PPT1 Chap 4 Zhang Student

Class information
Instructor: Dr. Jun Zhang

Office: CHEM 266
Email: zhanguab@uab.edu
Office hours: Thursdays 3-4 p.m. or by appointment
Teaching Assistant: Roxanne Robidoux
Email: rrobidoux13@gmail.com
Supplemental Instruction leader: Kaila Bree Wood
Email: kwood1@uab.edu
Textbook: Appling, Anthony-Cahill, & Mathews, Biochemistry, 1st Ed.
Pearson, 2016.
ISBN-13: 978-0-321-83992-3
Online homework: www.masteringchemistry.com
Course ID: MCZHANG52986
CH 460 Fundamentals of Biochemistry

Emphasis on: biological molecules
: structure, metabolism, chemistry
Objectives
: foundational knowledge
: preparation for specialization in Chemistry,
Biochemistry, health professions
: scientific thinking and communication
Why do you take this class?

(A) medical school health professions
(B) pharmacy school
(C) pursue research in the fields of biochemistry,
chemistry, structural biology and biophysics
(D) Not determined
Pearson Education Ltd 2015. Copying permitted for purchasing institution only.
Grade breakdown
Activity
In-Class Participation
(assignments, clicker)
On-Line Pre-lecture Homework
(Due 1 hour before the lecture)
On-Line Post-lecture Homework
Exams (3 @ 100 pts)
Final Exam
Total
Points
25
25
50
300
100
500
Read syllabus carefully.

Late submission of homework will result in 50% grade reduction.
Contact Roxanne Robidou and Kaila Wood if you have questions about the
homework.
The lecture schedule or the homework due date are subject to change.
The class materials can be updated.
in-class participation
For in-class participation, your participation score
will be calculated as follows:
70-100% of the sessions
25 pts
60-70%
20 pts
50-60%
15 pts
<50%
0 pts
A session is simply a class period during which
the clickers are used. You do not have to answer
questions correctly to indicate participation.
Online homework
Submit on the web with MasteringChemistry
http://www.pearsonmylabandmastering.com/northamerica/masteringchemistry/
Helps you become familiar with concepts,

provides practice problems.
Do problems BEFORE lecture as much as
possible.
Note the due dates for homework
Requirements for the class

Submit pre-lecture homework 1 hour before the
lecture.
Submit post-lecture homework on time.
Late pre-lecture and post-lecture homework
reduce your homework score by 50%.
Syllabus
Biochemistry:
Concepts and Connections

Dean R. Appling
Spencer J.
Anthony-Cahill
Christopher K.
Mathews
Chapter 4 Nucleic Acids

Electron micrograph of DNA released

from bacteriophage T2
DNA is a linear molecule, or polymeric molecule, or polymer.

The human genome contains three billion of bases. When stretched, the
length of all DNA from one human cell is 2 meters.
Chemical
structures of
ribonucleic
acid (RNA)
and deoxyribonucleic
acid (DNA)
The two types of nucleic acid
Basic components of
DNA/RNA:
Base: A, G, C, U/T
Sugar: ribose, deoxyribose
phosphate
Purine and pyrimidine bases found in DNA and RNA

5-methyl-U
Tricks to help you memorize:

Ring backbone of purines (A, G) are the same; ring backbone of pyrimidines (C, T, U) are the same.
The Chemical Differences Between DNA

and RNA Have Biological Significance
DNA: A, G, C, T
RNA: A, G, C, U
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

Deamination of cytosine forms uracil.

The 5-methyl group on

thymine labels it as a
special kind of uracil.
Nucleoside and nucleotide structures

and naming (purines)
Nucleosides have no phosphate group

Nucleotides have phosphate group at the 5 end.
Nucleoside and nucleotide structures

and naming (pyrimidines)
Nucleosides have no phosphate group

Nucleotides have phosphate group at the 5 end.
Nomenclature:
Bases, Nucleosides, Nucleotides
Base
Nucleoside
5-nucleotide
Adenine
Adenosine
Adenosine 5-monophosphate
Guanine
Guanosine
Guanosine 5-monophosphate
Cytosine
Cytidine
Cytidine 5-monophosphate
Uracil
Uridine
Uridine 5-monophosphate
Thymine
Thymidine
(deoxythymidine)
Deoxythymidine 5-monophosphate
Tautomeric forms of the bases
Small amount
Small amount
UV absorption spectra of
ribonucleotides
UV light absorption is used to detect and quantitate nucleic acids.

Usually, absorption at 260 nm was used for DNA/RNA quantification.
denoted atoms can undergo

proton
dissociation/association.
Acid-base titration curves
Consider the dissociation of

a weak acid:
HA H+ + A
Ka= [H+][A]/[HA]
pH = log[H+]
pKa = log Ka
pH = pKa + log [A]/[HA]
When:
pH < pKa, [HA] > [A-]
pH = pKa, [HA] = [A-]
pH > pKa, [HA] < [A-]
Example: charge of AMP At pH 3.8
If the pH is more than 1 unit higher than pKa, the group is

considered as completely deprotonated.
If the pH is more than 1 unit lower than pKa, the group is considered as
completely protonated. Note that pKa is at the logarithm scale.
First consider the phosphate group, at pH 3.8, phosphate has
primary ionization, therefore has -1 charge.
Second consider the base group, at pH 3.8, half of N-1 will be
protonated (+1) and half of N-1 will be deprotonated (0
charge).
In total, the net charge is -1+1X50%=-0.5.
In-class question:
charge of GMP At pH 6.1?
1
A. -2
B. -1.5
C. -1
D. -0.5
E. dont know
A mixture of nucleotides can be separated using electrophoresis or ionexchange chromatography by choosing a pH so that different nucleotides have
different net charges
Each nucleotide in DNA or RNA has a net charge of about 1 (at physiological
pH (pH 7.4) the base amino group is unprotonated)
Now focus on the sugar group

Chemical
structures of
ribonucleic
acid (RNA)
and deoxyribonucleic
acid (DNA)
Basic components of
DNA/RNA:
Base: A, G, C, U/T
Sugar: ribose, deoxyribose
phosphate
Ribose and Deoxyribose

Note: numbers are primed on sugars but not bases
Numbering begin from the Carbon connected to two O atoms,

or hemiacetal group.
An oxygen makes a many differences!
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 (DNA) must
be more stable
RNA is designed to be used and then broken down
2-OH makes RNA impossible to take B-form structure
due to steric hindrance.
2-OH makes RNA more flexible.
Alkaline hydrolysis of RNA proceeds through

a cyclic intermediate not possible in DNA
Alkaline hydrolysis yields a mixture of nucleoside 2 and 3-monophosphate.

Nucleotide Functions
1. Adenosine
2. Coenzyme components
NAD+ and NADP+ ; FMN and FAD
3. Regulatory molecules
cAMP and cGMP
4. Provide energy for reactions:
ATP === > ADP + Pi
ATP === > AMP + PPi
5. Substrates for making DNA and RNA
All NTPs and dNTPs
Adenosine: Nucleoside with

Physiological Activity
Adenosine functions as an
autacoid, or local hormone
Influences blood vessel dilation,
muscle contraction,
neurotransmitter release, fat
metabolism
Adenosine is also a sleep
regulator. Adenosine promotes
eventual sleepiness.
Caffeine promotes wakefulness
by blocking binding of adenosine
to its neuronal receptors.
NADP /NADPH, Coenzyme

+
Coenzyme: nonprotein
component of an enzyme
that provides a chemical
functionality not provided
by the protein.
Often these are vitamins
or contain vitamins.
The Nicotinamide Coenzymes NAD+/NADH and NADP+/NADPH
-carry out hydride (H:-) transfer reactions. All reactions involving
these coenzymes are two-electron transfers.
cAMP functions
Signal the effects of hormones
(adrenaline, glucagon)
Activate protein kinases
regulate glycogen and lipid
metabolism
Activate ion channels
Regulate cyclic nucleotidebinding proteins
Regulation of gene expression
(prokaryotes)
ATP, a 5-Nucleotide
(base + sugar + phosphate)
Nucleoside 5'-Triphosphates Are Carriers

of Chemical Energy
Nucleoside 5'-triphosphates are indispensable agents in
metabolism because their phosphoric anhydride bonds
are a source of chemical energy
ATP is central to energy metabolism
GTP drives protein synthesis
CTP drives lipid synthesis
UTP drives carbohydrate metabolism
ATP contains two pyrophosphate linkages. The hydrolysis of

phosphoric acid anhydrides is highly favorable.
Phosphoric Acid Anhydrides

ADP and ATP are examples of phosphoric acid
anhydrides
Note the similarity to acyl anhydrides
Large negative free energy change on hydrolysis is
due to:
electrostatic repulsion
stabilization of products by ionization and
resonance
entropy factors
solvation
Hydrolysis of Phosphoric Anhydrides is

Highly Favorable
Electrostatic repulsion and resonance in acetic anhydride
Hydrolysis of ATP to ADP

(and hydrolysis of ADP to
AMP) relieves electrostatic
repulsion.
Why are certain compounds energy rich?
Resonance stabilization of
phosphate ion product
Charge repulsion in
reactants
Tautomerization and/or
solvation of products
Entropy contribution
High-energy Carrier Molecules
Metabolically activated nucleotides

provide the energy needed for
polynucleotide synthesis
Primary, secondary and tertiary

structure of nucleic acids
Primary: Sequence of nucleotides
This strand has a 5-to-3 direction; can also be drawn:

pApCpGpTpT-OH or pApCpGpTpT (-OH is assumed)
or ACGTT, conventional representation is 5 end to the left
Secondary: 3D arrangements of nucleotide residues with respect

to one another. Short-term folding interactions such as the double
helix
Tertiary: Longer range 3D interactions. Superhelical forms: Overand underwinding, cruciforms

Early evidence
that DNA is the
carrier of genetic
information
Avery and his colleagues showed
that nonpathogenic pneumococci
could be made pathogenic by the
transfer of DNA from a pathogenic
strain.
Hershey & Chase 1952
Hershey & Chase 1952

Radiolabeled T2 Bacteriophage
In first experiment, all radioactivity was in the liquid where
cells were originally suspended
In second experiment, radioactivity was in the bacteria.
Since radioactivity was in the bacteria, the genetic
information was contained in the DNA and not the protein.
Rosalind Franklins fiber diffraction

pattern of DNA
1920 1958
X-ray fiber diffraction image
Information that can be

determined from the diffraction pattern
DNA double-helix structure: base pairing
Chargaffs rules; DNA base composition: mole percent A = T and G = C
DNA double-helix structure: stacking
36 angle between stacked base pairs

means 10 bp/turn (360)
The rise of the helix (0.34 ) is

the distance between successive
base pairs (twice the van der
Waals thickness of a planar ring)
Space-filling
model of DNA
Space-filling model of DNA
shows both close packing of
atoms in the structure and
major and minor grooves
DNA replication is
semiconservative
Each strand of the parent molecule acts as a template for a

new, complementary strand.
The MeselsonStahl experiment

(1958)
Growth in medium containing N15 as
the sole N source gives DNA of
density 1.724 g/ml.
Growth of E. coli in standard medium
gives DNA of density 1.710 g/ml.
After transfer of dense bacteria
(1.724) to light medium for one
generation, all of the DNA had
intermediate density (1.717
g/ml=0.5X[1.724+1.710]). As shown
by CsCl density equilibrium
centrifugation.
Succeeding generations cultured in
light medium show both the hybrid
and light DNA.
B-DNA
Two forms of
the DNA double
helix
Two forms of the DNA double
helix: A form, in low humidity,
and B form, in high humidity.
DNA in cells is mostly B form.
The A form is seen in doublestranded RNA and in DNA-RNA
hybrids. The oxygen atom at
C2 (in ribonucleotides)
imposes steric constraints.
A-DNA
Crystal structure of
B-DNA
The Dickerson dodecamer

~10.4 base pairs/turn, some
bases are tilted slightly, DNA
helix is sometimes slightly bent.
Watson-Crick parameters are
average values, local deviations
from these values are seen.
Double Helix
The stability of the DNA double helix is due to:
Hydrogen bonds between base pairs
estimated at 2-6 kJ mol-1 per hydrogen bond
Base-pair stacking interactions estimated at 4
kJ mol-1 per base pair
Covalent and noncovalent interaction

energies
Types of noncovalent interactions
Hydrogen
bonding
Hydrogen bonding stabilizes

structural elements
Hydrogen bonding stabilizes

structural elements
H-bond donors
H-bond acceptors
Electron
micrograph of
three DNA
molecules
Supercoiled
Relaxed
Each DNA molecule is

identical in sequence and
length.
Creating
supercoiled
circular DNA
L=T+W
L, linking number: Number
of times one strand
crosses the other. L can
only be changed by
breaking DNA strand.
T, twist: Number of turns
W, writhe: Number of
superhelical turns
L = T + W is constant so
long as no DNA strand is
broken.
More about L, T and W
L, linking number. Let count how many times the

red strand goes above the blue one? 2
How many times the blue goes above the red one?
More about L, T and W
T, Twist number. Let count how many times (turns)

the red strand goes around the blue one? Or how
many times (turns) the red strand goes around an
imaginary central helical axis? 0
Positive and Negative Supercoiling

L, linking number: Number of times
one strand crosses the other
T, twist: Number of turns
W, writhe: Number of superhelical
turns
L = T + W so long as no DNA strand
is broken
Superhelicity of DNA defined in terms
of , the superhelix density
where L0 is the linking number for
DNA in the relaxed state. L is the
change in linking number caused by
supercoiling.
https://www.youtube.com/watch?v=az2c6UbEdug
Biochemistry tool:
electrophoresis
cathode,
The terminal
negatively
charged
Anode,
The terminal
positively
charged
1. Gel matrix materials are usually porous materials, such as agarose and
polyacrylamide.
2. Particles that are positively charged will migrate to cathode direction;
Particles that are negatively charged will migrate to anode direction;
The velocity or distance of the migration depends on:
(1)How much charges the particle carry (2) the size of the particle: large one move
slower.
3. By convention, the anode is usually shown as red, and cathode as black.
4. By convention, A gel result is shown in such a way that migration
direction points downward.
Agarose gel electrophoresis of DNA in

various states of supercoiling
Topoisomerases are
enzymes that modify
state of DNA
supercoiling.
Topoisomerase action
can be followed by gel
electrophoresis on
agarose gels in the
presence of ethidium
bromide (a fluorescent
dye).
The smaller or more
compact a DNA
molecule, the more
rapid its migration
during electrophoresis
on agarose gels.
Conformations of single-stranded
nucleic acids
Most DNA molecules found in cells are double-stranded, but most

naturally occurring RNA molecules are single-stranded.
Self-complementarity and tRNA structure
Most RNA is single-stranded and could be a random coil except for areas of
internal complementarity. Transfer RNA molecules used in protein synthesis (75-80
nucleotides long) have extensive regions of intramolecular complementarity. These
regions fold in upon one another as shown here for a yeast transfer RNA.
A-form double helix

The A-form of the DNA double
helix.
The pitch of the A-form helix is
2.46; thus the A-form is a
shorter, wider structure than the
B-form.
One turn in A-form DNA requires
11 bp to complete.
A-DNA in Biology
Forms in dehydrated random-sequence DNA
Stronger tendency for certain sequences e.g.
alternating GC base pairs, high GC content
e.g. binding site for the transcription factor Sp1
Only structure formed by dsRNA
RNA-DNA hybrids formed during replication,
transcription
Comparison of B and A form DNA
A-form characteristics
C2-endo sugar pucker (B: C3-endo)

Steric hindrance of 2-OH reduced
Tilt of bases away from helix
axis
Fewer water molecules bound
Shorter and wider
Stryer, Biochemistry
Figure 29-10b
Nucleotide sugar
conformations. (b) The C2-endo conformation,
which occurs in B-DNA.
Voet & Voet, Biochemistry
Figure 29-10a
Nucleotide sugar conformations. (a)
The C3-endo conformation (on the same side of the sugar
ring as C5), which occurs in A-RNA and RNA-11.
Voet & Voet, Biochemistry
Z-DNA and base conformation
In A and B DNA helices all nucleotides are in

anti orientation
In the left-handed Z DNA helix,
pyrimidines are anti and purines are
syn
Comparison of A, B, Z DNA
See table
A: right-handed, short and broad, 2.3 rise, 11
bp per turn
B: right-handed, longer, thinner, 3.32 rise, 10
bp per turn
Z: left-handed, longest, thinnest, 3.8 rise, 12 bp
per turn
Comparison of A, B, Z DNA
A palindromic DNA sequence
Palindromic DNA sequences are symmetrical with respect to nucleotide

sequence (the same read in both directions). In this case, the palindromic sequences
to the right form a double hairpin, or cruciform conformation.
Triple-stranded DNA structures
G-quadruplexes
G-quadruplexes probably exist in telomeres, the protective structures at the

end of linear DNA molecules.
DNA denaturation
a) Heating dsDNA denatures it into two higher energy

strands
b) As T increases, -TS overcomes a

positive H, making G negative
c) Native DNA absorbs less light than denatured DNA
d) This change in absorbance can be

used to follow DNA denaturation
DNA denaturation
DNA denaturation is promoted by:

Electrostatic repulsion (phosphate group negative charge)
between chains (charge neutralized to some extent by anions:
Na+, K+, Mg2+); high ionic strength stabilizes duplex structure
High entropic factor in comparing flexible single-strand chain
with ordered duplex
Alkali
Heat
Enthalpic component stabilizes duplex DNA: H-bonding between

complementary bases and van der Waals attraction between
stacked bases
DNA denaturation is reversible: renaturation promoted by slow

cooling; rapid cooling creates a population of single-stranded
random coils
Helix-coil transition is cooperative: early structural changes

promote later ones, and melting of the helical structure occurs
over a small temperature range; an abrupt transition
Biological functions of nucleic acids
DNA polymerase is one of a host of proteins in the replisome, a

complex that replicates DNA
Transcription (the
production of an RNA
transcript) is the first
process in the readout of
information encoded in DNA
Biological functions of nucleic acids

In translation, the sequence of nucleotides in a messenger RNA molecule
provides the information for the synthesis of a protein at the ribosome.
Flow of genetic
information in a
typical cell
Creation of a recombinant DNA

molecule in vitro
Example of EcoRI
1. Over 3000 restriction enzymes

have been found and more than 600
are commercially available.
2. They are the most important tools
for molecular biology.
3. Recognition sites of restriction
enzymes are around 4-8 base pairs.
4. Many of them are palindromic.
Examples of restriction
enzymes
BamHI
Bacillus amyloliqu
efaciens
HindIII
Haemophilus influ
enzae
NotI
Nocardia otitidis
5'GGATCC
3'CCTAGG
5'---G GATCC---3
3'---CCTAG G---5'
5'AAGCTT
3'TTCGAA
5'---A AGCTT---3
3'---TTCGA A---5'
5'GCGGCCGC
3'CGCCGGCG
5'---GC GGCCGC---3'
3'---CGCCGG CG---5'
Cloning a DNA fragment into a plasmid vector
Site directed
mutagenesis
Site-directed mutagenesis using
M13 phage as a cloning vector.
M13 is a single-strand DNA phage
that replicates via a doublestranded DNA replicative
intermediate.
Single restriction enzyme versus

double restriction enzyme in cloning
Problems of single enzyme digestion and ligation
Plasmid self-linking
Direction of integrated gene
Self-linking of integrated gene
Solid-phase DNA synthesis
Coupling and oxidation steps in the solid-phase

synthesis of oligonucleotides by the phosphoramidite
method.
DNA nanotechnology
DNA
sequencing:
Sanger
method
3 5
5 3
Without 3 OH, strand

cannot be extended
How to read DNA sequence

from a sequencing gel
3 5
5 3
1. Start from the very bottom

end of a gel.
2.Read the corresponding di deoxy
nucleotide, this will be the First
nucleotide (5) of the
complementary strand.
3.Move one nucleotide up and
read the corresponding di deoxy
nucleotide. This will be the second
nucleotide of the complementary
strand.
4.Repeat the process.
5.Reverse complementary to the
complementary strand will be the
analyzed strand.
Automated DNA sequencing
All four base-specific termination reactions are analyzed in one gel

by use of ddNTPs modified with fluorescent dyes; each ddNTP
receives a unique color.
X-ray diffraction model
X-ray fiber diffraction
Crystal diffraction
Molecules in a unit cell
Diffraction pattern of a
Small DNA crystal
Partial electron
density map derived
from the diffraction
pattern
Models of GroEL at various resolutions

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Class information

Instructor: Dr. Jun Zhang

CH 460 Fundamentals of Biochemistry

Why do you take this class?

Read syllabus carefully.

Helps you become familiar with concepts,

Note the due dates for homework

Requirements for the class

Concepts and Connections

Chapter 4 Nucleic Acids

Electron micrograph of DNA released

DNA is a linear molecule, or polymeric molecule, or polymer.

The two types of nucleic acid

Purine and pyrimidine bases found in DNA and RNA

Tricks to help you memorize:

The Chemical Differences Between DNA

Repair enzymes recognize these "mutations" and replace these

The Chemical Differences Between DNA

Deamination of cytosine forms uracil.

The Chemical Differences Between DNA

The 5-methyl group on

Nucleoside and nucleotide structures

Nucleosides have no phosphate group

Nucleoside and nucleotide structures

Nucleosides have no phosphate group

Tautomeric forms of the bases

UV light absorption is used to detect and quantitate nucleic acids.

denoted atoms can undergo

Acid-base titration curves

Consider the dissociation of

pH = pKa + log [A]/[HA]

Example: charge of AMP At pH 3.8

If the pH is more than 1 unit higher than pKa, the group is

Now focus on the sugar group

Ribose and Deoxyribose

Numbering begin from the Carbon connected to two O atoms,

An oxygen makes a many differences!

DNA & RNA Differences

Vicinal -OH groups (2' and 3') in RNA make it more

Alkaline hydrolysis of RNA proceeds through

Alkaline hydrolysis yields a mixture of nucleoside 2 and 3-monophosphate.

Adenosine: Nucleoside with

NADP /NADPH, Coenzyme

Nucleoside 5'-Triphosphates Are Carriers

ATP contains two pyrophosphate linkages. The hydrolysis of

Phosphoric Acid Anhydrides

Hydrolysis of Phosphoric Anhydrides is

Electrostatic repulsion and resonance in acetic anhydride

Hydrolysis of ATP to ADP

Why are certain compounds energy rich?

High-energy Carrier Molecules

Metabolically activated nucleotides

Primary, secondary and tertiary

Primary: Sequence of nucleotides

This strand has a 5-to-3 direction; can also be drawn:

Secondary: 3D arrangements of nucleotide residues with respect

Tertiary: Longer range 3D interactions. Superhelical forms: Overand underwinding, cruciforms

Hershey & Chase 1952

Hershey & Chase 1952

Rosalind Franklins fiber diffraction

X-ray fiber diffraction image

Information that can be

DNA double-helix structure: base pairing