CH 03

Lecture Notes for
Chapter 3
From Genes to Proteins
Essential Biochemistry
Third Edition
Charlotte W. Pratt | Kathleen Cornely
Copyright 2014 John Wiley & Sons, Inc. All rights reserved.
KEY CONCEPTS: Section 3-1
DNA and RNA are polymers of
nucleotides, each of which consists of a:

Purine or pyrimidine base

Deoxyribose or ribose

Phosphate

2014 John Wiley & Sons, Inc. All rights reserved.
DNA and RNA have four nitrogenous
bases.
Purines: 2 examples
Pyrimidines: 3 examples
Notice the similarities in structure.
Notice differences in numbering around ring.
Adenine and guanine are purines in
both DNA and RNA.
Learn how to draw purines.
Adenine, A
Draw ring system
Two more steps
Add amino group (C6)
Fill in bonds
Guanine, G
Draw ring system
Three more steps
Add oxo group (C6)
Fill in bonds
Cytosine and thymine are
pyrimidines found in DNA.
Cytosine and uracil are pyrimidines
found in RNA.
Learn how to draw pyrimidines.
Cytosine, C
Draw ring system
Two more steps
Fill in bonds

Uracil, U and Thymine, T
Draw ring system
Two, three more steps
Add oxo group(C4)
Add H on N3
Add methyl group (C5)
for thymine

DNA and RNA contain sugar groups.
Found in RNA Found in DNA
Notice:
Ring
numbering on
the sugars
contain primes!
Ribose sugars have 2-OH groups, while deoxyribose sugars lack a 2-OH.
Phosphates can attach to the sugar
at C3 and/or C5.
Monophosphates are shown in examples here.
There can be up to 3 phosphates at a given terminus.
Nucleoside/nucleotide nomenclature
is distinct from that of bases.
Nucleoside = base + sugar

Nucleotide = base + sugar + phosphate

Nomenclature Summary
Base Nucleoside Nucleotide (example using monophosphates)

Adenine Adenosine Adenosine Monophosphate

Guanine Guanosine Guanosine Monophosphate

Thymine Thymidine Thymidine Monophosphate

Cytosine Cytidine Cytidine Monophosphate

Uracil Uridine Uridine Monophosphate
Some nucleotides have functions other
than encoding genetic information.
Nucleotides can be fused with vitamins
to form molecules that aid in
enzyme-catalyzed reactions.

(vitamin)
How do nucleotides
link?
Via sugar in
phosphodiester
backbone

Read sequence of
bases from 5 3

Nucleotides are
connected via
phosphodiester bonds.

Bases form specific hydrogen bonds.
A and T
form 2 H-bonds.
G and C
form 3 H-bonds.
Base pair width is similar
within a given conformation
of DNA.
Chargaffs rule
gives a clue to base pairing.
Chargaffs rule: the amount of A+G = C+T.

Base pairs contain a purine and a pyrimidine.

A base pairs with T or U; G base pairs with C.
A DNA molecule contains two antiparallel
strands that wind around each other to
form a double helix in which A and T bases
in opposite strands, and C and G bases in
opposite strands, pair through hydrogen
bonding.

DNA forms a double helix.
Ball-and-stick
representation
Space-filling
representation
with phosphate
backbone
emphasized in
white.
Strands are
antiparallel.
What drives DNA to form
a double helical shape in
3D?
Water plays a role in dictating
biomolecular structure via the
hydrophobic effect.
The hydrophobic effect is the phenomenon by which
nonpolar molecules aggregate to avoid contact with
hydrophilic molecules, particularly water.
From
Chapter 2
Unfavorable
Many H
2
O molecules
are ordered around the
nonpolar molecules.
Preferred
Fewer H
2
O molecules
are ordered around the
nonpolar molecules.
As H
2
O molecules
are freed, entropy
of the system
increases!
Axial View
of DNA
Base pairs form
the core of the
double helix.

Phosphate backbone
forms the periphery.

Double helical
conformation of DNA
is driven by entropic
forces that induce base
stacking.
Viewing 3D Structures
of Macromolecules
Go to http://www.rcsb.org/pdb/home/home.do

Search for a molecule of interest.

Select its link (e.g. 355D).

Select download files PDB file (text).

Open the file in molecular viewing freeware.
DNA is stabilized by different forces.
Predominant force: hydrophobicity, base
stacking and entropy

Hydrogen bonding (in base pairs)

Ionic interactions
Cations (e.g. Mg
2+
, Na
+
, K
+
)
Polyamines

Double-stranded nucleic acids are
denatured at high temperatures.

At lower temperatures, complementary
polynucleotides anneal.

DNA can denature (unfold).
Native state

Denatured
state

Abs = 260 nm

T
m
= melting
temperature
DNA can renature (refold, anneal).
DNAs ability to re-anneal
is very important in nature
and in biochemical research!
The biological information encoded by a
sequence of DNA is:
Transcribed to RNA
Then, translated into the amino acid
sequence of a protein
What does it mean to go
from genes to proteins?
Genes are sequences of DNA.
Replication: copying DNA
Transcription: converting DNA into RNA
Reverse transcription: converting RNA into DNA
Translation: making proteins from an RNA
template
DNA replication is critical to life.
In vivo
DNA must be copied in order to sustain life.
Excessive DNA replication can be indicative
of cancer.

In vitro
Technology known as the polymerase chain
reaction or PCR has revolutionized
researchers capability to study nucleic acids
and genes.
Transcription of DNA is critical to
life.
Cells cannot thrive if transcription is shut down!
Transcription
RNA polymerase (not shown here) unwinds and separates dsDNA at the position
where transcription occurs.
Notice that the RNA transcript is complementary to the
antisense (noncoding) strand!
There are three fundamental types of
RNA.
Messenger RNA: encodes for polypeptide
sequences

Transfer RNA: carries amino acids to
ribosome

Ribosomal RNA: aids in polypeptide
synthesis
tRNA is single-stranded and
forms unique conformations.
Translation results in protein
synthesis.
Translation occurs in
the ribosome, which
contains rRNA and
many other proteins.

In translation, tRNA
carries amino acids
to the ribosome and
binds to its
complement
in the mRNA
template.

Amino acids are
dictated
by the Genetic Code.
The genetic code is used to translate
mRNA into an amino acid sequence.
Read mRNA
sequence in the
Genetic Code
by the position
of 3 nucleotides.

Notice the
redundancy!

The genomes of different species vary in
size and number of genes.

Genes can be identified by their nucleotide
sequences.

Analysis of genetic data can provide
information about gene function and risk of
disease.
Gene size is roughly correlated with
organismal complexity.
A DNA molecule can be sequenced or amplified
by using DNA polymerase to make a copy of a
template strand.

Linking together DNA fragments produces
recombinant DNA molecules that can be used:
To study gene function
To express genes in host organisms
To engineer genes for commercial and
therapeutic purposes
DNA can be sequenced via
the Sanger method.
ddNTPs lack a
3-OH group

No more NTPs
can attach.
Dideoxy DNA Sequencing
Sanger Method
Dideoxy DNA Sequencing
Sanger Method
DNA can be amplified or copied
using DNA polymerase.
A process called the Polymerase Chain
Reaction or PCR can be used to make
billions of copies of DNA efficiently and
accurately.

PCR was developed by Kary Mullis, who
won the 1993 Nobel Prize in Chemistry for
his discovery.
What is required to make PCR work?
Heat-stable DNA polymerase (e.g., Taq
DNA polymerase)
Primers (DNA oligonucleotides)
Deoxynucleotide triphosphates
dATP, dGTP, dCTP, dTTP
DNA template
Buffer containing Mg
2+
, among others
Also required:

Thermal Cycler
a device that heats and
cools samples with
speed, precision, and
reproducibility.
PCR repeats three chemical reactions.
Step 1: Denaturation 9295C
dsDNA separates at high temp to form ssDNA.

Step 2: Annealing ~55C
Primers can base pair to ssDNA.

Step 3: Extension 72C
Optimal temp for heat-stable DNA polymerases to
work
New strand is synthesized.
PCR is conducted
over many cycles
until millions of
copies are
produced.
Restriction enzymes role in nature
is to aid in defense for bacteria.
Bacteria methylate their own DNA

Bacteriophages (viruses that infect bacteria)
have unmethylated DNA.

Bacterial restriction enzymes can recognize
and excise viral DNA.
Type II restriction enzymes are used
to selectively cut DNA.
Type Activity Cleavage Site
I Endonuclease & 1000 bp from
Methylase recognition sequence

II Endonuclease Within or near
recognition sequence

III Endonuclease & ~24-26 bp from
Methylase recognition sequence
EcoRV
5-G-A-T-A-T-C-3
3-C-T-A-T-A-G-5
EcoRI
5-G-A-A-T-T-C-3
3-C-T-T-A-A-G-5
Characteristics of Type II
Restriction Enzymes
Cleave 4-8 bp segment of dsDNA

Palindromic recognition sequences
Madam, Im Adam
Race Car

Remember:
Always read from
5' 3'
Axis of symmetry
Type II Restriction enzymes form
blunt or sticky ends.
EcoRI recognizes 5'-GAATTC-3'.
Complimentary sequence is implied.
EcoRI cuts both strands after the 5'-G.
Each fragment has sticky ends.
5'
5'
3'
3'
Type II Restriction enzymes form
blunt or sticky ends.
EcoRV recognizes 5'-GATATC-3'.
Complimentary sequence is implied.
EcoRV cuts both strands after the 5'-T.
Each fragment has blunt ends.
DNA fragment sizes can be
quantified from gel images.
Gel electrophoresis is a method
of separating DNA fragments by
size.

Gel is made of agarose.

DNA bands can be stained with
chemicals such as ethidium
bromide for fluorescent detection.
What is a DNA Plasmid?
Double-stranded, extra-chromosomal DNA
Size: 1200 kbases
Covalently closed, circular, superhelical
Bacterial
Dependent on host cells proteins for replication
and transcription machinery
Site-Directed
Mutagenesis
A method of introducing
a single amino acid
change in a protein as a
result of a mutation at a
specific site in the DNA.

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