Lecture II-1,2
Required Reading:
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pp. 41-55; 725-746 in FOB
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pp. 100-110 in MCB
Suggested Questions to Answer:
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Study Exercises 1-3 (review), 4&5 and Problem 2 on p. 75 of FOB
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Study Exercises 1, 2, 4, 5 and Problems 2, 6, 7 on p. 771 of FOB
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Problems 5 & 6 on p. 135 of MCB
Lecture Outline - Overview of Nucleic acids &
Molecular Biology; Nucleic acid structure
I. Significance of Nucleic acids
The "Central Dogma" of molecular biology:
Genes contain the information that specifies protein sequences
--> The flow of genetic information is from DNA to RNA to protein
Replication (DNA synthesis) ensures the faithful transmission of genetic
information from generation to generation
At least two additional processes are required for gene expression (the
functional manifestation of genetic information):
1) transcription (RNA synthesis)
2) translation (protein
synthesis)
Nucleic acids play two kinds of roles in these processes:
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Informational
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DNA and RNA are uniquely suited as repositories for the storage/retrieval
of genetic information
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Both molecules serve primarily or exclusively as templates (DNA
directs DNA & RNA synthesis, mRNA directs protein synthesis)
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DNA and mRNA are not part of the catalytic machinery for these processes
-
The sequence per se is what really matters: 3-D structures are of
secondary importance
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Dynamic
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rRNAs are integral parts of the ribosome - direct catalysis of peptide
bond synthesis
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tRNAs are central to translation: provides means for decoding mRNA
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Additional RNAs function in splicing/ RNA processing
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Certain sequences in DNA and (m)RNA function as recognition sites for components
that regulate gene expression
II. Review of Nucleotide
Structure (see Figs 4-1, 4-2 in
MCB)
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Bases
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Purines and pyrimidines
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Common ring atoms, numbering
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Variable substituents, H-bonding potential (donor/acceptor)
(Fig. 23-1, FOB)
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Tautomeric shifts and effects on H-bonding (Fig.
3-7, FOB)
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Nucleosides
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Base + sugar (ribose or deoxyribose) linked by N-glycosidic
bond (Fig. 23-5, FOB)
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1'-carbon of (deoxy)ribose (beta anomer)
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Ring N of base (#1 - pyrimidines, # 9 - purines)
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Nucleotides:
-
Nucleoside monophosphates: phosphoester bond
links P to sugar (usu. 5' carbon)
-
Nucleoside di- and triphosphates: additional P linked
via
high energy phosphoanhydride bond(s)
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Nomenclature (Table
3-1, FOB or 4-1, MCB)
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bases: adenine, cytosine, guanine, thymine, uracil
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nucleosides: (deoxy)adenosine, (deoxy)cytidine,
(deoxy)guanosine, thymidine, uridine
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nucleotides - (d)NMPs (nucleoside monophosphates)
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(d)AMP, (d)CMP, etc
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(deoxy)adenosine monophosphate, (deoxy)cytidine monophosphate,
etc.
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adenylate, cytidylate, guanylate, thymidylate, uridylate
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(d)NDPs and (d)NTPs:
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GDP=guanosine diphosphate, dATP = deoxyadenosine
triphosphate, etc.
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DNA and RNA
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ribose (RNA) vs. 2'-deoxyribose (DNA)
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Thymine/thymidine (DNA) vs. uracil/uridine
(RNA)
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DNA is typically double-stranded (ds), RNA is usu.
single-stranded (ss)
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Relative size: DNA usually enormous/continuous, RNA
comes in smaller, discrete molecules
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Chemical stability: RNA is subject to base hydrolysis
(2í OH acts catalytically)
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Functions of nucleotides
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Energy carriers: drive molecular pumps & motors;
link anabolism & catabolism
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Cell regulation and signaling (e.g., cAMP; ppGpp
in bacteria)
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Coenzyme structure (NAD, FAD, Coenzyme A)
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Carriers of "activated" molecules (e.g., UDP-sugars
for polysaccharide synthesis)
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Precursors/building blocks for DNA
and RNA
III. Covalent (primary) structure of Nucleic
acids (Fig. 3-6, FOB)
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Condensation" of (deoxy)nucleotides (NMP residues)
into linear chains
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Adjacent nucleotides are linked by phosphodiester
bonds
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Like proteins/polypeptides, nucleic acids have a
linear sequence with:
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Constant "backbone" (ribose-phosphate)
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Variable "side-chains" (bases)
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Polarity: 5' end is distinct from 3' end
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(Phosphate(s) often present at 5' end; 3' end typically
has free -OH group)
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By convention, base (nucleotide) sequences are specified
from 5' to 3':
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Direction of synthesis for both DNA and RNA
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Direction in which sequences are read/decoded during
protein synthesis (translation)
The structure & function of a DNA or RNA
molecule is determined by its sequence of BASES
--> so, why are these "nucleic acids",
rather than "nucleic bases"?
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Phosphate carries negative charges (~1 per nucleotide
at pH7)
-
Positive charge is negligible: the purine/pyrimidine
"bases" are not very basic
Note: In biology/biochemistry, "acidic"
and "basic" have somewhat different meanings than in chemistry. To
a biochemist, molecules or functional groups are classified based on whether
they are - or have the potential to become - negatively-charged (acids)
or positively-charged (bases). While chemists usually make a distinction
between the "acid" (proton donor) acetic acid and the "base" (proton acceptor)
acetate, both are "acidic" under the biochemical definition, which recognizes
these as different states of what is the essentially the same molecule.
Ultimately, the two definitions match if one considers only the NEUTRAL
(non-ionized) form of a molecule or functional group. However,
note that for a typical acidic or basic group, the ionized form may prevail
over the neutral form under physiological conditions (pH ~ 7). Paradoxically,
this means that in the cell, negatively charged "acids" (e.g., DNA) exist
primarily as (conjugate) bases, while positively charged "bases" (e.g.,
protonated amines/amino groups) are actually potential proton donors (acids
in the chemical sense).
IV. Higher order structure of DNA: the Double
Helix
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Antiparallel: 2 strands have opposite 5'-3' orientations
(Fig.
4-4b, MCB)
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Helix is right handed (except Z DNA) (Fig.
4-4a, MCB)
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Base Complementarity: bases are ìinsideî helix and
must match: G to C, A to T (Fig. 23-1, FOB)
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Chargaffís rules: for dsDNA, G/C and A/T ratios =
1, purines = pyrimidines
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G+C vs. A+T content is variable
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The sequence of one strand dictates the sequence
of the other
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For DNA sequencing projects, both strands are analyzed
to reduce errors
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Sequence of one strand is reported/published ñ other
strand is implied
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Base stacking: along each strand, bases overlie one
another
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Major and minor grooves expose opposite ìsidesî of
each base pair
Conformational variability in the double helix (Fig.
3-9 & 23-2, FOB)
-
Most DNA approximates the ìBî form proposed by Watson
and Crick
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10 bp/turn (so 36 degrees per bp) ñ DNA in solution
averages ~10.4 bp/turn
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Bases are ~ perpendicular to helix axis
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Bases are near the center of helix
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3.4 Angstroms/bp (= rise), 34 Angstroms/turn (pitch)
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Major groove is wide, minor groove is narrow
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A form :
-
For DNA, requires low water content (unlikely
in cells)
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Readily forms with dsRNA (Steric hindrance of 2í-OH
prevents B form)
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Right-handed, but less tightly wound than B form
(11 bp/turn)
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Helix is wider, bases are well off center & tilted,
rise & pitch are smaller
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Z DNA:
-
LEFT-HANDED
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Involves special sequences: alternating purine-pyrimidine
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Rarely encountered, but may be significant for certain
functions
V. Secondary structure: base pairing in ssRNA (or ssDNA) (Fig.
4-12, MCB; 23-23 FOB)
Single-stranded molecules may form:
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Hairpins
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Stem-loops
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More complex structures
Important for 3-D structure of tRNA, rRNA, ribozymes, etc
Result from ìpalindromicî sequences (aka inverted repeats)
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Complementary sequences must be present within the same strand
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The corresponding duplex (ds molecule) will have 2-fold symmetry, with
the same sequence on both strands; e.g.:
5í ..GCGAACGÖÖÖCGTTCGC.. 3í
3í ..CGCTTGCÖÖÖGCAAGCG.. 5í
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Palindromes may be ~perfect (hairpins) or interrupted (stem-loops)
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Helical regions may be imperfect due to:
-
Unusual (non-Watson-Crick) base pairs
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Unmatched bases on one strand
VI. Helix stability
Non-covalent interactions determine the stability of the double helix
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H-bonds mediate base-pairing: 3 bonds for G-C pairs, 2 for A-T(U)
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Little if any NET contribution to stability (water competes for these)
-
BUT absolutely critical: what would happen if base pairs lacked H-bonds?
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Base-stacking:
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van Der Waals & hydrophobic forces between bases of adjacent
(not paired) nucleotides
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G-C pairs contribute more than A-T
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Stacking (but not pairing) also occurs in ssDNA/RNA
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Ionic interactions:
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Intrinsic repulsion between negatively-charged phosphates
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Stabilized by counterions in solution: mono- and divalent cations
Helix Denaturation (melting) and Renaturation (annealing) (Fig.
23-15, FOB)
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Strand separation can be effected by:
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Thermal energy
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Denaturing solvents (e.g., formamide)
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Low salt
-
UV absorbance (at 260 nm) increases upon melting (Fig.
23-16, FOB)
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Melting curves: (Fig. 23-17, FOB; Fig. 4-9,
MCB)
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Relatively sharp transition indicates cooperativity (ìall or noneî)
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Tm = temp. required for 50% denaturation (at equilibrium)
-
Stability (melting temperature):
-
Increases with G-C content
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Decreases if mismatched bases are present
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Renaturation:
-
Requires temp. below (but near) Tm
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Basis for various hybridization methods (Southern blotting, cloning, etc)