Chapter 28: Nucleosides, Nucleotides, and Nucleic Acids.
Nucleic acids are the third class of biopolymers (polysaccharides
and proteins being the others)
Two major classes of nucleic acids
deoxyribonucleic acid (DNA): carrier of genetic information
ribonucleic acid (RNA): an intermediate in the expression
of genetic information and other diverse roles
The Central Dogma (F. Crick):
DNA
(genome)
mRNA
(transcriptome)
Protein
(proteome)
The monomeric units for nucleic acids are nucleotides
Nucleotides are made up of three structural subunits
1. Sugar: ribose in RNA, 2-deoxyribose in DNA
2. Heterocyclic base
3. Phosphate
340
Nucleoside, nucleotides and nucleic acids
phosphate
sugar
phosphate
phosphate
sugar
base
base
sugar
base
sugar
base
phosphate
nucleoside
nucleotides
sugar
base
nucleic acids
The chemical linkage between monomer units in nucleic acids
341
is a phosphodiester
28.1: Pyrimidines and Purines. The heterocyclic base; there
are five common bases for nucleic acids (Table 28.1, p. 1166).
Note that G, T and U exist in the keto form (and not the enol
form found in phenols)
7
N
NH2
6
5
N
8
9
N
H
4
1
N
2
N
H
N3
N
H
N
NH2
N3
2
N1
pyrimidine
H3C
N
N
H
O
cytosine (C)
DNA/RNA
NH
N
NH2
guanine (G)
DNA/RNA
O
4
6
N
N
adenine (A)
DNA/RNA
purine
5
O
O
NH
N
H
O
thymine (T)
DNA
NH
N
H
O
uracil (U)
RNA
28.2: Nucleosides. N-Glycosides of a purine or pyrimidine
heterocyclic base and a carbohydrate. The C-N bond involves
the anomeric carbon of the carbohydrate. The carbohydrates for
nucleic acids are D-ribose and 2-deoxy-D-ribose
342
Nucleosides = carbohydrate + base (Table 28.2, p. 1168)
ribonucleosides or 2’-deoxyribonucleosides
NH2
7
N
8
HO
5
N
9
5'
N
O
N
4
1
N
2
HO
X
HO
HO
RNA: X= OH, adenosine (A)
DNA: X= H, 2'-deoxyadenosine (dA)
5
N
6
X
H3C
3
O
NH
NH
1
N
O
2
O
HO
O
N
O
HO
O
N
O
1'
4'
HO
NH2
O
NH2
3'
N
RNA: X= OH, guanosine (G)
DNA: X= H, 2'-deoxyguanosine (dG)
4
5'
N
NH
2'
3'
HO
O
3
1'
4'
O
6
2'
X
RNA: X= OH, cytidine (C)
DNA: X= H, 2'-deoxycytidine (dC)
HO
DNA: thymidine (T)
HO
OH
RNA: R= H, uridine (U)
To differentiate the atoms of the carbohydrate from the base, the
position number of the carbohydrate is followed by a ´ (prime).
The stereochemistry of the glycosidic bond found in nucleic acids
343
is .
28.3: Nucleotides. Phosphoric acid esters of nucleosides.
Nucleotides = nucleoside + phosphate
HO
HO
O
HO P O
O
B
O
X
HO
HO
O
O
O
HO P O P O P O
O
O
O
B
O
X
B
O
O
O
HO
ribonucleotide 5'-diphosphate (X=OH, NDP)
deoxyribonucleotide 5'-diphosphate (X=H, dNDP)
X
ribonucleotide 5'-monophosphate (X=OH, NMP)
deoxyribonucleotide 5'-monophosphate (X=H, dNMP)
ribonucleoside (X=OH)
deoxyribonucleoside (X=H)
O
O
HO P O P O
O
O
B
O
X
ribonucleotide 5'-triphosphate (NTP)
deoxyribonucleotide 5'-triphosphate (X=H, dNTP)
B
O
P
O
O
OH
ribonucleotide
3',5'-cyclic phosphosphate (cNMP)
Kinase: enzymes that catalyze the phosphoryl transfer reaction
from ATP to an acceptor substrate. M2+ dependent
HO
HO
O
OH
Glucose
OH
NH2
N
O
O
O
O P O P O P O
O
O
O
OH
O
N
N
N
HO
OH
ATP
OPO32HO
HO
O
OH
OH
Glucose-6-phosphate
NH2
N
O
O
O P O P O
O
O
O
N
N
N
HO
OH
ADP
344
NH2
N
O
O
O
O P O P O P O
O
O
O
N
O
HO
O
N
N
O
-P2O7
OH
O
N
N
O
P
O
HO
O
NH2
-P2O7
GTP
N
O
O
N
N
OH
N
NH
NH2
OH
cGMP
O
N
O
O P O
O
H2O
N
O
N
AMP
O
P
O
O
HO
OH
cAMP
O
NH
OH
N
N
guanylate
cyclase
N
O
NH2
N
O
O P O
O
H2O
N
ATP
O
O
O
O P O P O P O
O
O
O
NH2
N
adenylyl
cyclase
O
N
NH
N
HO
OH
NH2
GMP
1971 Nobel Prize in Medicine or Physiology:
Earl Sutherland
28.4: Bioenergetics. (Please read)
28.5: ATP and Bioenergetics. (Please read)
NH2
N
O
O
O
O P O P O P O
O
O
O
O
N
N
N
HO
OH
ATP
H2O
NH2
N
O
O
O P O P O
O
O
N
O
N
N
HO
+
HPO4 2-
+ ~31 KJ/mol
(7.4 Kcal/mol)
OH
ADP
345
28.6: Phosphodiesters, Oligonucleotides, and
Polynucleotides. The chemical linkage between
nucleotide units in nucleic acids is a phosphodiester,
which connects the 5’-hydroxyl group of one
nucleotide to the 3’-hydroxyl group of
the next nucleotide.
3'
O
O P
O
O
O
5'
Base
3'
O
X
O P
O
O
O
5'
Base
3'
By convention, nucleic acid sequences are written
from left to right, from the 5’-end to the 3’-end.
Nucleic acids are negatively charged
O
X
5'
RNA, X= OH
DNA, X=H
28.7: Nucleic Acids.
1944: Avery, MacLeod & McCarty - Strong evidence that DNA
is genetic material
1950: Chargaff - careful analysis of DNA from a wide variety of
organisms. Content of A,T, C & G varied widely according
to the organism, however: A=T and C=G (Chargaff’ Rule)
1953: Watson & Crick - structure of DNA (1962 Nobel Prize with
346
M. Wilkens, 1962)
28.8: Secondary Structure of DNA: The Double Helix.
Initial “like-with-like”, parallel helix:
Does not fit with with Chargaff’s Rule: A = T
G=C
H
H N
N
N
dR
N
N
N
O
H
H
O
N
N
dR
N
N
N H
N
N
dR
N
N
H
H
O
N
N
dR
dR
N
O
N
N
H
H
dR
N
O
N
H N
N
O
dR
H
N
H
H
N
H
N
O
N
N
dR
O
N H
H
purine - purine
pyrimidine - pyrimidine
Wrong tautomers !!
Watson, J. D. The Double Helix, 1968
347
Two polynucleotide strands, running in opposite directions
(anti-parallel) and coiled around each other in a double helix.
The strands are held together by complementary hydrogenbonding between specific pairs of bases.
Antiparallel C-G Pair
Hydrogen Bond
Donor
N4
N
Hydrogen Bond
N3
Acceptor
Hydrogen Bond
Acceptor
H
O
N
5'
N
5'
O2
Bond
O6 Hydrogen
Acceptor
H
RO
H
N
N
N
N
O
O
H
N1 Hydrogen Bond
Donor
RO
O
N
H
N2
Hydrogen Bond
Donor
N6
Hydrogen Bond
Donor
OR
3'
OR
3'
Antiparallel T-A Pair
Hydrogen Bond
Acceptor
H
O4
Hydrogen Bond
N3
Donor
O
H N
N
5'
5'
RO
N H
N
N
O
OR
3'
N
N
RO
O
N1 Hydrogen Bond
Acceptor
O
OR
3'
348
DNA double helix
major
groove
12 Å
one
helical
turn
34 Å
minor
groove
6Å
backbone: deoxyribose and phosphodiester linkage
bases
349
28.9: Tertiary Structure of DNA: Supercoils. Each cell contains
about two meters of DNA. DNA is “packaged” by coiling around
a core of proteins known as histones. The DNA-histone
assembly is called a nucleosome. Histones are rich is lysine
and arginine residues.
350
Pdb code 1kx5
“It has not escaped our attention that the specific pairing we have postulated immediately
suggests a possible copying mechanism for the genetic material.” Watson & Crick
28.10: Replication of DNA.
The Central Dogma (F. Crick):
DNA
replication
DNA transcription mRNA translation Protein
(genome)
(transcriptome)
(proteome)
Expression and transfer of genetic information:
Replication: process by which DNA is copied with very high
fidelity.
Transcription: process by which the DNA genetic code is read
and transferred to messenger RNA (mRNA). This is an
intermediate step in protein expression
Translation: The process by which the genetic code is converted
to a protein, the end product of gene expression.
The DNA sequence codes for the mRNA sequence, which
codes for the protein sequence
351
DNA is replicated by the coordinated efforts of a number of
proteins and enzymes.
For replication, DNA must be unknotted, uncoiled and the
double helix unwound.
Topoisomerase: Enzyme that unknots and uncoils DNA
Helicase: Protein that unwinds the DNA double helix.
DNA polymerase: Enzyme that replicates DNA using each strand
as a template for the newly synthesized strand.
DNA ligase: enzyme that catalyzes the formation of the
phosphodiester bond between pieces of DNA.
DNA replication is semi-conservative: Each new strand of DNA
contains one parental (old, template) strand and one
daughter (newly synthesized) strand
352
Unwinding of DNA by helicases expose the DNA bases
(replication fork) so that replication can take place. Helicase
hydrolyzes ATP in order to break the hydrogen bonds between
DNA strands
DNA replication
353
DNA Polymerase: the new strand is replicated from the 5’  3’
(start from the 3’-end of the template)
DNA polymerases are Mg2+ ion dependent
The deoxynucleotide 5’-triphosphate (dNTP) is the reagent for
nucleotide incorporation
dNTP
OH
5'
O
O O
O P O P O P OO- O- O-
O
T
A
O
O
O
O
G
P
-O
O
template
strand
(old)
Mg2+
OH
O
O
O
C
(new)
5'
O
3'
3’-hydroxyl group of the growing DNA strand acts as a nucleophile
and attacks the -phosphorus atom of the dNTP.
354
Replication of the leading strand occurs continuously in the
5’  3’ direction of the new strand.
Replication of the lagging strand occurs discontinuously. Short
DNA fragments are initially synthesized and then ligated together.
DNA ligase catalyzes the formation of the phosphodiester bond
between pieces of DNA.
animations of DNA processing: http://www.wehi.edu.au/education/wehi-tv/dna/
355
DNA replication occurs with very high fidelity:
Most DNA polymerases have high intrinsic fidelity
Many DNA polymerases have “proof-reading”
(exonuclease) activity
Mismatch repair proteins seek out and repair base-pair
mismatches due to unfaithful replication
28.11 Ribonucleic Acid
RNA contains ribose rather than 2-deoxyribose and uracil rather
than thymine. RNA usually exist as a single strand.
There are three major kinds of RNA
messenger RNA (mRNA):
ribosomal RNA (rRNA)
transfer RNA (tRNA)
DNA is found in the cell nucleus and mitochondria; RNA is more
356
disperse in the cell.
Transcription: only one of the DNA strands is copied (coding
or antisense strand). An RNA polymerase replicates the DNA
sequence into a complementary sequence of mRNA (template
or sense strand). mRNAs are transported from the nucleus to
the cytoplasm, where they acts as the template for protein
biosynthesis (translation). A three base segment of mRNA
(codon) codes for an amino acid.The reading frame of the
codons is defined by the start and stop codons.
357
5'-cap
5'-UTR
start
Coding sequence
stop
3'-UTR
The mRNA is positioned in the ribosome through complementary
pairing of the 5’-untranslated region of mRNA with a rRNA.
Transfer RNA (tRNA): t-RNAs carries an amino acid on the
3’-terminal hydroxyl (A) (aminoacyl t-RNA) and the ribosome
catalyzes amide bond formation.
Ribosome: large assembly of proteins and rRNAs that catalyzes
protein and peptide biosynthesis using specific, complementary,
anti-parallel pairing interactions between mRNA and the
anti-codon loop of specific tRNA’s.
358
Although single-stranded, there are complementary sequences
within tRNA that give it a defined conformation.
The three base codon sequence of mRNA are complementary
to the “anti-codon” loops of the appropriate tRNA. The basepairing between the mRNA and the tRNA positions the tRNAs
for amino acid transfer to the growing peptide chain.
variable loop
TC loop
D loop
aminoacyl t-RNA
359
28.12: Protein Biosynthesis. Ribosomal protein synthesis
SCH3
OHC
SCH3
H
N
OHC
O
O
H
N
O
OH
H2 N
O
O
O
A site
P site
U A C
U A C
A U G U A U G C U C
U U
3'
5'
A U A
A U G U A U G C U C
U U
3'
5'
SCH33
CH3S
CH3S
OHC
OH
N
H
OJHC
OHC
HN
O
OH
OHC
OH
N
H
HN
OH O
O
HN
O
HN
O
O
OH
O
HHN
2N
OH
O
O
O
OH
CH33
CH
O
O
A U A
U A C
A
A C
A U
C G
U A
G A
U A C A
U A
A U G U A U G C U C U U
5'
A
U U
A U
U G
G U
U A
A U
U G
G C
C U
U C U
3'
5'
5'
3'
E site
360
28.13: AIDS. (please read)
28.14: DNA Sequencing.
Maxam-Gilbert: relys on reagents that react with a specific DNA
base that can subsequent give rise to a sequence
specific cleavage of DNA
Sanger: Enzymatic replication of the DNA fragment to be
sequenced with a DNA polymerase, Mg+2, and
dideoxynucleotides triphosphate (ddNTP) that truncates
DNA replication
Restriction endonucleases: Bacterial enzymes that cleave
DNA at specific sequences
5’-d(G-A-A-T-T-C)-3’
3’-d(C-T-T-A-A-G)-5’
5’-d(G-G-A-T-C-C)-3’
3’-d(C-C-T-A-G-G)-5’
5'
3'
3'
5'
EcoR I
restriction
enzyme
2-O PO
3
OH
3'
5'
BAM HI
5'
3'
2-O PO
3
OH
361
Sanger Sequencing:
key reagent: dideoxynucleotides triphosphates (ddNTP)
NH2
N
N
O
-O
O
O
N
P O P O P O
O-
O-
N
N
O
O
O
P O P O P O
O-
OddATP
3'
O
O-
OddTTP
N
O
O
O
-O
O
O
N
P O P O P O
O-
O-
N
NH
NH
-O
NH2
O
O
N
O
O
N
P O P O P O
O-
O-
O-
ddCTP
Anti-Viral Nucleosides
When a ddNTP
is incorporated
elongation of
the primer is
terminated
O
O
N
NH
HO
The ddNTP is
specifically
incorporated
opposite its
complementary
nucleotide base
N
O
HO
NH
N
O
O
N
N3
DNA polymerase
AZT
ddI
NH2
HO
O
H2N
N
N
O
O
N
N
OH
O
S
d4C
(-)-3TC
5'
Template
unknown sequence
O
O
O-
ddGTP
primer
Mg2+, dNTP's
+ one ddATP
-O
O
5'-32P
3'
O
NH2
362
Sanger Sequencing
ddA
ddG
ddC
ddT
5'
Larger
fragments
Smaller
fragments
C
A
T
T
G
C
A
T
T
A
G
T
G
T
C
G
T
A
A
C
G
T
A
A
T
C
A
C
A
G
5'
3'
32P
3'
32P-5'
primer
3'
template
363
28.15: The Human Genome Project. (please read)
28.16: DNA Profiling and Polymerase Chain Reaction (PCR).
method for amplifying DNA using a DNA polymerase, dNTPs and
cycling the temperature.
Heat stable DNA Polymerases (from archaea):
Taq: thermophilic bacteria (hot springs)- no proof reading
Pfu: geothermic vent bacteria- proof reading
Mg 2+
two Primer DNA strands (synthetic, large excess)
one sense primer and one antisense primer
one Template DNA strand (double strand)
dNTP’s
1 x 2 = 2 x 2 = 4 x 2 = 8 x 2 = 16 x 2 = 32 x 2 = 64 x 2 = 128 x 2 = 256 x 2 = 512 x 2
= 1,024 x 2 = 2,048 x 2 = 4,096 x 2 = 8,192 x 2 = 16,384 x 2 = 32,768 x 2 = 65,536 x 2
= 131,072 x 2 = 262,144 x 2 = 524,288 x 2 = 1,048,576
In principle, over one million copies per original, can be obtained after just twenty cycles
KARY B. MULLIS, 1993 Nobel Prize in Chemistry for his invention of the
polymerase chain reaction (PCR) method.
364
Polymerase Chain Reaction
5'
3'
3'
5'
5'
3'
3'
5'
95 °C
denaturation
5'
3'
72 °C
Taq, Mg 2+, dNTPs
55 - 68 °C
3'
3'
anneal
(+) and (-) primers
extension
5'
3'
5'
3'
3'
5'
5'
3'
5'
3'
3'
5'
5'
3'
3'
5'
95 °C
3'
denaturation
5'
2nd cycle
2 copies of DNA
repeat temperature cycles
amplification of DNA
For a PCR animation go to: http://www.blc.arizona.edu/INTERACTIVE/recombinant3.dna/pcr.html
http://users.ugent.be/~avierstr/principles/pcrani.html
365