Essential Biochemistry

Third Edition
Charlotte W. Pratt | Kathleen Cornely

Chapter 3
From Genes to Proteins

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

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DNA and RNA have four nitrogenous

bases.
Purines: 2 examples
Pyrimidines: 3 examples

Similarities in structure.
Differences in numbering around ring.

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Adenine and guanine are purines in

both DNA and RNA.

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Cytosine and thymine are

pyrimidines found in DNA.

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Cytosine and uracil are pyrimidines

found in RNA.

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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.
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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.
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Nucleoside/nucleotide nomenclature
is distinct from that of bases.

Nucleoside = base + sugar

Nucleotide = base + sugar + phosphate
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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

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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)

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How do nucleotides
link?
Via sugar in
phosphodiester
backbone
Read sequence of
bases from 5 3
Nucleotides are
connected via
phosphodiester bonds.
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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.
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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.

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KEY CONCEPTS: Section 3-1

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.

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DNA forms a double helix.

Strands are
antiparallel.

Space-filling
representation
with phosphate
backbone
emphasized in
white.

Ball-and-stick
representation

What drives DNA to form

a double helical shape in 3D?
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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.
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DNA is stabilized by different forces.

Predominant force: hydrophobicity, base
stacking and entropy
Hydrogen bonding (in base pairs)
Ionic interactions
Cations (e.g. Mg2+, Na+, K+)
Polyamines
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KEY CONCEPTS: Section 3-1

Double-stranded nucleic acids are
denatured at high temperatures.
At lower temperatures, complementary
polynucleotides anneal.

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DNA can denature (unfold).

Native state
Denatured
state
Abs = 260 nm
Tm = melting
temperature
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DNA can renature (refold, anneal).

DNAs ability to re-anneal

is very important in nature
and in biochemical research!
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KEY CONCEPTS: Section 3-2

The biological information encoded by a
sequence of DNA is:
Transcribed to RNA
Then, translated into the amino acid
sequence of a protein

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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
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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.
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Transcription of DNA is critical to

life.

Cells cannot thrive if transcription is shut down!

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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!
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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
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tRNA is single-stranded and

forms unique conformations.

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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.

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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!

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Genetic Code

DNA Replication
Sequence data

DNA

5
3
GGTCTTATGGGGTCAGAAAAACTTTAA sense (coding)
anti-sense
CCAGAATACCCCAGTCTTTTTGAAATT
3
5 (non-coding)

GGUCUUAUGGGGUCAGAAAAACUUUAA

RNA Synthesis (Transcription)

Sequence data

DNA

5
3
GGTCTTATGGGGTCAGAAAAACTTTAA sense (coding)
anti-sense
CCAGAATACCCCAGTCTTTTTGAAATT
3
5 (non-coding)
Transcription of anti-sense strand
RNA polymerase

RNA
GGUCUUAUGGGGUCAGAAAAACUUUAA
5
3

Protein Synthesis (Translation)

GGUCUU-AUG-GGG-UCA-GAA-AAA-CUU-UAA RNA
START
STOP
5
3

Translation of RNA into amino acids

Protein

Genetic Code

Protein Synthesis (Translation)

GGUCUU-AUG-GGG-UCA-GAA-AAA-CUU-UAA RNA
START
STOP
5
3

Translation of RNA into amino acids

* * M

G S E K L *

Protein

KEY CONCEPTS: Section 3-3

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.
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Gene size is roughly correlated with

organismal complexity.

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KEY CONCEPTS: Section 3-4

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
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DNA can be sequenced via

the Sanger method.

ddNTPs lack a
3-OH group
No more NTPs
can attach.

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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.
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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

Also required:
Thermal Cycler
a device that heats and
cools samples with
speed, precision, and
reproducibility.

Buffer containing Mg2+, among others

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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.
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PCR is conducted
over many cycles
until millions of
copies are
produced.

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Polymerase Chain Reaction (PCR)

Allows rapid polymerization of multiple copies of a specific DNA

sequence.
Produce more than a billion copies of a DNA sequence in less
than 3 hours.
Three major steps (called cycles), repeated about 25 to 35 times:
1.
2.
3.

DNA denaturation at high temperatures.

Attachment of single-stranded DNA primers at a lower temperature.
DNA synthesis by a DNA polymerase.

PCR Reagents
All reagents are mixed in a microcentrifuge
tube:

DNA Template (sample)

Control DNA
Oligonucleotide Primers
Deoxyribonucleotide triphosphates:
ATP, GTP, CTP, TTP

Reaction Buffer containing a cationic salt:

(MgCl2)

thermostable DNA polymerase:

Taq (Thermus aquaticus)

Primer Extension

http://www.youtube.com/watch?v=jx-Q-i7FsRw

PCR Reaction Cycle

DNA amplification cycle
1. Denature ~ 95C / 1.0 min
2. Annealing 45-65C / 30 .0 seg
3. Elongation ~ 72C / 1.0 - 2. 0 min

PCR

http://www.youtube.com/watch?v=eEcy9k_KsDI

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.

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Type II restriction enzymes are used

to selectively cut DNA.
Type
I

Activity
Endonuclease &
Methylase

Cleavage Site
1000 bp from
recognition sequence

II

Endonuclease

Within or near
recognition sequence

III

Endonuclease &
Methylase

~24-26 bp from
recognition sequence

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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'

EcoRI

EcoRV

5-G-A-A-T-T-C-3
3-C-T-T-A-A-G-5

5-G-A-T-A-T-C-3
3-C-T-A-T-A-G-5

Axis of symmetry
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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'

3'

3'

5'
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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.

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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.
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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

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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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