DeoxyriboNucleic Acid (DNA) - 5

Order of Subtopics Covered:

1. A Review
a. Nucleotides
b. DNA
2. DNA Replication
a. Steps
b. About DNA Replication
c. Enzymes involved in Replication
3. Ribonucleic Acid the Molecule
4. Protein Synthesis
a. Transcription/Translation Overview
b. Translation Steps
c. Protein synthesis overview
5. Gene Mutations
a. Types of Base Changes
6. Recombinant DNA
a. Steps in making rDNA
b. How it works
c. Methods of Formation
d. Uses of rDNA
Nucleotides A Review
Page 463-465
DNA and RNA are made up of NUCLEOTIDES. Each nucleotide is composed of:
1) PENTOSE sugar
2) NITROGENOUS base

N
N

3) PHOSPHATE GROUP

The bases may be PURINES (2 rings, include adenine and guanine) or

PYRIMIDINES (1 ring, include cytosine and thymine or URACIL)

Strict Rules of bonding: Chargaffs Rule of complementary base pairing

In DNA

In RNA

A ---T

A---U

C:::::G

C:::::G

Bases bond using HYDROGEN bonds.

The sugar-phosphate backbone may be generalized this way:

5

5 = FIVE PRIME
3 = THREE PRIME
Ensures nucleotides are connected
& read in the correct way just like reading a
Sentence from left to right.

3
DNA A Review
Functions:
1. Controls CELLULAR ACTIVITIES - DNA carries a code that are genetic
instructions.
o Encoded in a sequence of BASES strung together.
o Instructions used in building PROTEINS.
2. Replicates: DNA makes COPIES of itself to pass onto other cells.
3. Mutates: CHANCE changes in and/or recombination of DNA which allow for
GENETIC DIVERSITY.

Characteristics:
o Bases: A T G C
o Double STRANDED
o Sugar: DEOXYRIBOSE
o Remains in the NUCLEUS

 Structure:

RUNG

Hydrogen bonds

o This is found in a spiral shaped called the double HELIX

o Together, all the H-bonds hold the 2 strands together TIGHTLY
o Each rung or base PAIR consists of a purine and a PYRIMIDINE
( a 2-ringed and a 1-ringed structure).
o DNA has POLARITY always read in one direction. This is important because
REPRODUCTION of DNA and READING of DNA must occur in a particular
direction just like reading a sentence in from left to right is important.
o Inherent in DNAs structure is a mechanism for REPRODUCTION. Before a cell can
divide, DNA must be DUPLICATED. The basis of the continuity of life is that DNA
can REPLICATE itself (cell replacement and heredity)

Page 467-468
DNA replication: (Making a COPY).
STEPS:
1) 2 strands of DNA become ladder-like.

2) The enzyme HELICASE breaks H-bonds between bases and UNZIPS the ladder.
o DNA is opened at REPLICATION origin (A-T rich)

3) Free floating nucleotides in the NUCLEOPLASM move to appropriate position via

COMPLEMENTARY base-pairing
o The enzyme DNA POLYMERASE facilitates BASE pairing (A-Tand C-G)
between S.S. DNA and nucleotides.
.

4) Adjacent nucleotides become JOINED through sugar-phosphate bonds.

5) Once complete, 2 DNA molecules wind up. 2 new DNA molecules are IDENTICAL to
each other.

OLD MOTHER STRAND

NEW DAUGHTER STRAND

ABOUT DNA REPLICATION:

o Called SEMICONSERVATIVE replication because each DNA molecule contains one
NEW and one original strand.
Since half of each original molecule is CONSERVED, in each of the new
molecules. This ensures VERY, VERY ACCURATE replication.
o Complimentary is crucial to REPLICATION, each strand acting as a TEMPLATE for a
new strand.
o Replication takes about 8 HOURS for the entire genome, with only 1 or 2 errors!!
(comparable to about 1000 textbooks)
o Bacterial genome consists of a single circular RING of DNA, has 1 replication origin.
In humans, the genome has about 10,000 replication origins, allowing DNA replication
to begin at many places at onceTHIS IS FASTER !

ENZYMES INVOLVED IN DNA REPLICATION

o Helicase: causes DNA to UNZIP
o Topoisomerase: holds strands APART
o DNA Polymerase: At heart of replication MACHINE
- Synthesizes new DNA STRAND using old strand as a TEMPLATE
- Catalyses the ADDITION of nucleotides to the growing DNA strand,
linking them with PHOSPHODIESTER bonds
- PROOFREADS: checks pairing as soon as it happens before making
phosphodiester bonds

Ribonucleic Acid the molecule

There are 3 TYPES of RNA, each with a DIFFERENT function, but all are involved in
PROTEIN SYNTHESIS.
1. MESSENGER RNA (mRNA)
2. RIBOSOMAL RNA (rRNA)
3. TRANSFER RNA (tRNA)

MESSENGER RNA (mRNA) is a COPY of the genetic code contained in the

sequences of bases in the cells DNA.
o Acts as a go-between for DNA in the nucleus and the RIBOSOMES of the
CYTOPLASM.
o 5 10% of the cells RNA

RIBOSOMAL RNA (rRNA) is a STRUCTURAL part of the RIBOSOMES.

o Associates with proteins to form ribosomes.
o Ribosomes are the site of PROTEIN SYNTHESIS.
o Vary in size
o 85 90% of the cells RNA.

TRANSFER RNA (tRNA) delivers AMINO ACIDS from the CYTOPLASM to the
RIBOSOME.
o There is a DIFFERENT tRNA for each amino acid.
o ~80 nucleotieds and are shaped in a CLOVERLEAF pattern.
o 5% of the cells RNA.

AMINO ACID

ANTICODON

H-BONDS

Page 468-475
Protein Synthesis:
Central Dogma of Molecular Biology

DNA info mRNA info PROTEIN

*One Gene, one PROTEIN*
1) DNA Transcription (DNA RNA): Synthesis of MRNA (messenger RNA):

Photocopying stage DNA doesnt leave NUCLEUS and protein is made in

the CYTOPLASM.

TRANSCRIPTION STEPS:
i. 2 DNA strands SEPARATE .
ii. The 1 strand that makes sense & acts as a TEMPLATE &
complementary RNA bases are brought in.
*Note URACIL, instead of THYMINE, binds with adenine
CYTOSINE binds with GUANINE.
iii. RNA POLYMERASE catalyses the addition of RIBONUCLEOTIDES by
forming SUGAR-PHOSPHATE bonds between adjacent RNA
nucleotides. New RNA strand GROWS as bases on DNA strand form Hbonds with RNA bases one at a time, forming a CHAIN.
iv. mRNA is released from DNA.
v. DNA REWINDS, and returns to normal DOUBLE HELIX form.
vi. Assembled mRNA strand moves to the CYTOPLASM from the nucleus
via a NUCLEAR PORE.

2) RNA Translation (RNA PROTEINS) process that changes RNA into PROTEIN;
Occurs on the surface of a RIBOSOME; Baking or reading recipe stage

The order of the bases in DNA, and subsequently the mRNA, determines the
ORDER of AMINO ACIDS in the protein being made.
o RNA is like a sentence and made of WORDS a set of letters; RNAs
words are called CODONS which are a set of 3 nucleotides.
o Each amino acid is coded for by a set of 3 nucleotides a CODON. This is
called the TRIPLET CODE.

In fact, the SAME amino acid is often specified by MORE THAN ONE codon.
However (and this is very important), the reverse is never true: that is, any ONE
CODON only specifies ONE AMINO ACID -- there is no vagueness in the code
(e.g. CCU will always produce proline).
The code also contains PUNCTUATION. It tells when to START reading the
gene for a particular protein and when to STOP.
Each codon corresponds to an amino acid, or a "start" or "stop" synthesis signal.
The genetic code is UNIVERSAL: the same codons stand for the same amino
acids in all living things (well, almost all living things). This "Biochemical Unity"
suggests that all living things have a COMMON EVOLUTIONARY ANCESTOR.
And here it is, the most important chart in all of Biology: the GENETIC CODE!

The genetic code is DEGENERATE, as a single a.a. can be coded for by more
than one codon.
o Example: CAA and CAG both code for GLUTAMINE
o Start codon, AUG is the first to be translated and codes for METHIONINE
o Terminator codon: tRNA with no a.a. triggers ribosome to RELEASE
polypeptide chain, terminating translation.

TRANSLATION STEPS:
i.

Initiation: Proteins called initiation factors bring together:

1. MRNA, 2 RIBOSOMAL SUBUNITS, TRNA (WITH AN A.A.)
2. rRNA (RIBOSOMAL RNA) makes up large and small subunits of the
ribosome (made in the nucleolus).
3. tRNA (TRANSFER RNA) brings AMINO acids to the ribosome
o What happens during initiation:
1) Small ribosomal subunit binds MRNA
2) Large subunit, with A and P sites, binds to the small subunit.
3) The ribosome moves along the mRNA molecule until the start CODON
(AUG) reaches the P-site.
4) The tRNA has a binding site of 3 bases called an ANTICODON that is
COMPLEMENTARY to the mRNA codon. Therefore, the codon of mRNA
of AUG is "read" by a tRNA that has a UAC anticodon. tRNA, carrying
METHIONINE anitcodon (UAC) enters the P-site.
o This methionyl-tRNA is in the P site of the ribosome. The A site
next to it is available to the tRNA bearing the NEXT amino acid.
o There is a SPECIFIC tRNA for each mRNA codon that codes for
an amino acid.

ii. Elongation: More AMINO acids are added one by one, with the help of proteins
called elongation factors, to form a POLYPEPTIDE.
o What happens during elongation:
1)A second COMPLEMENTARY tRNA carrying the next a.a. to bind to the
A-Site.
2)codon on mRNA forms an H-bond with ANTICODON on tRNA, which
carries an a.a.

3)A PEPTIDE bond forms between the two adjacent AMINO acids.
4)Ribosome shifts along mRNA to the next CODON.
5)Ribosome releases the tRNA from the P-site.
6)tRNA in A-site moves to the P-SITE.
7)This process repeats with the addition of each amino ACID.

P-Site

A-Site

1) Termination: Termination codon signals for translation to STOP

1) Terminator sequence (UAA, UAG, UGA) enters the A-site
2) Ribosome breaks apart tRNA and a.a. in P-site
3) tRNA without an a.a. enters A-site
4) Protein called release factor causes H2O molecule to be added to the
POLYPEPTIDE chain, freeing it from the ribosome.
5) Ribosomal subunits SEPARATE
6) Released polypeptide chain folds and COILS, some a.a. may be
removed, it may be CLEARED , and several chains may come
together to form a LARGER PROTEIN
(4o STRUCTURE).

About Translation:
o 1 mRNA may have many ribosomes attached, forming a POLYSOME.

DNA
mRNA

Protein Synthesis Overview:

TTACGGCTATGCGGTGCTATATTTAGCACATGCTCGCATT

Protein
Gene Mutations & Affects on Protein Production

A mutation is any ALTERATION in a gene or its EXPRESSION.

o Change will first be reflected in the RNA copy, then in the enzyme or other
PROTEIN that the RNA codes for, and finally in the appearance of new TRAITS
Pg 478,wiki in the living organism.

Mutations occur within a GENOMIC LIBRARY. These can be:

o Caused by MUTAGENS
o Occur spontaneously during DNA REPLICATION, or repair.

There are two main categories of mutations:

1. GENE MUTATIONS (affect only one gene)
2. CHROMOSOMAL MUTATIONS (affect many genes because they affect entire
chromosomes or parts of chromosomes).

Mutagens are PHYSICAL or CHEMICAL agents that cause changes to DNA.

o Ionizing RADIATION (X-RAYS, gamma rays or alpha particles)
o UV light NATURAL mutagen
o Exposure to chemicals like those found in CIGARETTES, DDT, ASBESTOS.

TYPES OF BASE CHANGES (a.k.a. POINT MUTATION):

o Substitution: change in a nucleotide BASE
may change AMINO ACID (MISSENSE mutation)
may not change amino acid (SILENT mutation)
may change location of STOP codon (NONSENSE mutation)
o Deletion: REMOVAL of a nucleotide.
FRAMESHIFT: changes reading frame and all amino acids
after deletion (Nonsense mutation)
o Addition: addition of a nucleotide
FRAMESHIFT: changes reading frame and all amino acids after
addition. (Nonsense mutation)

EXAMPLES
It takes only a single different pair of bases to produce a different or imperfect
organism.

Consider an analogy of a mutation to a sentence in English.

EXAMPLE OF THE EFFECT OF A MUTATION:

ORIGINAL MESSAGE:

THE BIG DOG BIT TED AND RAN OFF

DELETION/FRAME SHIFT:

THE BID OGB ITT EDA NDR ANO FF

Try it for yourself: Here is a section of DNA before a mutation.

DNA
mRNA
a.a.

G
C

G G
C C
Proline

C T C
G A G
Glut. acid

T A G
A U C
Isoleucine

C
G

G A
C U
Alanine

G
C

A T
U A
Leucine

DNA
a.a.

T T
A A
Stop

T A C
A U G
Methionine

G
C

G G
C C
Proline

G C T
C G A
Arginine

C
G

T A
A U
Valine

G C G
C G C
Arginine

A
U

G A
C U
Serine

T A T
A U A
Isoleucine

Here the same section is after two bases have been switched from the original
sequence.

DNA
mRNA

T A C
A U G
Methionine

C
G

G G
C C
Alanine

C T C
G A G
Glut. Acid

T A G
A U C
Isoleucine

C
G

G G
C C
Alanine

G
C

A T
U A
Leucine

Notice the different effects that different POINT mutations can have!

If there is a change in the DNA that causes a change in the significant part of the
mRNA codon(s), a different amino acid will be translated, and a different protein will
be made. Usually random changes are HARMFUL (frequently mutations are
lethal). About one time in million, the change might actually improve the protein
(this is called a BENEFICIAL MUTATION. Beneficial mutations, while infrequent,
drive the evolution of species!

Occasionally, a mutations will be NEUTRAL that is it will have no effect on the

protein produced (as in the case of the second mutation in the second example
above), or it will change an amino acid on a non-vital part of the protein.

Recombinant DNA
Recombinant DNA (rDNA) is DNA in which DNA from one SPECIES (e.g. a gene
coding for the protein Insulin) is inserted into the DNA of a SECOND species (e.g.
bacteria).
o The second species can then go on to produce proteins of the first species, and
when it reproduces, it will copy the other species' DNA and pass it onto its
OFFSPRING. The gene can now said to be "CLONED."

A
U

Here the same section is after one extra base (a G in the third codon) has been
added to the original sequence.

mRNA

a.a.

T A C
A U G
Methionine

The "recombining" of the DNA from different species has allowed the production of
RARE PROTEINS in large quantities (e.g. insulin). Isolating, modifying, and
reinserting DNA sequences, called "Human gene therapy" is aimed at controlling or
curing genetic disorders.

A
U

T T
A A
Stop

As DNA is so fundamental to life processes, DNA technology raises many social,

legal, ecological, and ethical questions.

The uses of recombinant DNA include:

1. CLONING GENES: viruses and bacteria can be used to make copies of the
gene(s) of another species. Whole organisms can now be cloned as well!
2. PRODUCING BIOTECHNOLOGY PRODUCTS: genetically engineered
prokaryotic and eukaryotic cells can be used to MASS produce once rare
medicinal PROTEINS and HORMONES as well as VACCINES to prevent
disease (e.g. hepatitis B).
3. MAKING TRANSGENIC ORGANISMS: we can alter the DNA of bacteria, plants,
and farm animals to make them more valuable and less susceptible to disease.
- BACTERIA is very useful in this capacity
4. GENE THERAPY: to replace DEFECTIVE genes in a living organism (especially
humans) with healthy genes, and is used to treat genetic disorders and diseases.
- Can be Ex Vivo (outside living organism) or In Vivo (inside living
organism).

STEPS IN MAKING Rdna:

A "VECTOR" is something that can get the DNA from one

species into the other species' DNA. Often, this can be a
"PLASMID", a circular piece of DNA found in some bacteria.

PLASMID

Main Bacterial DNA

A human gene, such as the gene for INSULIN, is inserted into
the plasmid and then the plasmid is taken up by BACTERIA.
The bacteria reproduces the plasmid along with its own DNA when it reproduces,
and translates the human gene, producing human PROTEIN.