Lecture 6

Lecture 6
Nucleotides and Nucleic
Acids

Clarification for the previous
lessons
 2,3-disphosphoglycerate (2,3-DPG ) =
2,3-bisphosphoglycerate (2,3-BPG)
 Hemoglobin saturation curve =
oxygen–hemoglobin dissociation curve

oxygen–hemoglobin dissociation curve
 CO2 ?

Sample question
 The level of carbon dioxide in the blood affects the
oxygen carrying capacity of hemoglobin in two ways.
Describe the dual effect of CO2 on Hb.
 Hints: (1) H2O + CO2  H2CO3  H+ + HCO3
- ; alter blood pH
(the Bohr Effect);
(2) Hb·NH2+CO2  Hb·NH·COOH ; carbamino
 Generally, CO2 pressure increase  curve right shift
(Low oxygen binding affinity)

 Other factors interfering with O2 loading:
Carbon monoxide - displaces oxygen from hemoglobin
Methemoglobinemia Fe2+ → Fe3+ (doesn't combine with O2)

Sample question
 What is the shape of the oxygen hemoglobin
dissociation curve?
 How does the shape of the curve relate to the
cooperative binding of O2?
 How does its shape influence loading of oxygen
at the lung and unloading of oxygen at the tissue
level?
 What causes oxygen movement into and out of
the blood?

Information Transfer in Cells
 Information encoded in a DNA molecule is
transcribed via synthesis of an RNA
molecule
 The sequence of the RNA molecule is
"read" and is translated into the sequence
of amino acids in a protein.

Nucleic Acids
 Compound contained C, N, O, and high
amount of P.
 Was an acid compound found in nuclei
therefore named nucleic acid

Nucleic Acids
 Nucleic acids are long polymers of
nucleotides.
 Nucleotides contain a 5 carbon sugar, a
weakly basic nitrogenous compound
(base), one or more phosphate groups.
 Nucleosides are similar to nucleotides but
have no phosphate groups.

Nitrogenous Bases
 Pyrimidines
Cytosine (DNA, RNA)
Uracil (RNA)
Thymine (DNA)
 Purines
Adenine (DNA, RNA)
Guanine (DNA, RNA)

Properties of Pyrimidines and
Purines
 Keto-enol tautomerism
 Strong absorbance of UV light

Pentoses of Nucleotides
 D-ribose (in RNA)
 2-deoxy-D-ribose (in DNA)
 The difference - 2'-OH vs 2'-H
 This difference affects secondary structure
and stability

 L-ribose and L-deoxyribose not found in nature
 D-amino acids is rare.

Nucleosides
Linkage of a base to a sugar
 Base is linked via a glycosidic bond
 The carbon of the glycosidic bond is anomeric
 Named by adding -idine to the root name of a
pyrimidine or -osine to the root name of a
purine
 Conformation can be syn or anti
 Sugars make nucleosides more water-soluble
than free bases

Nucleotides
Nucleoside phosphates

Functions of Nucleotides
 Nucleoside 5'-triphosphates are carriers of
energy
 Bases serve as recognition units
 Cyclic nucleotides are signal molecules and
regulators of cellular metabolism and
reproduction
 ATP is central to energy metabolism
 GTP drives protein synthesis
 CTP drives lipid synthesis
 UTP drives carbohydrate metabolism

Nucleic Acids - Polynucleotides
 Polymers linked 3' to 5' by
phosphodiester bridges
 Ribonucleic acid and deoxyribonucleic
acid
 Sequence is always read 5' to 3'
 In terms of genetic information, this
corresponds to "N to C" in proteins

 Nucleotide monomers are joined by 3’-5’
phosphodiester linkages to form nucleic acid
(polynucleotide) polymers

Classes of Nucleic Acids
 DNA - one type, one purpose
 RNA - several types, several purposes
ribosomal RNA - the basis of structure and
function of ribosomes
messenger RNA - carries the message
transfer RNA - carries the amino acids
microRNA - regulates gene expression

Messenger RNA
Transcription product of DNA
 In prokaryotes, a single mRNA contains
the information for synthesis of many
proteins
 In eukaryotes, a single mRNA codes for
just one protein, but structure is composed
of introns and exons

Eukaryotic mRNA
 DNA is transcribed to produce
heterogeneous nuclear RNA
mixed introns and exons with poly A
intron - intervening sequence

Ribosomal RNA
 Ribosomes are about 2/3 RNA, 1/3 protein
 rRNA serves as a scaffold for ribosomal
proteins
 23S rRNA in E. coli is the peptidyl
transferase

Transfer RNA
 Small polynucleotide chains - 73 to
94 residues each
 Several bases usually methylated
 Each a.a. has at least one unique
tRNA which carries the a.a. to the
ribosome
 3'-terminal sequence is always
CCA-a.a.
 Aminoacyl tRNA molecules are the
substrates of protein synthesis

DNA & RNA Differences?
Why does DNA contain thymine?
 Cytosine spontaneously deaminates to
form uracil
 Repair enzymes recognize these
"mutations" and replace these Us with Cs
 But how would the repair enzymes
distinguish natural U from mutant U?
 Nature solves this dilemma by using
thymine (5-methyl-U) in place of uracil

DNA & RNA Differences?
Why is DNA 2'-deoxy and RNA is not?
 Vicinal -OH groups (2' and 3') in RNA
make it more susceptible to hydrolysis
 DNA, lacking 2'-OH is more stable
 This makes sense - the genetic material
must be more stable
 RNA is designed to be used and then
broken down

The Structure of DNA
 Diameter of 2 nm
 Length of 1.6 million nm (E. coli)
 Compact and folded (E. coli cell is only
2000 nm long)
 Eukaryotic DNA wrapped around histone
proteins to form nucleosomes
 Base pairs: A-T, G-C

DNA
 Structure level 1- Linear array of
nucleotides
 Structure level 2- double helix
 Structure level 3- Super-coiling,
stem-loop formation
 Structure level 4- Packaging into
chromatin

The DNA Double Helix
Stabilized by hydrogen bonds
 "Base pairs" arise from hydrogen bonds
 Erwin Chargaff had the pairing data, but
didn't understand its implications
 Rosalind Franklin's X-ray fiber diffraction
data was crucial
 Francis Crick knew it was a helix
 James Watson figured out the H-bonds

Base pairing evident in DNA
compositions

Bases from two adjacent DNA strands
can hydrogen bond
•Guanine pairs with
cytosine
•Adenine pairs with
thymine

H-bonding of adjacent antiparallel DNA strands
form double helix structure

Properties of DNA Double Helix
 Hydrophillic sugar phosphate backbone winds around
outside of helix
 Noncovalent interactions between upper and lower
surfaces of base-pairs (stacking) forms a closely
packed hydrophobic interior.
 Hydrophobic environment makes H-bonding between
bases stronger (no competition with water)
 Cause the sugar-phosphate backbone to twist.

View down the Double Helix
Sugar-phosphate
backbone
Hydrophobic
Interior with base
pair stacking

Factors stabilizing DNA double Helix
 Hydrophobic interactions – burying hydrophobic
purine and pyrimidine rings in interior
 Stacking interactions – van der Waals
interactions between stacked bases.
 Hydrogen Bonding – H-bonding between bases
 Charge-Charge Interactions – Electrostatic
repulsions of negatively charged phosphate
groups are minimized by interaction with cations
(e.g. Mg2+)

DNA Secondary structure
 DNA is double stranded with
antiparallel strands
 Right hand double helix
 Three different helical forms (A, B
and Z DNA.

Comparison of A, B, Z DNA
• A: right-handed, short and broad, 2.3 A, 11 bp
per turn
• B: right-handed, longer, thinner, 3.32 A, 10 bp
per turn
• Z: left-handed, longest, thinnest, 3.8 A, 12 bp
per turn

Z-DNA • Found in G:C-rich
regions of
DNA
• G goes to syn
conformation
• C stays anti but
whole C
nucleoside
(base and
sugar) flips 180
degrees

DNA sequence Determines Melting Point
 Double Strand DNA can be
denatured by heat (get strand
separation)
 Can determine degree of
denturation by measuring
absorbance at 260 nm.
 Conjugated double bonds in
bases absorb light at 260 nm.
 Base stacking causes less
absorbance.
 Increased single strandedness
causes increase in absorbance

DNA sequence Determines Melting Point
 Melting
temperature
related to G:C and
A:T content.
 3 H-bonds of G:C
pair require higher
temperatures to
denture than 2 H-bonds
of A:T pair.

DNA Structure Level 3
 Super coiling
 Cruciform structures (cross shape)

Supercoils
• In duplex DNA, ten bp per turn of helix (relaxed
form)
• DNA helix can be over-wound.
• Over winding of DNA helix can be compensated by
supercoiling.
• Supercoiling prevalent in circular DNA molecules
and within local regions of long linear DNA strands
• Enzymes called topoisomerases or gyrases can
introduce or remove supercoils
• In vivo most DNA is negatively supercoiled.
• Therefore, it is easy to unwind short regions of the
molecule to allow access for enzymes

Each super coil compensates for one + or – turn of
the double helix

•Cruciforms occur in
palindromic regions of DNA
•Can form intrachain base
pairing
•Negative supercoiling may
promote cruciforms

DNA Structure level 4
 In chromosomes, DNA is tightly
associated with proteins

Chromosome Structure
• Human DNA’s total length is ~2 meters!
• This must be packaged into a nucleus that
is about 5 micrometers in diameter
• This represents a compression of more
than 100,000!
• It is made possible by wrapping the DNA
around protein spools called nucleosomes
and then packing these in helical filaments

Nucleosome Structure
• Chromatin, the nucleoprotein
complex, consists of histones and
nonhistone chromosomal proteins
• major histone proteins: H1, H2A,
H2B, H3, and H4
• Histone octamers are major part of
the “protein spools”
• Nonhistone proteins are regulators
of gene expression

Histones H2A, H2B, H3 and H4 are known as the core histones,
while histones H1 are known as the linker histones.

•4 major histone (H2A,
H2B, H3, H4) proteins for
octomer
•200 base pair long DNA
strand winds around the
octomer
•146 base pair DNA
“spacer separates
individual nucleosomes
•H1 protein involved in
higher-order chromatin
structure.
•Without H1, Chromatin
looks like beads on
string

Solenoid Structure of Chromatin

Hydrolysis of Nucleic Acids
 RNA is resistant to dilute acid
 DNA is depurinated by dilute acid
 DNA is not susceptible to base
 RNA is hydrolyzed by dilute base

Restriction Enzymes
 Bacteria have learned to "restrict" the possibility of attack
from foreign DNA by means of "restriction enzymes"
 Type II restriction enzymes cleave DNA chains at selected
sites
 Type II restriction enzymes cut DNA about 20-30 base pairs
after the recognition site.
 Type I enzymes cut at a site that differs, and is a random
distance (at least 1000 bp) away, from their recognition site.
 Enzymes may recognize 4, 6 or more bases in selecting sites
for cleavage
 An enzyme that recognizes a 6-base sequence is a "six-cutter"

Type II Restriction Enzymes
 No ATP requirement
 Recognition sites in dsDNA usually have a
2-fold axis of symmetry
 Cleavage can leave staggered or "sticky"
ends or can produce "blunt” ends

Type II Restriction Enzymes
 Names use 3-letter italicized
code:
 1st letter - genus; 2nd,3rd -
species
 Following letter denotes strain
 EcoRI is the first restriction
enzyme found in the R strain
of E. coli

DNA sequencing---Chain
Termination Method
• Based on DNA polymerase reaction
• 4 separate rxns
• Each reaction mixture contains dATP, dGTP,
dCTP and dTTP
• Each reaction also contains a small amount of
one dideoxynucleotide (ddATP, ddGTP, ddCTP
and ddTTP).
• Each of the 4 dideoxynucleotides are labeled with
a different fluorescent dye.
• Dideoxynucleotides missing 3’-OH group. Once
incorporated into the DNA chain, chain
elongation stops)

N
N
NH2
N N
H H
O
H H
H H
H H
H
NH
N
N
O
NH2
N
O
H
O
O P
O
HO
O-N
N
NH2
N N
H H
O
H H
H H
H H
O H
O P
O
O-NH
N
N
O
NH2
N
O
H
O
O P
O
HO
O-NH
N
N
O
NH2
N
O
H H
H H
H
OH
OH
OH
O PH
O
O-NH
N
N
O
NH2
N
O
H H
H H
H
OH
OH
O P
O
O P
O
O-No
Chain Elongation

Chain Termination Method
• Run each reaction mixture on electrophoresis gel
• Short fragments go to bottom, long fragments on
top
• Read the "sequence" from bottom of gel to top
• Convert this "sequence" to the complementary
sequence
• Now read from the other end and you have the
sequence you wanted - read 5' to 3'

 The polymerase
chain reaction
(PCR) is a method
to rapidly amplify
sequences of
DNA.

Lab for next week
 Activity Determination of Serum Glutamate
Pyruvate Transaminase

Lecture 6

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Lecture 6