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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 informationribonucleic acid (RNA): an intermediate in the expression
of genetic information and other diverse rolesThe Central Dogma (F. Crick):
DNA mRNA Protein (genome) (transcriptome) (proteome)
The monomeric units for nucleic acids are nucleotidesNucleotides are made up of three structural subunits
1. Sugar: ribose in RNA, 2-deoxyribose in DNA2. Heterocyclic base3. Phosphate
2
sugar base
sugar base
phosphate
sugar base
phosphate
sugar base
phosphate
sugar base
phosphate
nucleoside nucleotides
nucleic acids
Nucleoside, nucleotides and nucleic acids
The chemical linkage between monomer units in nucleic acidsis a phosphodiester
3
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)
N
NH N
N
N
N
purine
pyrimidine
N
NH N
N N
NH N
NH
NH2 O
NH2
NH
N
O
NH2
NH
NH
O
O
NH
NH
O
O
H3C
adenine (A)DNA/RNA
guanine (G)DNA/RNA
cytosine (C)DNA/RNA
thymine (T)DNA
uracil (U)RNA
1
2
34
567
8
9
1
2
3
4
5
6
28.2: Nucleosides. N-Glycosides of a purine or pyrimidine heterocyclic base and a carbohydrate. The C-N bond involvesthe anomeric carbon of the carbohydrate. The carbohydrates for nucleic acids are D-ribose and 2-deoxy-D-ribose
4
Nucleosides = carbohydrate + base (Table 28.2, p. 1168) ribonucleosides or 2’-deoxyribonucleosides
OHO N
HO
N
N
N
X
NH2
OHO N
HO
N
N
NH
X
O
NH2
RNA: X= OH, adenosine (A)DNA: X= H, 2'-deoxyadenosine (dA)
RNA: X= OH, guanosine (G)DNA: X= H, 2'-deoxyguanosine (dG)
OHO
HO X
N
N
O
NH2
OHO
HO
N
NH
O
O
H3C
RNA: X= OH, cytidine (C)DNA: X= H, 2'-deoxycytidine (dC)
DNA: thymidine (T)
OHO
HO
N
NH
O
O
RNA: R= H, uridine (U)
1
2
34
567
89
1'
2'3'
4'
5'
OH
1'
2'3'
4'
5' 21
3
45
6
To differentiate the atoms of the carbohydrate from the base, theposition number of the carbohydrate is followed by a ´ (prime).
The stereochemistry of the glycosidic bond found in nucleic acids is .
5
28.3: Nucleotides. Phosphoric acid esters of nucleosides.Nucleotides = nucleoside + phosphate
O
HO X
HO B
ribonucleoside (X=OH)deoxyribonucleoside (X=H)
ribonucleotide 5'-monophosphate (X=OH, NMP)deoxyribonucleotide 5'-monophosphate (X=H, dNMP)
O
HO X
O BPHO
O
O
O
HO X
O BPO
O
O
PHO
O
O
O
HO X
O BPO
O
O
PO
O
O
PHO
O
O
ribonucleotide 5'-diphosphate (X=OH, NDP)deoxyribonucleotide 5'-diphosphate (X=H, dNDP)
ribonucleotide 5'-triphosphate (NTP)deoxyribonucleotide 5'-triphosphate (X=H, dNTP)
O
O OH
O B
PO
O
ribonucleotide3',5'-cyclic phosphosphate (cNMP)
Kinase: enzymes that catalyze the phosphoryl transfer reactionfrom ATP to an acceptor substrate. M2+ dependent
OHOHO
OHOH
OH
O N
N
NN
NH2
HO OH
OPO
O
O
P
O
O
O
PO
O
O
OHOHO
OHOH
OPO32-
ATP
O N
N
NN
NH2
HO OH
OPO
O
O
P
O
O
O
ADPGlucose-6-phosphateGlucose
6
O N
N
NN
NH2
HO OH
OPO
O
O
P
O
O
O
PO
O
O
ATP
O N
N
NN
NH2
HO OH
OPO
O
O
AMP
O
O OH
O
PO
ON
N
NN
NH2
-P2O7
adenylylcyclase H2O
cAMP
O N
N
NNH
O
HO OH
OPO
O
O
P
O
O
O
PO
O
O
GTP
O N
N
NNH
O
HO OH
OPO
O
O
GMP
O
O OH
O
PO
ON
N
NNH
O
-P2O7
guanylatecyclase H2O
cGMP
NH2 NH2 NH2
1971 Nobel Prize in Medicine or Physiology: Earl Sutherland
28.4: Bioenergetics. (Please read)28.5: ATP and Bioenergetics. (Please read)
+ HPO4 2-O N
N
NN
NH2
HO OH
OPO
O
O
P
O
O
O
PO
O
O
ATP
H2O O N
N
NN
NH2
HO OH
OPO
O
O
P
O
O
O
ADP
+ ~31 KJ/mol (7.4 Kcal/mol)
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28.6: Phosphodiesters, Oligonucleotides, and Polynucleotides. The chemical linkage betweennucleotide 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.
By convention, nucleic acid sequences are writtenfrom left to right, from the 5’-end to the 3’-end.
Nucleic acids are negatively charged
28.7: Nucleic Acids. 1944: Avery, MacLeod & McCarty - Strong evidence that DNA
is genetic material1950: Chargaff - careful analysis of DNA from a wide variety of
organisms. Content of A,T, C & G varied widely accordingto the organism, however: A=T and C=G (Chargaff’ Rule)
1953: Watson & Crick - structure of DNA (1962 Nobel Prize with M. Wilkens, 1962)
RNA, X= OHDNA, X=H
O
O
O X
Base
POO
O
O
O X
Base
P
OO
O
3'
3'
5'
5'
3'
5'
8
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
Wrong tautomers !!
Watson, J. D. The Double Helix, 1968
N
N
NN
N
H
dR
N
N
NN
N
H
dR
HN
N
dR
OH
O
N
N
dR
OH
O
purine - purine pyrimidine - pyrimidine
N
N
dR
NH
O
N
N
dR
NH
O
H
N
N
NN
O
N
H
dR
N
N
NN
O
H
dR
HH
H
NH
H
H
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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 hydrogen- bonding between specific pairs of bases.
O
OR
RON
N
N
O
H
H
O
OR
RO
N
N N
NO
N
H
H
HHydrogen Bond
Donor
Hydrogen BondDonor
Hydrogen BondAcceptor
Hydrogen BondAcceptor
3'
5'
Hydrogen BondAcceptor
Hydrogen BondDonor
5'
O2
N4 O6
3'
Antiparallel C-G Pair
N3 N1
N 2
O
OR
RON
N
O
O
O
OR
RO
N
N N
NN
H
H
H
5'
3'
3'
5'
Hydrogen BondDonorN6
Hydrogen BondAcceptor
Hydrogen BondAcceptor O4
Hydrogen BondDonor
Antiparallel T-A Pair
N1N3
10
DNA double helix
one helical
turn34 Å
major groove12 Å
minor groove
6 Å
backbone: deoxyribose and phosphodiester linkagebases
11
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.
Pdb code 1kx5
12
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, whichcodes for the protein sequence
“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
13
DNA is replicated by the coordinated efforts of a number of proteins and enzymes.
For replication, DNA must be unknotted, uncoiled and thedouble helix unwound.
Topoisomerase: Enzyme that unknots and uncoils DNAHelicase: 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 DNAcontains one parental (old, template) strand and onedaughter (newly synthesized) strand
14
Unwinding of DNA by helicases expose the DNA bases (replication fork) so that replication can take place. Helicasehydrolyzes ATP in order to break the hydrogen bonds betweenDNA strands
DNA replication
15
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
G
O
O
OPO
-O
A
O
O
O
T
O
OH
O P OO
C
O
OH
O
O-PO-
OO P
O-
OO-
Mg2+
5'
3'
5'templatestrand(old)
(new)
3’-hydroxyl group of the growing DNA strand acts as a nucleophile and attacks the -phosphorus atom of the dNTP.
dNTP
16
Replication of the leading strand occurs continuously in the 5’ 3’ direction of the new strand.
Replication of the lagging strand occurs discontinuously. ShortDNA 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/
17
DNA replication occurs with very high fidelity:Most DNA polymerases have high intrinsic fidelityMany DNA polymerases have “proof-reading”
(exonuclease) activityMismatch 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
disperse in the cell.
18
Transcription: only one of the DNA strands is copied (codingor 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.
19
5'-cap 5'-UTR start stop 3'-UTRCoding sequence
The mRNA is positioned in the ribosome through complementarypairing 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 catalyzesprotein and peptide biosynthesis using specific, complementary,anti-parallel pairing interactions between mRNA and the anti-codon loop of specific tRNA’s.
20
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 base-pairing between the mRNA and the tRNA positions the tRNAs for amino acid transfer to the growing peptide chain.
aminoacyl t-RNA
TC loop
D loop
variable loop
21
28.12: Protein Biosynthesis. Ribosomal protein synthesis
P site
U G U AA U C U CG U U
3'5'
A site
A CU
OO
HN
SCH3
OHC
U G U AA U C U CG U U
3'5'
A CU
OO
HN
SCH3
OHC
U AA
OO
H2N
OH
U G U AA U C U CG U U
3'5'
U AA
OO
HN
OHONH
CH3S
A CU
OH
OHC
U G U AA U C U CG U U
3'5'
U AA
OO
HN
OHONH
CH3S
A CU
OH
OHC
U G U AA U C U CG U U
3'5'
U AA
OO
HN
OHONH
CH3S
A CU
OH
OHC
E siteE site
U G U AA U C U CG U U
3'5'
U AA
OO
HN
OHONH
CH3S
OHC
G AC
O O
CH3H2N
U G U AA U C U CG U U
3'5'
O
HN
OH
O
HN
G AC
O O
CH3HN
SCH3
U AA
OH
OJHC
U G U AA U C U CG U U
3'5'
O
HN
OH
O
HN
G AC
O O
CH3HN
SCH3
U AA
OH
OHC
U G U AA U C U CG U U
3'5'
O
HN
OH
O
HN
G AC
O O
CH3HN
SCH3
U AA
OH
OHC
22
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 cleaveDNA 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’
EcoR I
BAM HI
5'
3'
3'
5'
5'
3'
3'
5'
OH 2-O3PO
2-O3PO OH
restrictionenzyme
23
Sanger Sequencing:key reagent: dideoxynucleotides triphosphates (ddNTP)
N
NN
N
NH2
OOPO
O-
O
P
O
O-
OP
O-O
O-
NH
NN
N
O
OOPO
O-
O
P
O
O-
OP
O-O
O-
ddATP
NH2
ddGTP
OOPO
O-
O
P
O
O-
OP
O-O
O-
OOPO
O-
O
P
O
O-
OP
O-O
O-
ddTTP ddCTP
N
NH
O
ON
N
NH2
O
When a ddNTPis incorporatedelongation of the primer is terminated
The ddNTP isspecificallyincorporatedopposite itscomplementarynucleotide base
DNA polymerase
3'
5'-32P3'
5'
primer
Templateunknown sequence
Mg2+, dNTP's+ one ddATP
O
N3
HO N
NH
O
OOHO N
N
N
NH
O
S
O OHN
N
H2N
OOHO N
N
NH2
O
ddIAZT
(-)-3TC
Anti-Viral Nucleosides
d4C
24
Sanger Sequencing
Larger fragments
Smallerfragments
GTAACGTAATCACAG
ddA ddG ddC ddT
CATTGCATTAGTGTC
5'32P
3'
5'
3'
32P-5' 3'
primer template
25
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 readingPfu: 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.
26
Polymerase Chain Reaction
For a PCR animation go to: http://www.blc.arizona.edu/INTERACTIVE/recombinant3.dna/pcr.html http://users.ugent.be/~avierstr/principles/pcrani.html
5'
3'
3'
5'
95 °C
denaturation
5' 3'
3' 5'
anneal (+) and (-) primers
55 - 68 °C
5' 3'
3' 5'
3'3'
72 °CTaq, Mg 2+, dNTPs
extension
5'
3'
3'
5'
5'
3'
3'
5'
95 °C
denaturation
2nd cycle
5'
3'
3'
5'
5'
3'
3'
5'
amplification of DNA
2 copies of DNA
repeat temperature cycles
