This document provides an overview of nucleotides, nucleic acids, and DNA/RNA structure. It discusses the components of nucleotides, including sugars, phosphates, and nitrogenous bases. Nucleic acids are polymers of nucleotides linked by phosphodiester bonds. The two main nucleic acids are DNA and RNA. DNA contains the sugar deoxyribose and thymine, while RNA contains ribose and uracil. DNA generally takes the form of a double helix with base pairing between adenine-thymine and guanine-cytosine. RNA can have various structures and functions such as mRNA, tRNA, and rRNA.
4. 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)
5. Other factors interfering with O2 loading:
Carbon monoxide - displaces oxygen from hemoglobin
Methemoglobinemia Fe2+ → Fe3+ (doesn't combine with O2)
6. 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?
7. 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.
8.
9. Nucleic Acids
Compound contained C, N, O, and high
amount of P.
Was an acid compound found in nuclei
therefore named nucleic acid
10. 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.
17. 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
18.
19. L-ribose and L-deoxyribose not found in nature
D-amino acids is rare.
20. 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
25. 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
26.
27.
28.
29. 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
30. Nucleotide monomers are joined by 3’-5’
phosphodiester linkages to form nucleic acid
(polynucleotide) polymers
31.
32. 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
33. 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
34. Eukaryotic mRNA
DNA is transcribed to produce
heterogeneous nuclear RNA
mixed introns and exons with poly A
intron - intervening sequence
35.
36. 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
37.
38. 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
39. 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
40. 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
41. 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
42. 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
43. 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
47. 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.
48. View down the Double Helix
Sugar-phosphate
backbone
Hydrophobic
Interior with base
pair stacking
49. 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+)
50. DNA Secondary structure
DNA is double stranded with
antiparallel strands
Right hand double helix
Three different helical forms (A, B
and Z DNA.
51. 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
53. 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
54. 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
55. 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.
57. 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
58. Each super coil compensates for one + or – turn of
the double helix
59. •Cruciforms occur in
palindromic regions of DNA
•Can form intrachain base
pairing
•Negative supercoiling may
promote cruciforms
60. DNA Structure level 4
In chromosomes, DNA is tightly
associated with proteins
61. 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
62. 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
63. Histones H2A, H2B, H3 and H4 are known as the core histones,
while histones H1 are known as the linker histones.
64. •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
67. 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
68.
69.
70.
71.
72.
73. 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"
74. 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
75. 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
76. 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)
77.
78. 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
79. 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'