DNA base sequences

Nucleic Acid Chemistry

Native
___________
DNA solutions at physiological pH and
_____
is very viscous Carefully prepaired
RT
Double stranded DNA and RNA can become denatured
80
by raising the temperature to above ____C
or alter the
pH to extreme acid or base there is a physical change to
the DNA
Melting
Denaturation or ___________
of the double-helical DNA
_______
temperatures and ____
influence the H-bonds
PH
High
between bases on the two complimentary strands and
on the _______
stacking on one strand of DNA
Base

Nucleic Acid Chemistry

Completely
partially
DNA can __________
or ________________
unwind
Covalent
No ____________
backbone bonds are _________
Broken

during the unwinding

Part
DNA can undergo renaturation if _______
of the double

strands (minimum _____

Intact
10-12 bases) are still ________
Returned
When pH and/or temperature are _________
to

physiological conditions, DNA will reanneal (or rewind)

Nucleic Acid Chemistry

If the DNA is __________
denatured into two separate
Completely
2
strands, then renaturation requires ____
steps
Strands will attempt to find each other through
Random
________
collisions and start to form short segments of
______________
DNA
Complimentary
If the complimentary section is appropriate and long
enough then the _________
of the strand will ________
Rewind
rest
to form the double-helix __________
Quickly

Annealing and
Denaturation of
double stranded DNA

Fig 8.26

UV Absorbance of DNA
Stacked
________
bases in nucleic acids decrease the absorbance

of UV light relative to ______

nucleotides in solution
Free
Presence of double stranded DNA ________
the
Decreases

absorbance

of

UV

light

even

more

known

as

Hyperchromic
__________________
effect

Denatured single stranded DNA __________

the
Increases
Hyperchromic
absorbance of light known as _____________
effect

tm - melt temperature
Denature
Viral and bacterial DNA _________
slowly in solution if
Heated
________
Specific
tm is species _________
and is the point at which ______
Half
the DNA is single stranded
Higher ______
content the higher the melt temperature
Glc
Stacking
tm influenced by the _________
interactions of
neighbouring bases and by H-bond ___________
Interactions
Ionic
PH
tm can be determined under fixed ____
and ________
strength conditions (concentration of Na+)
Under carefully controlled conditions, only ____
A/T rich
Melt
regions will _____
and the G/C conditions will remain
Intact
_________

tm - melt temperature
Denatured regions are called bubbles
In vivo, strand separation is initiated at A/T rich areas for
replication and transcription
regions will melt and the G/C conditions will remain
intact

Fig 8.27

tm - melt temperature
Denatured regions are called bubbles
In vivo, strand separation is initiated at A/T rich areas for
replication and transcription
regions will melt and the G/C conditions will remain
intact
Partial denatured DNA
and then fixed for viewing
Red arrows point to some
bubbles
Electron micrograph with
5-fold enhancement of
DNA

RNA and RNA/DNA hybrids

Duplexes
RNA can form ________
and they are ______
stable to
More
heat denaturation than _____
DNA duplexes
Higher
So require _______
temperatures for denaturation
(about 20C than for comparable DNA duplex sequences)
Hybrid
RNA and DNA can form _______
and denature at
Intermediate
_____________
temperatures between pure RNA
duplexes and DNA duplexes
Using the complimentary nature of DNA, complimentary
sequences may be found between ___________
Species
Mouse
_________
and human DNA were isolated, denatured
and then mixed and allowed to sit for many hours at
25C below tm, some of the DNA will anneal

RNA and RNA/DNA hybrids

Concentration
Temperature, DNA composition (length, ___________,
complexity and _____
content), ____
concentration
Salt
G/C
influence the ability of DNA to anneal
Low
______
temperature results in short sequences annealing
to any complimentary sequence which will prevent
10
_________
sequences from properly annealing
Human
Most Human DNA will anneal to form ________
DNA
duplexes and same with the mouse DNA
A small portion will form a mixture of DNA
human/mouse _______
which shows the part of the DNA
Hybrid
Evolutionary
that has ______________
heritage
larger
________
portion of human/mouse DNA will form
hybrids than human/yeast DNA hybrids

DNA from two sources and the presence of hybrids

Fig 8.29

Uses of Hybridization Procedures

Relationship
evolutionary _________
between two species influence
Much
how ______
hybridization will occur
A specific gene or sequence can be found if there is a
________
complimentary sequence that is labelled
known
(fluorescently or with chemical tags)
Strand
Complimentary ______
can be from the same species,
Synthesized
different species or chemically __________
Slight modifications are made to find RNA sequences
This procedure can be used to ID a person from hair left
at a crime scene or to predict the onset of disease (that
may exist in modifications within certain genes a
sequence of DNA)

Nonenzymatic transformation of Nucleotides

Changes
Cells do not want ________
in their genetic material
Cell
Too many changes will lead to ________
death or species
Extinction
_________
Spontaneous
Purine and pyrimidine can undergo ___________
alterations on a very slow time basis.
Mutations
Alterations lead to __________
(permanent changes in
the DNA)
Too many alterations / mutations leads to ________
and
Aging
possibly carcinogenesis

Deamination
Exocyclic
Loss of ____________
amino groups
1 cytosine will be converted to ________
in every 107
Uracil
cytosine bases in ____
hours
24
Result is about _____
spontaneous events per day
100
Slower
Rate for deamination of A and G is about 1/100th ______
T not ____
DNA contains ___
U
Foreign DNA which is removed
Presence of U appears as ______
and repaired
If U was present in the DNA than it would be harder to
find the spontaneous deamination events and this would
Permanent
lead to _________
changes in the genetic material

Deamination
C deamination leads to
Drop
_____
in G/C content and
would _______
the A/U
Increase
base pairing in DNA cells
Over a millennia, C
deamination could lead to
_______
of C/G pairing
loss
and its genetic code
Using Thymine as a Base
_____
was a important turning
point for _____
term
Long
storage
of
genetic
information
Fig 8.30

Depurination
Loss
Glucose
Hydrolysis of N-b-_________
bond leads to the ______
of a ________
from the pentose
Base
Creates a DNA ________
called ____
site (apurinic or
Lesson
AP
apyrimidinic depending on which base was lost) or
________
site
Abasic
Rate is faster for _________
(1 in 105 purines in
Purine
mammalian cells for 24 hours) in typical cell conditions
than in pyrimidines
Add _______
acid to purines in a test tube and the rate
Dilute
of reaction is faster
DNA incubated in pH __
3 will result in a selective removal
apurims
of purine bases to yield __________
acid
Fig 8.30

Depurination

UV-Radiation
Radiation causes condensation of 2 ethylene groups to
6-4
form a ___________
ring or ______
photoproduct
Cyclobutane
X-rays and gamma rays (ionizing radiation) initiate the
ring opening
Yeild fragmentation of bases and breaks in covalent
backbone of nucleic acids
Bends and _______
Kinks
______
Solar spectra (200 nm to 400 nm) in near UV radiation
cause pyrimidine dimer formation or other chemical
changes in the DNA in human ______
cells and bacteria
Skin
UV and ionizing radiation make up __%
DNA damage by
10
environmental agents
Fig 8.30

Radiation damage
products (dimers and
photoproduct) to DNA
and their result to
DNA structure (kink)

Fig 8.31

Industrial Activity

DNA can be damaged by industrial products or their side

reactions
____________
agents (nitrous acid (HNO2) or
Deanimating
compounds which can generate nitrous acid or nitrites)
Used as preservatives in food to prevent bacterial growth
_______
health hazard with them in food (and
lower
modifications to DNA) than them not in food (food
spoilage)
Fig 8.32a

Industrial Activity
Alkylating agents (will alter certain bases)
Dimethylsulfate (DMSO) will methylate guanine at O6
which prevents base pairing with cytosine (if in the
correct tautomer)
Other reactions do occur naturally in cells such as the Sadenosyl methionine

Fig 8.32b and pg 302

Oxidative species and damage to DNA

Hydrogen peroxide, hydroxyl radicals and superoxide
radicals are products of irradiation or byproduct of
aerobic metabolism
Cells are able to destroy oxidative species through
catalase or superoxide dismutase but some destruction is
done
DNA is able to self repair most of the damage before it is
permanently passed on to the next generation

Methylation of some DNA bases

A
_________
and _______
are more often methylated then
C
guanine or thymine
Methylation occurs more prominently in some sequences
or sections of genes
Not all reasons for methylation are understood
DNA methylases (enzyme) use S-adenoylmethionine for
the ________
source
Methyl
Ecoli uses methylation for two reasons:
Foreign
To label its DNA so that _______
DNA can be more
easily found
To label a _______
Sequence where DNA repair must be made
due to ____________
bases
Mismatched

Methylation of some DNA bases

Adenine and cytosine are more often methylated then
guanine or thymine
Methylation occurs more prominently in some sequences
or sections of genes
Not all reasons for methylation are understood
DNA methylases (enzyme) use S-adenoylmethionine for
the methyl source
Ecoli uses methylation for two reasons:
To label its DNA so that foreign DNA can be more
easily found
To label a sequence where DNA repair must be made
due to mismatched bases

Sequencing DNA
Before 1970s very time consuming process
1977 two new techniques were developed (Alan
Maxam and Walter Gilbert) and (Frederick Sanger)
Used improved understanding of nucleotide chemistry
and DNA metabolism and electrophoresis to separate
DNA strands one nucleotide at a time
Polyacrylamide gel used for shorter DNA sequences
Agarose used for longer DNA sequences

Sequencing DNA
Basic principle reduce the DNA to four sets of labelled
fragments
Reaction producing each fragment is nucleobase specific
Fragments are radioactively labelled on the 5 end
Mixtures undergo electrophoresis and the fragment can
be sequenced based on where the bands appear

Sanger method: desired sequence

has a primer to which the
nucleotides are added following
the template strand

Sanger method: desired sequence

has a primer to which the
nucleotides are added

Sanger method: dideoxynucleotides (ddNTP)

are used interrupt the bonding sequence of the
addition of one ddNTP at a time to the
template

Sanger method:
mixture of dNTP
and ddNTP are
added, the template
grows a
complementary
sequence which has
a labeled primer,
sequence is read
after mixture of
templates are
passed through the
electrophoresis gel

Sequencing DNA
Each nucleotide is added as dNTP and ddNTP and the
fragments will stop growing with ddNTP is placed in the
growing strand
Shorter fragments will migrate faster in the gel
Knowing the location of the nucleotide in the gel, the
sequence can be read one base at a time
The sequence that is read from the gel is the
complementary sequence to the one that was being
analyzed

Human Genone Project (HGP)

HGP
accelerated
DNA
sequencing methods
Each nucleotide would be
labeled with a fluorescent tag to
allow large sequences to be
analyzed in a few hours
Each fragment of DNA would
have a tag and be read by a
detector as it passed through
electrophoresis gel

Fig 8.34

Creating DNA on a solid silica support

Phosphoramidite method
Growing strand is attached to a solid support
Protecting groups prevent unwanted reactions
Polynucleotides up to 70 or 80 bases in length can be
easily made
Progress can be followed spectrophotometrically if the
base is coloured with a tag
Extra care must be taken to protect 2 OH in ribose when
RNA is being produced

Creating DNA on a solid silica support:

Phosphoramidite method

Fig 8.35

Summary: Nucleic Acid Chemistry-1

DNA can melt at high temperatures or extreme pH
G/C rich areas melt at higher temperatures than A/T rich
areas
Single stranded DNA that is denatured can form duplexes
with a complimentary strand of DNA from the same
species or different species (hybrid)
Hybridization concept used to isolate certain genes and
RNA

Summary: Nucleic Acid Chemistry-2

DNA is fairly stable but can undergo some chemical
reactions at very slow rates but still influence stability of
genetic material
deamination C to U (so foreign U base substitutions
can be repaired)
Hydrolysis of base sugar bonds
Radiation forming pyrimidine dimers or photoproducts
Oxidative damage
Sequence of DNA can be determined by several different
manners
Oligonucleotides can be synthesized rapidly and
accurately

Other Functions of Nucleotides

Nucleotides have other functions besides being subunits
in nucleic acids
Energy carriers
Components of enzyme cofactors
Chemical messengers
Ribonucleotides can have 1 to 3 phosphates linked to the
ribose
Known as a, b or g depending
on distance from ribose

Fig 8.36

Nomenclature of Nucleotides

Fig 8.36

Other Functions of Nucleotides

Hydrolysis of phosphate groups provides chemical energy
to drive other chemical reactions within the cell
_______
(adenosine 5triphosphate) is the most
ATP
__________
energy form
Common
GTP, UTP and CTP are also used as forms of energy
__________ are activated precursors of DNA and RNA
synthesis

Other Functions of Nucleotides

________ linkage between a and ribose yields 14 kJ/mol
____________ linkage between a/b and b/g yields about
30 kJ/mol each
Hydrolysis of ATP is often _______ to other biochemical
reactions to provide positive free-energy change and will
result in product formation

Fig 8.37

Adenosine Participates in Many Reactions

__________ is an non protein component of an enzyme
(inorganic ion or co-enzyme) required for catalysis
Enzyme co-factors use _______ as part of their structure
Adenosine appears in the _________ structure but most
cofactors do not look like nucleotides
Adenosine does not participate in the ________ function
of the co-factor
_____ of adenosine reduces the activities of the co-factor
by _____ amount
Loss of 3-phosphoadenosine diphosphate from
acetoacetyl-Co-A (reduces its reactivity by a factor of
106

Regulatory Molecules
First _____________ are hormones or external chemical
signals which interact with receptors on cell surfaces
First messengers can lead to formation of
________messengers inside cells
Second messengers will trigger _________ changes in
the cell interior
_______ = adenosine 3,5-cyclic monophosphate
Formed from ATP in a reaction catalyzed by adenylyl
cyclase (enzyme located in the inner face of plasma
membrane)
cAMP has ____________ functions for every cell
outside the plant kingdom

Coenzyme A (CoA) functions

in acyl group transfer reactions
acyl group is attached it CoA through the thioester
linkage of b-mercaptoethylamine

Fig 8.38

Flavin adenine
dinucleotide (FAD)
active form of Vitamin B2
works in electron transfers

Fig 8.38

Nicotinamide
adenine dinucleotide
(NAD+)
works in hydride transfers

Fig 8.38

Why use Adenosine?

Adenosine may be used due to evolutionary ________
Adenosine can _________ potential binding energy
Other nucleobases can offer ________ potential binding
energy
Using one compound for many purposes reduces down
the need for many additional systems and is an
____________ advantage
Presence of _______ amounts of adenosine led to its
incorporation into other _________
Single protein ________ that can bind adenosine can be
used in many _________ enzymes
__________ binding fold present in many enzymes that
use ATP and nucleotide cofactors

Regulatory Molecules
cGMP = guanosine 3,5- cyclic monophosphate
Exists in many cells and also has _________ functions
ppGpp = guanosine 5 diphosphate, 3 diphosphate
Found in ___________ cells
Produced when there is a _________ in protein
synthesis during amino acid __________
___________ the synthesis of rRNA and tRNA
molecules ________ for protein synthesis (to prevent
unwanted production of nucleic acids)

Regulatory Molecules

Fig 8.39

Summary: Other Functions of Nucleotides

ATP is important commodity in cells for energy
Adenosine is used in many cofacotors that involve energy
binding requirements
cAMP is used as a second messenger to triggered by
hormones or other chemical signals

Learning Outcomes
Structure and function of nucleic acids
Draw and ID purine and pyramidine nitrogeneous bases
Draw and understand the mechanism for formation of ring in
pentose ring in nuclotides
Draw the mechanism for formation and cleavage of Nglycosidic bond
ID and draw the mechanism for the formation of
phosphodiester bond which links nucleotides together in DNA
What are the major characteristics of DNA that highlight the
important chemical interactions that impart structure and
functionality

Can you define the terms?

Gene (pg 281)

Ribosomal RNA (rRNA) (pg 281)
Messenger RNA (mRNA) (pg 281)
Transfer RNA (rRNA) (pg 281)
Nucleotide (pg 281)
Nucleoside (pg 281)
Pyrimidine (pg 282)
Purine (pg 282)
Deoxyribonucleotides (pg 283)
Ribonucleotides (pg 283)
Phosphodiester linkage (pg 285)
5end (pg 285)
3end (pg 285)
Oligonucleotide (pg 286)
Polynucleotide (pg 286)
Base pair (pg 287)

Major groove (pg 289)

Minor groove (pg 289)
B-form DNA (pg 291)
A-form DNA (pg 291)
Z-form DNA (pg 291)
Palindrome (pg 291)
Hairpin (pg 292)
Cruciform (pg 292)
Triplex DNA (pg 292)
G tetraplex (pg 292)
Transcription (pg 294)
Monocistronic mRNA (pg 294)
Polycistronic mRNA (pg 294)
Mutation (pg 299)
Second messenger (pg 308)
Adenosine 3, 5 cyclic monophosphate
(cyclic AMP, cAMP) (pg 308)

Things to ponder for next lecture

Try to determine the correct answer
Results taken up in next lecture

What other name can be given to a nucleotide?

A. Glycosylated nucleoside
B. Purinated pentose
C. Deoxyribonucleotide
D. Nucleoside phosphate
E. Pyrimidinate

 Some effort has been made to extract DNA from

fossilized dinosaur bones that have been buried for
many millions of years in arid climates. In what form is
this DNA most likely to be found?

A. A
B. B
C. Z
D. Tetraplex
E. Completely denatured

 What function is served by the major groove in DNA?

A. It is the binding site for ethidium bromide.
B. It is a binding site for regulatory proteins.
C. It is a region of denatured DNA to which restriction
endonucleases bind.
D. It is a region of DNA that can form a hybrid duplex
with DNA of another species.
E. It is the site of guanine tetraplex formation.

 Which structure is found in RNA, but would be the

result of a mutation or faulty annealing in DNA?

A. Hairpin
B. Double-stranded helix
C. Cruciform
D. Sequence complementation
E. Bulge

 Telomeres are non-coding sequences on the ends of

chromosomes. They contain a high concentration of
guanosine. Given this information, what other structure
may be in telomeres?

A. Cyclobutane dimers
B. A high concentration of triplexes
C. Cruciform
D. Tetraplexes
E. A-form DNA