Structures

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Structures
The FOUR basic groups of biological molecules are
In each case, they are built from simple
carbon-based building blocks.
Of these four, only proteins and nucleic acids
carry information in the sequence of their
building blocks.
Although proteins are, by far, the most abundant
of the four, nucleic acids are the most
fundamental in the sense that they alone carry
the remarkable potential for self-duplication.
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The two types of Nucleic Acid

• There are two types of nucleic acid, ribonucleic
  acid (RNA) and deoxyribonucleic acid (DNA).

• Each monomer unit contains
• A phosphate group
• A 5-carbon sugar
• Ribose in RNA
• 2-deoxyribose in DNA
• A purine or pyrimidine base
• Adenine (A)
• Guanine (G)
• Cytosine (C)
• Thymine (T) DNA only
• Uracil (U) RNA only

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Important Terminology
The carbon atoms are numbered with primes to
distinguish them from atoms in the bases.
Repeating unit of ribonucleic acid (RNA)
Repeating unit of deoxyribonucleic acid (DNA)
2-OH on ribose sugar has been hydrolyzed to a
2-H
HO ?
GLYCOSIDIC BOND- Bond formed between the C1 on
the sugar and the base nitrogen.
? H
A NUCLEOTIDE 5-monophosphorylated
derivative of a nucleoside, the repeating unit in
nucleic acids
A NUCLEOSIDE The non-phosphorylated sugar base
adduct.
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The phosphodiester link
Oligonucleotide Small polymers containing only a
few residues
Polynucleotide Large polymers containing many
residues e.g. DNA and RNA
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The Bases
PYRIMIDINES
PURINES
For purine bases, it is the N9 that attaches via
the glycosidic bond to the C1 on ribose to form
the resultant nucleoside
For pyrimidine bases, it is the N1 that attaches
via the glycosidic bond to the C1 on ribose to
form the resultant nucleoside.
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Nucleotides and Naming
Base Adenine (A)
ATP Adenosine 5-triphosphate
base sugar (nucleoside or nucleotide?)
Adenosine
nucleoside phosphate grp Adenosine
5-monophosphate (AMP)
sugar is deoxyribose, not ribose deoxyadenosine
5-monophosphate (dAMP)
nucleoside diphosphate grp Adenosine
5-diphosphate (ADP)
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Naming
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H-bonding between bases

• Also related to the conjugated double bond
  system.

Potential H-bonds
pKa 3.8
To C-1 of sugar
To C-1 of sugar
Potential H-bonds
pKa9.4
pKa4.5
But more on this later.
To C-1 of sugar
To C-1 of sugar
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Phosphodiester Linkage

• VIA DEHYDRATION

To the free 3OH of a POLYNUCLEOTIDE
?Gº 25 kJ/mol
H
H
H20
The addition of a NUCLEOTIDE
Hydrolysis of polynucleotides to nucleotides is
the thermodynamically favored process.
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Polynucleotide Stability

• Luckily, although polynucleotides are
  thermodynamically unstable in vivo,their
  hydrolysis is exceedingly slow unless catalyzed.

Metastable

• When catalysts are present, however, hydrolysis
  is rapid.
• Acid catalyzed yields mixture of nucleotides
• Extreme acid conditions glycosidic bond is also
  hydrolyzed
• Base catalyzed works only for RNA, not DNA
• yielding mixture of 2 and 3
  nucleotides
• Nuclease catalyzed in vivo catalysis of
  phophodiester bonds

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Polynucleotide Synthesis

• If not by the direct elimination of water, how
  are polynucleotides synthesized in vivo?

?Gº -31 kJ/mol 25 kJ/mol ________
- 6 kJ/mol
Can be further hydrolyzed to 2 x Pi with a ?Gº
33 kJ/mol to remove one product and thereby
drive the reaction to the right.
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Primary Structure of Nucleic Acids

• Two important features of all polynucleotides
• Directionality of polynucleotide chain 5PO4?3OH
  ?5PO4 (N1)?3OH(N1).
• Individuality of polynucleotide chain determined
  by sequence of its bases, i.e. the nucleotide
  sequence. (Primary Structure)
• Primary structure notation, e.g. ACGTT, assumes
• The sequence of nucleotides are by their one
  letter code
• All phosphodiester links are 3?5. By
  convention, with the 5 end to the left and the
  3 end to the right. Could write pApCpGpTpT to be
  more specific.
• There is an unreacted phosphate on the 5 end and
  an unreacted hydroxyl on the 3 end. If not, e.g.
  if phosphate on 3 end and hydroxyl on 5 end
  then write ApCpGpTpTp

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DNA as the GENETIC stuff

• Genetic information is stored in the primary
  structure of DNA.
• Genomic DNA ? total genetic information of an
  organism, some of which can be transcribed ?
  GENE.
• A gene is nothing more than a particular DNA
  sequence that encodes information in a
  four-letter language.
• Wow? And how do we know this?

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History of Nucleic Acids

• 1. The Swiss biologist, Friedrich Miescher,
  isolated DNA from salmon sperm in 1868.
• 2. In 1944, Oswald Avery, Colin MacLeod, and
  Maclyn McCarty showed that DNA from pathogenic
  strains of the bacterium Pneumococcus could be
  transferred into nonpathogenic strains, making
  them (and any succeeding generations) pathogenic
  (Figure 4.8a).
• 3. Erwin Chargaff reported in 1947 that the
  quantitities of adenine and thymine in DNA were
  very close to the same value. Similarly, he
  observed that cytosine and guanine were also very
  close to equal in quantity.
• 4. In 1952, Alfred Hershey and Martha Chase
  showed T2 bacteriophage inject only DNA into
  cells and this is sufficient to make more T2
  bacteriophage (Figure 4.8b).
• 5. James Watson and Francis Crick proposed the
  model of the double helix of DNA in 1953 (Figure
  4.10).

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Secondary Structure

• The Double Helix, Watson and Crick 1953
• Rosalind Franklins DNA diffraction pattern
  clearly showed that
• there was a regular, repetitive 3-D structure
• typical of a helical secondary structure
• with 10 residues/turn
• Two DNA strands/ helical molecule
• Were the phosphates or the bases facing each
  other in the middle of the helix?

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Watson and Crick 1953

• Key insight was that the two-stranded helix could
  be stabilized by h-bonding between the bases
  paired in one particular way

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Key Features of W-Cs DNA model

• Distance between base pairs 0.34 nm
• Closely packed within helix (van der Waals
  thickness of a planar ring 0.17 nm)
• Bases, although inside helix, can be approached
  through major and minor grooves.
• Hydrophilic phosphate-deoxyribose backbones of
  helix are on outside in contact with H2O
• The two DNA strands run in opposite directions
• Right-handedness to helix

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Data explained by WCs model

• Chargaffs rule When DNA was isolated from many
  organisms A and T were almost always present in
  nearly equal quantities, as were G and C.
• Self-replication is the property that genetic
  material must have and complementarity allows for
  this.But how?

• Each strand must act as a template for a new,
  complementary strand.

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DNA Replication

• Meselson and Stahl 1958
• N15-labeled parent DNA was allowed to replicate
  through one generation of growth in N14 media.
• Then the DNA was isolated and separated according
  to its density using density gradient
  centrifugation with a CsCl gradient.
• In this way, the three models posed on the left
  were distinguished.

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Meselson and Stahl 1958
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Alternative Nucleic Acid Structures

• B-form DNA High humidity (Watson and Crick form)

• A-form DNA Low humidity
• RNA and DNA-RNA hybrids adopt the A-form

Right-handed Right-handed 10
residues/turn 11 residues/turn
0.34 nm rise/residue 0.255 nm rise/residue
Bases nearly ? to axis Bases strongly tilted
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Local Variations from Idealized structures
Dickersons (1983) Palindrome Crystal Structure
gave us much more detailed information concerning
the secondary structure of DNA 5CGCGAATTCGCG3
3GCGCTTAAGCGC5 Note the local distortions from
the idealized structures. Nucleic acid secondary
structure is not homogeneous. It varies in
response to the local sequence.
H2O spine
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DNA tertiary structure

• Many naturally occurring DNA molecules are
  circular.
• Circular DNA has no free 5 or 3 ends.
• Most circular DNA is supercoiled.
• Three-dimensional structure, such as supercoiling
  involves a higher order folding of elements of
  regular secondary structure is called tertiary
  structure.

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ssDNA secondary structures

• ssDNA can have some local secondary structure.

example tRNA
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Transcription/Translation

• Transcription DNA to RNA

Base Pairing Rules In DNA_____ is transcribed
?as ___ in RNA 1. T ? A
2. G ? C 3. A ? U 4. C ? G

• DNA contains the genetic information in cells,
  but proteins are not made directly from DNA.
• Instead, complementary RNA molecules must first
  be made from the DNA ? transcription.
• Transcription requires an enzyme called RNA
  polymerase.

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Transcription/Translation

• Translation RNA to PROTEIN
• There are 20 different amino acids and 4
  different nucleotides. Blocks of three
  nucleotides serve as the codon for one amino
  acid.

Free amino acids
5
Growing Protein Chain
7
6
AAtRNA
4
Anti-codon
free tRNA
8
1
mRNA
2
Ribosome
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Recombinant DNA technology

• Individual genes (or specific encoding DNA
  sequences) from higher organisms are isolated
  from their genome and CLONED into bacteria.
• Expression of these foreign genes in bacteria
  allows for growth of large amounts of some
  normally rare eukaryotic proteins.
• The cloned genes can also be modified slightly
  to answer specific structural or functional
  questions about the proteins they encode.

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Z-DNA
In Z-form, purines are always syn.
Anti- Always favored in A- or B- forms

• Z-DNA is a left-hand helix with alternate
  purine/pyrimidine bases in alternate syn/anti
  conformations.

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Nucleic Acid Denaturation

• Helix-Coil Transitions Competing factors create
  a balance between structured and unstructured
  forms of nucleic acids.
• Two factors favor dissociatin of double helicies
  into random coil ss chains
• Electrostatic repulsion between the chains
• At pH gt 2, every residue on a DNA or RNA molecule
  carries a negative charge sometimes two charges
  depending on pH.
• Often partially neutralized by small counterions,
  still there is a net negative charge/NA molecule.
• Entropy of the random coil
• Random coil structure has a higher entropy
• ?G ?H-T?S
• Since -T?S contribution to ?G in the direction of
  the random coil, ?H contributions must compensate
  to stabilize helical structure.

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Melting Temperature

• ?G ?H-T?S
• the sign of ?G will change with T.
• At low T, ?G gt 0 and helix is stable.
• As T is raised there is a transition where ?G
  becomes negative and helix will melt into
  random coil structures.
• Denatured DNA has a stronger UV
  absorbance so this transition can be
  followed by observing the increase in OD260 as
  a function of T.
• The helix-coil transition is SHARP. It occurs
  over a very small temperature range, refered
  to as the polynucleotides melting
  temperature or Tm.
• Tm increases with increasing G C content,
  Why?

Tm ?H/?S