212
418
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 Five-Membered Unsaturated Heterocycles (please read)28.2 Structures of Pyrrole, Furan, and Thiophene (please read)
28.3 Electrophilic Substitution Reactions of Pyrrole, Furan, and Thiophene (please read)
28.4 Pyridine, a Six-Membered Heterocycle (please read)28.5 Electrophilic Substitution of Pyridine (please read)28.6 Nucleophilic Substitution of Pyridine (please read)28.7 Fused-Ring Heterocycles (please read)
These sections contain some important concepts that werecovered previously.
NH
O S
Pyrole Furan ThiopheneCyclopentadienyl
anion
1
2
3
1
2
3
1
2
3
419
28.8 Nucleic Acids and NucleotidesNucleic 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) (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
213
420
Nucleoside, nucleotides and nucleic acids
The chemical linkage between monomer units in nucleic acidsis a phosphodiester
sugar base
sugar base
phosphate
sugar base
phosphate
sugar base
phosphate
sugar base
phosphate
nucleoside nucleotides
nucleic acids
421
The sugar: based on the furanose form D-riboseribose for RNA; 2-deoxyribose for DNA
OHOOH
HO OH
CHO
OHH
OHH
OHH
CH2OH
1
23
4
5
OHOOH
HO
D-ribose D-2-deoxyribose
The heterocyclic base; there are five common bases for nucleicacids
N
NH
N
N
N
N
purine
pyrimidine
N
NH
N
NN
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
5
67
8
9
1
2
3
4
5
6
214
422
Nucleosides: sugar + baseribonucleosides 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
3
4
567
89
1'
2'3'
4'
5'
OH
423
Nucleotides: nucleoside + phosphate
28.9 Structure of nucleic acids: The chemical linkage between nucleotide units in nucleic acids is a phosphodiester, whichconnects the 5’-hydroxyl groupof one nucleotide to the3’-hydroxyl group of the next.
DNA is negatively charged
O
HO X
HO B
ribonucleoside (X=OH)deoxyribonucleoside (X=H)
ribonucleotide 5'-monophosphate (X=OH)deoxyribonucleotide 5'-monophosphate (X=H)
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
nucleotide 5'-diphosphate nucleotide 5'-triphosphate
O
O OH
O B
P
O
O
ribonucleotide3',5'-cyclic phosphosphate
O
O
O X
Base
PO O
O
O
O X
Base
P
O
O O
3'
3'
5'
5'
5'
3'
215
424
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 ModelChargaff’s Rule: the number of A = T and G = C in DNATwo 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.
“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.”
425
O
OR
RON
N
N
O
H
H
O
OR
RO
N
NN
NO
N
H
H
H
Hydrogen BondDonor
Hydrogen BondDonor
Hydrogen BondAcceptor
Hydrogen BondAcceptor
3'
5'
Hydrogen BondAcceptor
Hydrogen BondDonor
5'
O2
N4 O6
3'
Antiparallel C-G Pair
N3 N1
N2
O
OR
RON
N
O
O
O
OR
RO
N
N N
NN
H
H
H
5'
3'
3'
5'
Hydrogen BondDonor
N6
Hydrogen BondAcceptor
Hydrogen BondAcceptor O4
Hydrogen BondDonor
Antiparallel T-A Pair
N1N3
Anti-parallel, complementary hydrogen-bonding of DNA base pairs
216
426
DNA double helix
one helical
turn34 Å
major groove12 Å
minor groove
6 Å
backbone: deoxyribose and phosphodiester linkagebases
427
DNA Grooves
cytidine guanosine
thymidine adenosine
minor groove
major groove
minor groove
major groove
217
428
28.11 Nucleic Acids and HeredityThe Central Dogma (F. Crick):
DNA replication DNA transcription mRNA translation Protein (genome) (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 isconverted 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
429
28.12 Replication of DNA: DNA is replicated by the coordinatedefforts of a number of proteins and enzymes.
Each cell contains about two meters of DNA. The DNA mustbe “packaged” into the cell nucleus by super-coiling and knotting.
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 replicates DNA using each strand as
a template for the newly synthesized strand.
DNA replication is semi-conservative: Each new strand of DNAcontains one parental (old, template) strand and onedaughter (newly synthesized) strand
218
430
Unwinding of DNA by helicases expose the DNA bases (replication fork) so that replication can take place
leading strandDNA polymerase δ
Lagging strandDNA polymerase ε
DNA replication
431
DNA Polymerase: the new strand is replicated from the 5’ → 3’(start from the 3’-end of the template) DNA polymerases are Mg2+ ion dependentThe deoxynucleotide 5’-triphosphate (dNTP) is the reagent
for nucleotide incorporation
G
O
O
OP
O
-O
A
O
O
O
T
O
OH
O P O
O
C
O
OH
O
O-PO-
O
O P
O-
O
O-
Mg2+
5'
3'
5'
templatestrand(old)
(new)
3’-hydroxyl group of the growing DNA strand acts as a nucleophile and attacks the α-phosphorus atom of the dNTP.
dNTP
219
432
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/
433
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
28.13 Structure and Synthesis of RNA: TranscriptionRNA contains ribose rather than 2-deoxyribose and uracil rather
than thymineThere 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 moredisperse in the cell.
220
434
Transcription: only one of the DNA strands is copied (codingor antisense strand). It sequence is converted to thecomplementary 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.
435
The “anticodon” region of of tRNA is complementary for ato the mRNA codon sequence.
The t-RNA carries an amino acid on the 3’-hydroxyl (aminoacylt-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
221
436
U G U AA U C U CG U U
3'
A CU
OO
H2N
H3CS
5'
U G U AA U C U CG U U
3'
A CU
OO
H2N
H3CS
5'
U AA
OO
H2N
OH
U G U AA U C U CG U U
3'
A CU
5'
U AA
OO
HN
OHOH2N
H3CS
HO
U G U AA U C U CG U U
3'
A CU
5'
U AA
OO
HN
OHOH2N
H3CS
HO
U G U AA U C U CG U U
3'5'
U AA
OO
HN
OHOH2N
H3CS
G AC
O O
CH3H2N
Ribosomal protein synthesis
437
DNA sequencingMaxam-Gilbert: relys on reagents that react with a specific
DNA base that can subsequent give rise to asequence 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 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
222
438
Sanger Sequencing dideoxynucleotides triphosphate (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
Template
unknown 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
439
Sanger Sequencing
Larger fragments
Smallerfragments
5'
3'
32P-5' 3'
primer template
G
T
A
A
C
G
T
A
A
T
C
A
C
A
G
ddA ddG ddC ddT
C
A
T
T
G
C
A
T
T
A
G
T
G
T
C
5'
32P
3'
223
440
Automated Sanger Sequencing with flourescent ddNTP's
Excitation: ~ 490 nM, Emission: ddT= 526 nm. ddC= 519 nm, ddA= 512 nm, ddG= 505 nm
N
NN
NH2
O
-3 H3O9P3O
HN
ON
CH3
O
OO O -
CH3H3C
NH
NN
O
O
-3 H3O9P3O
HN
ON
CH3
O
OO O -
NH2
OO O -
O
-3 H3O9P3O
N
N
NH2
O
HN O
NH3C
(CH2)2O
CH3CH3
OO O -
CH3H3CO
-3 H3O9P3O
HN
N
O
O
HN O
NH3C
(CH2)2O
CH3CH3
ddA ddGddT ddC
Sanger sequencing using flourescent ddNTP's: terminated DNA strands are separated by capillary electrophoresis, detected and identified by their fluorescence emission
441
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 awaynon-specific hybridization after 1-10 hrs. Raise temperature and “melt” awaypartially hybridized sequences.
3’-ACGGTGCG CGGTGCGA
GGTGCGAG GTGCGAGA TGCGAGAA GCGAGAAT etc
3’-ACGGTGCGAGAAT---5’ (from the chip)5’-TGCCACGCTCTTA---3’
224
442
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 nucleotidesolid phase DNA synthesis is from 3’→5’, which is theopposite direction from nature.
Protected nucleotide reagents: 5’-protecting group: 4,4’-dimethyoxytrityl (trityl is a triphenylmethyl group), abbreviated DMT
5’-DMT ether C O
OCH3
OCH3
O
HO
Base
Removes with mild acid(Cl3CCO2H)
443
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.
N
N N
NN
N N
NH
HN O
NH
N
N
O
HN
N
NH
O
O
H3C
G
R R R R
O
O
O
TCA
The 3’-phosphorous group: phosphoramidite
OHO B
HO
Base is protected
DMTr-Cl
pyridine
ODMTrO B
HO
NPO
Cl
CN
pyridine
ODMTrO B
O
PO N[CH(CH3)2]
NC
225
444
OH O
O
O
Si (CH2)3 NH2
O
O
O
Si (CH2)3 NH
C (CH2)2
O
C
O
OH
O
O
O
Si (CH2)3 NH
C (CH2)2
O
C
O
O
B1
O
DMT-O
S
O
B
O
PO
O
O
B1
O
DMT-O
CN
PN (iPr)2
O
O
B2
O
DMT-O
CN
OH
OH
O
B1
O
HO
SILICA
SILICA
SILICA
SILICA
Cl3CCOOH
S
Cl3CCOOH
NH3, H2O, !
I2, H2O
coupling
N
NN
NH
oxidation of P
5'- deprotection
O
B1
O
P
O
O
O
B2
O
DMT-O
OCN
S
Cl3CCOOH
5'- deprotection
repeat cycle
O
B1
O
P
O
O
O
B2
O
O
OCN
S
P
O
O
B3
O
DMT-O
OCN
O
B1
O
P
O
O
O
B2
O
O
OCN
S
P
O
O
B3
O
HO
OCN
5'- deprotection
O
B1
OH
P
O
O
O
B2
O
O
O
P
O
O
B3
O
HO
Odeprotect Pand the baseamino groups
Automated, solid-phase DNA synthesis
445
28.17 Polymerase Chain Reaction (PCR): method foramplifying DNA using DNA polymerase and cycling 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 stranddNTP’s O B
HO
OPO
O-
O
P
O
O
O-
P-O
O-
O
KARY B. MULLIS, 1993 Nobel Prize in Chemistry for his invention of the polymerase chain reaction (PCR) method.
226
446
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: 72 °C 1 min + 1 min per Kb (replication) 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
447
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 °C
Taq, 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
Top Related