Structure Determinaon by NMR - Jay Ponder Lab Home Page
39
Structure and Properties of Nucleic Acid Components
Transcript of Structure Determinaon by NMR - Jay Ponder Lab Home Page
Structure and Properties of Nucleic Acid Components
Human DNA: 3 Billion Base Pairs
Bdl001.pdb
Double Helical B DNA
N
N N
N
NH2
N
N
O
O
H CH3
N
N N
N
O
H2N
H
N
N
NH2
O
A
T
G
C
ACGT bases.cwg
COMPLEMENTARY WATSON CRICK BASE PAIRINGBETWEEN TWO ANTIPARALLEL STRANDS
A base recognizes T, and G recognizes C
N
N
O
N
H
H
N
N
O
OP
O
O
-O
H
H
O-
OP
O
O
O
H
O
N
NN
G C
A T
N
N
NN
N
O
OP
O
O
-O
HH
H
CH3
O-
OP
O
O
OO
O
NN
THE FOUR LETTER CODE: A, C, G, T
B DNA Structure
ACGTTGCA
ACGT
CA
GTTGCA
5'3'
3'5'
3'5'
3'5'
5'3'
ACGTTGCA
5'3'
3'5'
ACGTTGCA
5'3'
3'5'
DNA Replication: 5' to 3', template directedvia Watson Crick Base Pairing
DNA Polymerase
GCA
T
CAT G
5'
5'
3' 5'
3'
5'
O
O P O
O O-
OPO-O
O
O
OH
O
OPO-O O
O
OPO- O
O O
OO-P
O
OO
O P O
OTEMPLATE
PRIMER
B:
O
OO-P
O
O
OPO- O
O
O
OH
O
O- OP
P O
P O-
O
OO-
O-
OOP
O-O
O
O
OH
Replication of DNA
DNA Polymerase
A T G T T A
A - UU - A
G - CU - A
A - U
5'
5'
S
H
NO
O
O
H
ON
O
HH
3'
5'
5'
3'
5'pp
p
3'
TRANSCRIPTION
mRNA
aa-tRNA
DNA
DNA to RNA to PROTEIN
..
codo
n
anti-codon
TRANSLATION
tRNA
Ribosome
O
OH
OH
O
O- OP
P O
P O-
O
OO-
O-
O
O
OH
OH
O
OPO- O
O O
OO-P
O
B:
POLYMERIZATION
TEMPLATE
OO
O P O
O
O
OO-P
O
O
OPO- O
O
O
OPO-O O
O
OH
OHO
O P O
O O-
OH
3'
5'3'
5'
ACG U
CG U
A
PO
P-O
O O-O O-
O
O O-
P O
PO
P-O
O O-O O-
O
O O-
P O
DNA to RNA: Transcription by RNA polymerase
RNA Polymerase
fMetformylmethionine
mRNA to Protein: Translation
N
O
H
SCH3
H
O
NR2
O
B
OHO
O
O
HNH2
R3
O
B
OHO
O
OB
OHHO
O
O
N
O
H
SCH3
H
O
NR
O
B
OHO
O
O
H
shiftposition
O
O
O OH
B
O
R
NH2
O
H
SCH3
H
O
N
B
OHO
O
O
N'N'N'
O
O
O OH
B
O
RNH2
5'
U A C
O
O
O OH
B
ON
O
H
SCH3
H
5'
tailcoding region stopcodon
startcodon
3'U A G
leader
' A U G N N N
Translation
OHO OH
OHHO
CHO
OH
OHOH
OH O
OH
HO
OHHO
O
H
HO OH
HO
CHO
OH
OHOH
H O
OH
HO
HHO
OH
OHCHO
CH2OHOHH
OH
THE SUGAR SUBUNIT OF DNA AND RNA
+
+
D-(-)-ribose D-ribofuranoside
alpha-anomer beta-anomer
beta-anomer alpha-anomer
D-(+)-glyceraldehyde
FISHER PROJECTION
2-deoxyribose
D-deoxyribofuranoside
POP-OO- O-
O
O OO Base
HO OH
O Base
OH
HO
HO
OH
O
O
O-Base-O OP
HO
OH
P POPOO- O-
O
O OO Base
HO
O
O-
-O
α
α
αβ
β
γ
NTP or dNTP
guanylic acid
cytidylic acidadenylic acid
guanosinecytidineadenosine
NDP or dNDP
NMP or dNMPNucleotide monophosphate:
Nucleoside:
uradylic acid (RNA)
thymidylic acid (DNA)
uridine (RNA)thymidine (DNA)
NOTE: In DNA the OHin the brackets is absent
OH
OBaseO
OPO
-O
5'
3'
2',3'-cyclic AMP3',5'-cyclic AMP(cAMP)
Cyclic Nucleotide
Nucleosides and Nucleotides
O
OBaseHO
O
P-O O
3' 2'
O
O P
O-
O
O O O
O
H
O
O
N
N
H
O
O
N
N H
CH3
H
CH3
O
T
OP
O
T
OO
O-
dNpdNpdN = d(NpNpN) = d(NNN) for oligodeoxyribonucleotides
NpNpN = NNN for oligoribonucleotides
d(NNN)Nd(NNN) for mixed oligo ribo and deoxyribonucleotides (embedded substrates, such as embedded RNA)
When strands associate the two sequences are given, both in the 5'-3'
d(ACGTATG)·d(CATACGT)
Polymers.
poly (dA) : homopolymer d(AAA)n = d(AAAAAAA...)poly (dA-dT): heteropolymer d(AT)n = d(ATATATA...)poly (dA,dT): random copolymer d(ATTAATTTTATATTA...)
When polymer strands associate the stoichiometry is given: poly (A) · 2 poly(U)
Structure and Nomenclaturefor dinucleotides and polymers
Table. Rate of hydrolysis of the bases at pH 1. (Kochetkov and Budovskii 1972)
nucleoside rate (´ 104 sec) dA 4.3 dG 8.3 dC .0011 dU .001 A .0036 G .0093 C .00001 U .00001
O
O OP
O
OO- O-
O
O
P
OH B
HO H
O
O OP
O
OO- O-
O
O
P
O
N
O OP
O
OO- O-
O
O
P
NN
NH2N
H
O
N
O OP
O
OO- O-
O
O
P
NN
NH2N
File: acidhyd.cwg
Mechanism for the acid hydrolysis of theglycosidic bond in DNA.
O
Base
O OP
O
OO- O-
O
O
P
O
O OP
O
OO- O-
O
O
P
OH
O
O OP
O
OO- O-
OOP
H
OH H
H
hemiacetalabasic site
O
O OP
O
OO- O-
OOP
H
OH
B
O
OP
O
OO-
H
OH
H -O
O-O
O
P
B
OH
OH
-O
O-O
O
PO-P
O
OO-
3'-phosphate 5'-phosphate
enolization
β-elimination(retro-Michael Rxn)
β-elimination(retro-Michael Rxn)
hydrolysis
Mechanism of strand cleavagefollowing glycosidic bond hydrolysis
BB H
In the presence of piperidine, the b-elimination reactions may take place through the enamine.
Path B
Path A
3'-phosphate
2'-phosphateH
OH B
HB
O
O
OP
O
B
OO
O
P
H
H
O
O
OP
O
PO O
B
O
O
OP
O
PO O
B
HO
H
2',3'-cyclicphosphate
H
OHB
H B
O
O
OP
O
PO O
B
transesterificationO
O
O
P
P
O
OO O
R
H
B
B
B H
O
O
OP
O
PO O
B
H B
BO
H
2',3'-cyclicphosphate
RNA strand cleavage under basic condition
Table. Basic Hydrolysis of RNA. Substrate Conditions Time Temperature Result ApA 0.5 N NaOH 8 h 28ºC 47% hydrolysis RNA 0.1 N NaOH 20 min 100ºC mononucleotides RNA 10% piperidine 90 min 100ºC mononucleotides RNA 1.0 N NaOH 0.7 min 27ºC 1% cleavage RNA pH 9 30 min 70ºC 1% cleavage Table. Acidic Hydrolysis of RNA. Substrate Conditions Time Temperature Result ApA 0.3 N HCl 8 h 45ºC 69% hydrolysis RNA pH 1 100 min 20ºC 10% cleavage RNA pH 2 4 min 100 ºC 10% cleavage RNA pH 3 40 min 100 ºC 10% cleavage
Rates of Hydrolysis of RNA under basic and acidic conditions
NN
N
NH
H
H
H
NN
H
H
H
H
NN
NN
N
N
O
N
H
HH
H N
O H
H
HH
H
H
NN
N
N
N HH
H
H
H NN
O
O
CH3
HH
H
9
8
Bases are shown in their Watson Crick base-pairing arrangement
Gua Cyt
ThyAdothymineadenine
cytosineguanine
6
54
3
21
7
65
4
3 2
1
PyrimidinesPurines
Basic Constituents of Nucleic AcidsTHE BASES
The Four Letter Code: A, C, G, Twhere A recongizes T and C recognizes G
Minor RNA nucleosides, adenine derivatives
m1A m6A
m2A
N
NN
N
NH2
CH3
N
NN
N
NH2
CH3 N
NN
N
NCH3H
N
NN
N
N
O
N
CO2H
OH
CH3
H
H
N
NN
N
NH
N
NN
N
NH
S CH3
N
NN
N
N
O
N
OH
CH3
H
H
O N
OH OH
OH
H
N
NN
N
NHH
OHO
HO OCH3
N
NN
N
O
H
N
NN
N
O
CH3
I m1I
AmN+
t6Ams2t6Ai6A
(dm6A in DNA)
Minor RNA nucleosides, cytosine derivatives
N
N
O
CH3
NH
m3C
N
N
S
NH2
H3C
s2m5C
N
N
O
NH2
OHO
HO OCH3
Cm
s2C
N
N
S
NH2
N
N
O
N
O
CH3
H
m5C
N
N
S
NH2
H3C
ac4C
(dm5C in DNA)
N
N
O
NH2
OR
(DNA only, R=H or a sugar, foundin T even bacteriophages)
Minor RNA nucleosides, guanine derivatives
m1G
m2G
N
NN
N
O
CH3
NH2 N
NN
N
O
NCH3
H
H
N
NN
N
O
OHO
HO OCH3
NH2
H
Gm
N
NN
N
O
NCH3
CH3
H
m2G2
N
NN
N
O
NH2
HH3C
m7G
N
NN
N
O
CH3
N
N O
OCH3
OCH3O
H
CH3
Y
(dm2G in DNA)2
(dm7G in DNA)
(dm1G in DNA)
N
N
O
H
O
Minor RNA nucleosides, Uracil derivatives
D or hU
N
N
O
CH3
O
m3U rT or T
N
N
O
H
O
O
O
HO
V
N
N
O
H
O
RO
O
cm5U R=H, cmm5U, R=CH3
N
N
O
H
S
N
N
S
H
O
H3C
N
N
O
H
O
H3C
N
N
O
H
O
NCH3
HN
N
O
H
O
RO
O
N
N
S
H
O
RO
O
s2cm5U R=H, s2am5U, R=CH3
s2mam5Us2m5U s2U
N
N
O
O CO2H
NH2 N
N
O
H
O
OHO
HO OCH3
UmX
N N
O
H
O
OHO
HO OCH3
H
Y or YU
N
N
O
H
O
HO
(found in DNA of SP 8bacteriophage)
N
NN
N
O
NH
H
H
N
NN
N
HN
H
N
N
O
NHH
N
N
O
O
HCH3
Localization of lone pairs in bases.
N
NN
N
HN
H
N
N
O
NHH
N
NN
N
O
NH
H
H
N
N
O
O
HCH3
In plane lone pairs, σ type
out of plane lone pairs, p typeconjugated to π system
POH
O
O
O Base
HO OH
HO
OH
O
O
O
BaseHO OP
O
HO
OH
O
O
OBaseOP
N
NN
N
HN
H
H
N
N
O
NHH
H
N
NN
N
O
N
H
H
H
N
N
O
O
HCH3
N
N
O
O
H
OH
O
O
OBaseO OP
O
HO
OH
O
O
O
BaseOP
P
OO
O
O Base
HO OH
HO P
OO
O
O Base
HO OH
O
pKa=9.7
pKa=7pKa=1
pKa=9.4 pKa=9.3pKa=4.2pKa=3.5
+ +
pKa = 1
H
uracilthymineguaninecytosineadenine
ACID-BASE PROPERTIES
H H
N
N
O
O
CH3
H
N
NN
N
O
NH
H
H
N
NN
N
NH
H
N
NN
N
HN
H
N
NN
N
NH
H
etc
N
N
O
NHH
N
N
O
NH
H
N
N
O
NH
H
N
NN
N
O
NH
H
H
N
NN
N
O
NH
H H
N
N
O
O
HCH3
N
N
O
O
CH3
H
TAUTOMERS OF THE BASES
amino imino imino
keto enol keto
amino/keto imino/keto imino/enol
keto enol enol
No.
Figure
homo purine 7 homops1.cw2 pyrimdine 5 homopy1.cw2
hetero purine 4 hetpu1.cw2 pyrmidine 1 homopy1.cw2
purine-pyrimidine
AT 4 pupybp1.cw2
GC 2 Total 23
Base Pairing Combinations
ΝΝ
Ν
Ν
Ν ΗΗ
ΗΗ Ν
Ν
Ν
ΝΝ
homo purine - symmetric
Ν
Ν
Ν
Ν
Ν
Η
Η
Η
Η
Ν
ΝΝ
Ν
Ν
Ν Ν
Ν
Ν Ο
Η
ΝΗΗ
ΗΗΝ
Η
Ο Ν
Ν
Ν ΝΝ
Ν
ΝΝ
Ο
Η
Ν
Η
Η
Η
Η
ΝΗ
Ο
ΝΝ
Ν
Ν
homo purine - asymmetric
ΝΝ
Ν
Ν
Ν ΗΗ
Η
Η
Ν
Ν
Ν
Ν
Ν
Ν
Ν
Ν
Ν
Ο
Η
Ν Η
Η
Η
Η
ΝΗ
Ο
ΝΝ
Ν
Ν
ΝΝ
Ν
Ν
Ο
Η
Ν ΗΗ Η
Η
ΝΗ
Ο
ΝΝ
Ν
Ν
1 2
3 4
56
7
Base pairing schemes for identical purines
anti
anti
Hoogsteen
Reverse Hoogsteen
homo pyrimidine, symmetric
Ν Ν
Ο
Ο
Η
Η
Ο
Ο
Ν Ν
Ν
Ν
Ο
Ο
ΗΝ
Ν
Ο
Ο
Η
ΝΝ
Ν
Ο
Η
Η
Η
Η
Ο
Ν
ΝΝ
Ν
Ν
Ν
Ο
ΗΗ
ΗΗ
Ο
Ν
Ν
Ν
Ν
Ν
Ο
Ο
ΗΝ
Ν
Ν
Ο
ΗΗ
homo pyrimidine, asymmetric
12 13
1415
16
ΝΝ
Ο
Ο
ΗΝ
Ν
Ν
Ο
ΗΗ
Ν
Ν
Ο
Ο
Η
Η
Ο
Ο
Ν Ν
17
18
hetero pyrimidine, symmetric
Base pairing schemes for pyrimidines
anti
anti
ΝΝ
Ν
Ν
Ν ΗΗ
ΗΗ Ν
Η
Ο
Ν
Ν
ΝΝ
hetero purine symmetric heteropurine - asymmetric
89
10 11
ΗΗ Ν
Ν
Ν
ΝΝ
ΗΗΝΗ
Ο
ΝΝ
ΝΝ
Η
ΗΝ
Ν
Ν
Ν
ΝΝΝ
Ν
ΝΟ
Η
ΝΗ Η
ΗΗ
Ν Η
Ο
Ν Ν
ΝΝΗ
Η
Ν
Ν
Ν
Ν
Ν
Base Pairing Schemes for mixed purines
anti
anti
ΝΝ
Ν
Ν
Ν ΗΗ
Η
Ο
Ο
ΝΝΝ
Ν
Ν
Ν
Ο
Η
Ν ΗΗ
ΗΗ
Ο
Ν
ΝΝ
19 20
Pseudosymmetric, Watson-Crick Base Pair
Ν
ΝΝ
Ν
Ο
Η
Ν Η
Η
Ν
Ν
Ν
Ο
Η ΗΝ
Ν
Ν
Ν
Ν ΗΗ
ΝΝ
Ο
Ο
Η
21 22
Η
Ο
Ο
ΝΝ
Η
Η
Ν
Ν
Ν
Ν
Ν
23
Η
Ο
Ο
Ν
Ν
Ν
Ν
Ν
Ν
Ν
Η
Η
24
Hoogsteen Reversed Hoogsteen
Reversed Watson-Crick, assymetric
Ν Ν
Ν
ΝΟ
Η
Ν ΗΗ
ΝΝ
Ο
Ο
Η
ΝΝ
Ν
Ο
Η
Η
Η
Η
Ν
ΝΝ
Ν
Ν
Ν
Ν
Ο
Ο
Η
ΗΗΝ
Η
ΟΝ
Ν
ΝΝ
ΝΝ
Ν
Ο
Η
Η
ΝΝ
Ν
ΝΝΗ
Η
25 26
2728
Purine-Pyrimidine Base Pairs
anti anti
anti
anti
anti
Conformational Analysis of Nucleosides and Nucleotides
H5"H5'
OC4'
C5'
O
O3'
H3'
C3'
OH4'
PO2-
OH1'O
H1'
α β
γ δ
ε ζ
χ χ
Definition of Angles in Nucleotides
pyrimidines
C2
C6
C4
N8
purines
C4'O
H3'
H5'
H5" O5'H4'
C5'
O3'O
O
PO
PO2-
O5'O
O
O
C3'
OBase
OH
HO
τ1
τ2
τ3
τ4 τ0 The torsion bonds aredefined by the sugar ringatoms. Example, τ1 is defined by O4'-C1'-C2'-C3'
Definition of the bond angles in deoxyribosering system and the pseudorotation equation
Pseudorotation equation:
τi = τmax.cos(P+144.(i+3))
where P is the phase angle
OBaseOH
OH
OBase
OH
OH
OOH
HO
BaseO
BaseOH
OH
Twist FormEnvelope form
C2'-endo C2'-exo C2'-endo, C3'-exo C2'-exo, C3'-endoT2E 2E
4
5
32
1
4 atoms in plane1 atom out of plane (#5)
4
5
32 1
3 atoms in plane1 atom below plane (#4)1 atom above plane (#5)
O0 0 O O O
P=162
23 T2
3
P=0P=180P=342
Definition of envelope and twistforms of deoxyribose ring system
Pseudorotation Cycle for Ribose Ring
Potential Energy Surface for the Deoxyribose Ring System as a function of τ and P.
τ P
E = kcal, perpendicular to plane of paper
OBase
HO
HO O
BaseHO
HO O
BaseHO
HO
Bad VDWinteractionC2'
H
H
C1'
H
HOC4'
Bad VDWinteractiondue to eclipsedbond
Origin of the high energy of the O4'-exoconformation of deoxyribonucleosides
2' 1'
Figure: synant1.cw2
ON
RO
RO
N
N
N
NH2
ON
RO
RO
N
N
N
NH2
anti syn
Glycosyl Bond Angle Terms
O
PO
O
O-
σ*LUMO
nHOMO
anomericeffect:
Anomeric effect in phosphodiesters explainspreference for gauche conformations
A & B duplex DNA
N3
N N1
N
O N
O6N2
H
H
HN
N9
O
O
O
P
OO
OP
5'O O
O--O
H
5'
3'
H
3'
GO
OC
O
O
P
5'
P
5'3'
3'
O
O-O
-O
5'
3'
G
5'
3'
C
O
O
OP
N9 N3 HN2
3'
5'
OO-
O
O
N1O6
O
P
3'
5'
O O-
DIFFERENT PERSPECTIVES ON THE WATSON CRICK BASE PAIR (BP)
Looking into minor groove
minor groove side
MAJOR GROOVE SIDE
C2 pseudo axis of symmetry
TOP VIEW
SIDE VIEW
SCHEMATIC VIEW
5'-G-3'3'-C-5'
5' 3'G
5'3'C
