Download 212 Chapter 28 Biomolecules: Heterocycles and Nucleic Acids

Chapter 28 Biomolecules: Heterocycles and Nucleic Acids
Heterocycles: cyclic organic compounds that contain rings atoms
other than carbon (N,S,O are the most common).
28.1
28.2
Five-Membered Unsaturated Heterocycles (please read)
Structures of Pyrrole, Furan, and Thiophene (please read)
3
3
N1
H
Pyrole
28.3
28.4
28.5
28.6
28.7
3
2
2
O1
Furan
2
S1
Thiophene
Cyclopentadienyl
anion
Electrophilic Substitution Reactions of Pyrrole, Furan,
and Thiophene (please read)
Pyridine, a Six-Membered Heterocycle (please read)
Electrophilic Substitution of Pyridine (please read)
Nucleophilic Substitution of Pyridine (please read)
Fused-Ring Heterocycles (please read)
These sections contain some important concepts that were
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covered previously.
28.8 Nucleic Acids and Nucleotides
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
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
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212
Nucleoside, nucleotides and nucleic acids
phosphate
base
sugar
phosphate
phosphate
base
sugar
base
sugar
base
sugar
phosphate
nucleoside
nucleotides
base
sugar
nucleic acids
The chemical linkage between monomer units in nucleic acids
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is a phosphodiester
The sugar: based on the furanose form D-ribose
ribose for RNA; 2-deoxyribose for DNA
CHO
H
OH
H
OH
HO
5
OH
3
CH2OH
HO
H
HO
O 1 OH
4
O
OH
2
OH
HO
D-ribose
D-2-deoxyribose
The heterocyclic base; there are five common bases for nucleic
acids
7
N
5
N
H
4
NH2
6
N
8
9
N3
1
N
2
N
H
N
H
NH2
4
6
N
N
adenine (A)
DNA/RNA
purine
5
N
3
2
N1
pyrimidine
O
N
O
N
H
O
cytosine (C)
DNA/RNA
NH2
guanine (G)
DNA/RNA
H3C
N
NH
N
O
NH
N
H
O
thymine (T)
DNA
NH
N
H
O
uracil (U)
RNA
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213
Nucleosides: sugar + base
ribonucleosides or 2’-deoxyribonucleosides
NH2
7
5
N
8
5'
HO
O
6
N
9
N
4
N
N
2
HO
3
1'
4'
O
1
N
NH2
2'
3'
X
HO
RNA: X= OH, guanosine (G)
DNA: X= H, 2'-deoxyguanosine (dG)
NH2
N
O
HO
X
HO
O
O
H3C
N
O
X
HO
RNA: X= OH, adenosine (A)
DNA: X= H, 2'-deoxyadenosine (dA)
HO
O
NH
N
NH
NH
O
N
O
HO
O
DNA: thymidine (T)
O
OH
HO
HO
RNA: X= OH, cytidine (C)
DNA: X= H, 2'-deoxycytidine (dC)
N
RNA: R= H, uridine (U)
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Nucleotides: nucleoside + phosphate
HO
HO
O
HO P O
O
B
O
HO
X
ribonucleoside (X=OH)
deoxyribonucleoside (X=H)
O
O
HO P O P O
O
O
B
O
HO
X
nucleotide 5'-diphosphate
28.9
B
O
X
ribonucleotide 5'-monophosphate (X=OH)
deoxyribonucleotide 5'-monophosphate (X=H)
O
O
O
HO P O P O P O
O
O
O
B
O
O
B
O
O
HO
P
O
X
nucleotide 5'-triphosphate
Structure of nucleic acids:
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.
O
OH
ribonucleotide
3',5'-cyclic phosphosphate
3'
O
O P O
O
5'
Base
O
3'
O
X
O P O
O
5'
O
Base
3'
DNA is negatively charged
O
5'
X
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DNA sequences are written from left to right from the 5’ to 3’
5’-ATCGCAT-3’
or
5’-d(ATCGCAT)-3’
28.10 Base Pairing in DNA: The Watson–Crick Model
Chargaff’s Rule: the number of A = T and G = C in DNA
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.
“Molecular Structure of Nucleic Acids” Watson J. D.; Crick, F. H. C. Nature 1953, 171, 737-738
"Molecular Structure of Deoxypentose Nucleic Acids" Wilkins, M. H. F.; Stokes A.R.; Wilson, H. R.
Nature 1953,171, 738-740.
”Molecular Configuration in Sodium Thymonucleate,” Franklin, R.; Gosling, R. G. Nature 1953,
171, 740-741
1962 Nobel Prize in Medicine: F. H. C. Crick, J. D. Watson, Maurice F. H. Wilkins,
“for their discoveries concerning the molecular structure of nucleic acids and its
significance for information transfer in living material.”
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Anti-parallel, complementary hydrogen-bonding of DNA
base pairs
Antiparallel C-G Pair
Hydrogen Bond
Donor
N
5'
O2
H
O
N
5'
Hydrogen Bond
N3
Acceptor
Hydrogen Bond
Acceptor
Bond
O6 Hydrogen
Acceptor
H
N4
RO
N
H
N
O
H
N
N
N
N
O
N1 Hydrogen Bond
RO
Donor
O
Hydrogen Bond
N2
Donor
H
OR
3'
OR
3'
Antiparallel T-A Pair
Hydrogen Bond
Acceptor
H
O4
Hydrogen Bond
N3
Donor
O
5'
RO
N H
OR
N6
N
Hydrogen Bond
Donor
5'
N
N
N
N
O
H N
RO
O
N1 Hydrogen Bond
Acceptor
O
OR
3'
3'
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215
DNA double helix
major
groove
12 Å
one
helical
turn
34 Å
minor
groove
6Å
backbone: deoxyribose and phosphodiester linkage
bases
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DNA Grooves
major groove
guanosine
cytidine
minor groove
major groove
adenosine
thymidine
minor groove
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“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
28.11 Nucleic Acids and Heredity
The Central Dogma (F. Crick):
DNA
DNA transcription mRNA
(genome)
replication
translation
Protein
(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
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codes for the protein sequence
28.12 Replication of DNA: DNA is replicated by the coordinated
efforts of a number of proteins and enzymes.
Each cell contains about two meters of DNA. The DNA must
be “packaged” into the cell nucleus by super-coiling and
knotting.
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 replicates DNA using each strand as
a template for the newly synthesized strand.
DNA replication is semi-conservative: Each new strand of DNA
contains one parental (old, template) strand and one
daughter (newly synthesized) strand
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217
Unwinding of DNA by helicases expose the DNA bases
(replication fork) so that replication can take place
leading strand
DNA polymerase δ
DNA replication
Lagging strand
DNA polymerase ε
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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
OH
5'
O
O O
O P O P O P OO
O- O-
O
T
A
O
O
O
O P
-O
O
template
strand
(old)
Mg2+
OH
O
G
O
dNTP
O
C
5'
(new)
O
3'
3’-hydroxyl group of the growing DNA strand acts as a nucleophile
and attacks the α-phosphorus atom of the dNTP.
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218
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/
432
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
28.13 Structure and Synthesis of RNA: Transcription
RNA contains ribose rather than 2-deoxyribose and uracil rather
than thymine
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
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disperse in the cell.
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Transcription: only one of the DNA strands is copied (coding
or antisense strand). It sequence is converted to the
complementary sequence in mRNA (template or
sense strand), which codes for the amino acid
sequence of a protein (or peptide)
28.14 RNA and Protein Biosynthesis: Translation
• proteins are synthesized in the cytoplasm on ribosomes.
• mRNA is the template for protein biosynthesis.
• a three base segment of mRNA (codon) codes for a
an amino acid.
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The “anticodon” region of of tRNA is complementary for a
to the mRNA codon sequence.
The t-RNA carries an amino acid on the 3’-hydroxyl (aminoacyl
t-RNA) and the ribosome catalyzes amide bond formation.
Although single-stranded, the are complementary sequences
within tRNA that give it a defined conformation
aminoacyl t-RNA
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220
Ribosomal protein synthesis
OH
H3CS
H3CS
H2N
H2N
H2N
O
O
O
O
U A C
U A C
A U G U A U G C U C
O
A U A
A U G U A U G C U C
U U
U U
3'
5'
3'
5'
H3CS
H3CS
O
H2N
OH
O
U A C
O
H2N
HN
HO
O
OH
H2N
HN
O
HO
A U A
A U G U A U G C U C
U A C
U U
5'
O
O
O
A U A
C G A
CH3
O
A U G U A U G C U C U U
3'
3'
5'
3'
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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 DNA polymerase, Mg+2, and
dideoxynucleotides triphosphate (ddNTP) that truncate
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
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221
Sanger Sequencing dideoxynucleotides triphosphate (ddNTP)
NH2
N
O
-O
O
O
N
P O P O P O
O-
O-
-O
O
O
O
P O P O P O
O-
O-
O-
ddATP
3'
N
NH
O
N
N
NH2
O
O
N
O
O
O
-O
O
N
P O P O P O
O-
O-
O
O-
N
NH
N
O
NH2
-O
O
O
O-
O
O
O-
ddGTP
ddTTP
N
P O P O P O
O-
O-
O
ddCTP
5'-32P
primer
Anti-Viral Nucleosides
When a ddNTP
is incorporated
elongation of
the primer is
terminated
3'
DNA polymerase
O
HO
O
O
NH
N
N
N3
AZT
The ddNTP is
specifically
incorporated
opposite its
complementary
nucleotide base
Mg2+, dNTP's
+ one ddATP
N
O
O
N
NH
HO
ddI
NH2
HO
O
H2N
N
N
O
O
N
N
OH
O
S
(-)-3TC
d4C
5'
Template
unknown sequence
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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
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222
Automated Sanger Sequencing with flourescent ddNTP's
H
N
O
HN
O
-3
H3C
N
O
H3O9P3O
O
N
N
O
-3
(CH2)2
H3C
CH3
N
O
N
CH3
O
N
CH3 HN
CH3
NH2
N
N
H3O9P3O
O
O
O
NH
O-
N
-3
O
O-
O
O
CH3
N
H3O9P3O
N
NH2
O
CH3
ddA
ddC
Excitation: ~ 490 nM,
O
O-
O
O
-3
ddT
O
N
H3C
(CH2)2
O
O-
O
O
CH3 HN
H3C
H3O9P3O
CH3
O
O
H
N
NH2
O
ddG
Emission: ddT= 526 nm. ddC= 519 nm, ddA= 512 nm, ddG= 505 nm
Sanger sequencing using flourescent ddNTP's: terminated DNA
strands are separated by capillary electrophoresis,
detected and identified by their fluorescence emission
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Sequencing on a “chip” (Lagniappe)
Spatially addressable:
8-mer chip: 65, 536 different sequences
12-mer chip: 1,677,216 different sequences
DNA fragment
DNA–fluorophore
Place DNA on the chip, then wash away
non-specific hybridization after 1-10 hrs.
Raise temperature and “melt” away
partially hybridized sequences.
3’-ACGGTGCG
CGGTGCGA
GGTGCGAG
GTGCGAGA
TGCGAGAA
GCGAGAAT
etc
3’-ACGGTGCGAGAAT---5’
5’-TGCCACGCTCTTA---3’
(from the chip)
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223
28.16 DNA Synthesis: short segments of DNA can be efficiently
by automated, solid-phase methods.
Solid support: controlled pore glass (silica)
linked to the 3’-hydroxyl group of the first nucleotide
solid phase DNA synthesis is from 3’→5’, which is the
opposite direction from nature.
Protected nucleotide reagents:
5’-protecting group: 4,4’-dimethyoxytrityl (trityl is a
triphenylmethyl group), abbreviated DMT
OCH3
5’-DMT ether
C
O
O
Removes with mild acid
(Cl3CCO2H)
Base
HO
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OCH3
The base amino groups of dA, dG and dC must be protected,
usually as an amides. The base of T does not require
further protection.
O
O
HN
N
N
N
HN
O
N
R
N
N
NH
N
R
A
O
H3C
N
O
N
N
H
O
R
G
NH
N
O
R
C
T
The 3’-phosphorous group: phosphoramidite
HO
O
B
DMTr-Cl
pyridine
HO
Base is protected
DMTrO
O
B
N
P
O
CN
DMTrO
O
B
Cl
HO
pyridine
O
NC
O
P
N[CH(CH3)2]
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224
Automated, solid-phase DNA synthesis
S
I
L
I
C
A
S
I
L
I
C
A
OH
OH
OH
S
I
L
I
C
A
O
Si (CH2)3 NH2
O
O
O
O
O
Si (CH2)3 N C (CH2)2 C OH
H
O
O
DMT-O
B2
O
DMT-O
S
I
L
I
C
A
B1
N
O
O
HO
O
O
I2, H2O
CN
O
O
oxidation of P
N (iPr)2
CN
DMT-O
B2
B3
O
O
O
P
O
coupling
O
S
B1
P
O
NH
N
5'- deprotection
repeat cycle
O
O
N
O
DMT-O
DMT-O
B1
Cl3CCOOH
Si (CH2)3 N C (CH2)2 C O
H
O
O
O
O
P O
O
CN
Cl3CCOOH
5'- deprotection
B1
B
O
P O
CN
O
B2
O
O
O
S
S
O
O
O
O
P O
CN
O
B1
O
HO
B3
HO
B3
O
Cl3CCOOH
5'- deprotection
O
S
O
O
P O
CN
O
NH3, H2O, !
B2
deprotect P
and the base
amino groups
O
O
O
P O
CN
O
B1
O
O
P O
O
B2
O
O
O
P O
O
B1
O
S
O
O
O
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OH
28.17 Polymerase Chain Reaction (PCR): method for
amplifying DNA using DNA polymerase and cycling
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
O
O
O
-O P O P O P O
O B
dNTP’s
OOOHO
KARY B. MULLIS, 1993 Nobel Prize in Chemistry for his invention of
the polymerase chain reaction (PCR) method.
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225
A typical PCR temperature cycle
Denaturation:
94 °C
0.5 - 1 min
Annealing:
55-68 °C
0.5 - 1 min
5 °C below the
Tm of the primer
Extension:
(replication)
72 °C
1 min
+ 1 min per Kb
of DNA
# of cycles
25 - 35
Final extension
72 °C
10 min
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
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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'
denaturation
3'
5'
2nd cycle
5'
3'
3'
5'
5'
3'
3'
5'
95 °C
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
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