Nucleotides and nucleic acids I
Biochemistry 302
January 18, 2006
http://biochem.uvm.edu/courses/kelm/302
User: student
PW: nucleicacids
Central Dogma of Molecular Biology
(Cell as a factory analogy)
• DNA = permanent
repository which
stores master plans
• RNA = temporary
repository → “copy”
of certain plans
– Working RNAs (e.g.
rRNA, snRNA).
– Adapter RNAs (e.g.
tRNA, miRNA)
– Intermediary RNAs (e.g.
mRNA).
• Protein = working
machinery
Fig. 4.23
Basic chemical structure of DNA and
RNA (heteropolymers of nucleotides)
•
•
•
•
•
Monomer composition
(nucleotide)
– heterocyclic
pentose sugar
– phosphate
– nitrogenous base
RNA: polar ribose
phosphate backbone
DNA: polar deoxyribose
phosphate backbone
(no 2′-hydroxyl)
Nucleotides joined by
3′,5′- phosphodiester
linkages
Nitrogenous bases –
side chains
Lehninger Principles of Biochemistry, 4th ed., Ch 8
Major nitrogenous bases found in DNA
and/or RNA (purines & pyrimidines)
• DNA: A, G, C, T
• RNA: A, G, C, U
• N-β-glycosyl bond: 1′
carbon of ribose and
N9 of Pur base (A, G)
or N1 of Pyr base (C,
T, U)
• Pur or Pyr base +
ribose = nucleoside
parent compounds
Fig. 4.2
Nucleotide Nomenclature
DNA
RNA
Chemistry of nucleotide components
•
Phosphate group
– Strong acid
– pKa ~1 for primary ionization,
~6 for secondary
•
Purine/Pyrimidine (pKa ~2.4-9.5)
– Weak tautomeric bases
• Isomers differing in position of H
atoms & double bond.
• Less stable imino & enol forms
found in special base interactions.
– Conjugated double-bonds
• Resonance among ring atoms
• Absorb UV light
Lehninger Principles of Biochemistry, 4th ed., Ch 8
Fig. 4.4
Chemical stability of polynucleotides
(contribution of the ribose ring)
•
•
•
•
•
Hydrolysis of DNA and
RNA is thermodynamically
favorable but very slow.
Acid-labile bond (purine
glycosidic linkage in DNA
but not RNA
Base-labile bond (PDE
bond in RNA but not DNA)
Nucleases (endo & exo,
specific & non-specific)
promote rapid hydrolysis of
PDE bonds in DNA or RNA.
Dehydration-resistant (e.g.
DNA in fossils) but water
content (level of hydration)
affects secondary structure
Lehninger Principles of Biochemistry, 4th ed., Ch 8
DNA and genetics: a historical
perspective
•
•
•
•
•
~1868 – Friedrich Miescher isolates phosphorus-containing
substance “nuclein” from nuclei of leukocytes and salmon
sperm, noted 2 portions… Acidic (DNA), Basic (Protein)
CW 1860s to 1940s – Genetic inheritance dictated by
proteins → Nucleic acid too simple (4 nucleotides vs ~20
amino acids → DNA merely a structural material present in
the cell nucleus.
1944 to 1952 – DNA transfer & labeling studies point to DNA
as the repository of genetic information.
Late 1940s – Chargaff’s rules of DNA composition A = T; G
= C; A + G (purines) = C + T (pyrimidines)
1953 – Watson & Crick propose structure of DNA.
Avery, MacLeod, and McCarty, 1944
Hershey-Chase, 1952
T2 bacteriophage
infection
Viral T2
32P-DNA
(not 35Sprotein)
transferred
to and
propagated
in E. coli
Elucidation of DNA structure
Franklin and Wilkins 1953; King’s College
Watson and Crick 1953; Cambridge Univ.
•
R. Franklin & M. Wilkins – X-ray
diffraction pattern of wet DNA
fibers consistent with regular,
repetitive helical 3D structure w/ 2
distinct periodicities.
– Primary repeat ( 3.4 Å)
– Secondary repeat (34 Å)
•
J. Watson & F. Crick – Built best fit
model based on X-ray data,
Chargaff’s rules, DNA chemical
composition, & clever deduction.
– Ten residues/turn (34 Å)
– Helical rise (3.4 Å, distance betw
vertically stacked bases
– Two DNA strands/helix (fiber
density)
Cross pattern
typical of helix
R. E. Franklin and R. Gosling (1953)
Nature 171:740
Properties of nucleotide bases → 3D
structure of nucleic acid
•
pH-dependent tautomers
– Adenine and Cytosine (amino form at pH 7)
– Guanine and Thymine (keto form at pH 7)
•
Functional groups (H-bonding)
– Ring nitrogens
– Carbonyl groups
– Exocyclic amino groups
•
Highly conjugated → resonance
– Pyrimidines (planar)
– Purines (nearly planar slight pucker)
•
Hydrophobic character
– Hydrophobic stacking interactions
– van der Waals interactions between
uncharged atoms
Watson and Crick 1953
Intuition: H-bonding
between certain bases
on opposite strands
stabilizes the helix
~1.08 nm
Geometric Features:
• H-bonding between A=T, G≡C
base pairs → distance between
C-1′ atoms the same → constant
helical diameter
bp stacking
and rotation
36°
relative to
long axis
• Bases “stacked” & slightly
offset inside the double helix
• Deoxyribose-phosphate
backbone exposed to water
• Pentose ring in C-2′ endo
conformation (sugar pucker)
H-bonding
(different #
in A=T vs
G≡C bps)
Rise = 0.34 nm
Fig. 4.10
antiparallel
strands
H-bonding pattern in W-C base pairs
and numbering convention
A=T
(N6,N1) = (O4,N3)
(H-bond: two electronegative atoms,
such as nitrogen and oxygen,
interacting with the same hydrogen)
G≡C
(O6,N1,N2) ≡ (N4,N3,O2)
antiparallel strands
Lehninger Principles of Biochemistry, 4th ed., Ch 8
Other features of Watson-Crick model
•
•
•
5′
Right handedness
(counterclockwise rotation)
Antiparallel strands
Major/minor grooves
– Created by offset base
pairing of 2 strands
– Major groove allows direct
access to bases
– Minor groove faces ribose
backbone
•
Base-pairing explains
Chargaff’s rule → A/T or
G/C ~1 in organisms with
dsDNA genomes.
3′
van der Waals radius of atoms
Fig. 4.11
Other views of the Watson-Crick model
for the structure of DNA
Ribose and phosphate
oxygens are in blue.
Phosphorus atoms are in
yellow. Atoms comprising
bases are in gray.
Because B-DNA is really 10.5 bp/turn.
Lehninger Principles of Biochemistry, 4th ed., Ch 8
Were Watson and Crick right?
• Limitations of fiber
diffraction studies
– Fiber heterogeneity
– Modeling intensive
(idealized version)
• Enhanced precision of
crystallography
– Atom positions specified
– Structure of B-DNA more
distorted than WatsonCrick model
– Bending occurs wherever
≥ 4 adenosine residues
appear in a row in one
strand
DNA Bending
Fig. 4-16
R.E. Dickerson et al. 1983
Secondary structural variants (deduced
from fiber diffraction and crystal structures)
• B-form
– DNA fibers prepared under
high humidity
– Form found in cells
• A-form (compact)
– DNA fibers prepared under
low humidity
– RNA-RNA and RNA-DNA
hybrids
A-DNA:
deeper
narrow
major
groove
Z-DNA:
deeper
narrow
minor
groove
• Z-form (zigzag)
– elongated left-handed DNA
– alternating C (or 5-meC) &
G residues in alternating
anti and syn glycosyl bond
conformation
Each structure has 36 bp.
Properties of the three forms of DNA
Pitch = (Helix rise)(base pairs/turn)
Lehninger Principles of Biochemistry, 4th ed., Ch 8
Structural variation in DNA & dsRNA→
nucleotide conformation
H
Steric constraints restrict rotation about
bonds 4 (sugar pucker) and 7 (C-1′-N-β
glycosyl bond) in different DNA structural
variants (A, B, Z).
Lehninger, Principles of Biochemistry, 4th ed., Ch 8
Structural variation in DNA & dsRNA→
β-furanose or sugar pucker
B-DNA
Lehninger Principles of Biochemistry, 4th ed., Ch 8
A-DNA
(or RNA)
What drives B-DNA into an A-DNA
conformation?
• Similarities
– Helical sense
– W-C base pairing
• Differences
– Position of bases with
respect to helical axis
– Base “tilt”
– Groove width and depth
(shallower minor groove
in A-DNA but deeper
major groove)
– 11 bp/turn in A-DNA
– Rise, pitch (repeat), and
rotation per residue are
smaller in A-DNA
Fig. 4-15
No
H2O