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Notes RNA and DNA

Background:
In the 1950s, monumental race in the scientific community was run between to research groups
striving for the same goal, determining the structure of DNA. Dr. Watson and Dr. Crick head a
lab in England while Linus Pauling ran his group at Cal-Tech. Watson and Crick where very
intimidated by Pauling, he had just won the 1954 Nobel Prize in chemistry. They were David
and Pauling was Goliath. Like David, Watson and Crick won. And like Davids slaying of
Goliath, Watson and Crick used a tactic unfamiliar to Pauling. They were not releasing their
findings. While Pauling regularly published his findings, which Watson and Crick led them to
their structure of DNA. They ensured the only people they spoke of about their research would
not be talking to anyone in Paulings group. Yes, scientist are people too and they can play
dirty. If you dont think they played dirty read their book, The Double Helix written by Dr.
James D. Watson.
For all organisms, DNA, deoxyribonucleic acid, is composed of the same components. The
components of DNA are sequenced a specific side-by-side arrangements termed base pairs,
along the DNA strands. This order blueprints the exact instructions required to create a
particular organism with its own unique traits.
A genome is an organisms complete set of DNA. Genomes vary widely in their number of base
pairs. The smallest known genome for an organism, a bacteria, contains about 600,000 base
pairs. The human genome consists of approximately 3 billion base pairs. All the cells in a the
human body contain the genome, except for mature red blood cells.
DNA in the human genome is arranged into 24 distinct chromosomes. 22 of which are found in
all human, the last two are gender specific. Each chromosome is a physically separate
molecule that ranges in length from roughly 50 million to 250 million base pairs. A chromosome
is large enough to be viewed with an electron microscope, the figure on the left in the picture
below is a chromosome.
The chromosomes are divided into sections which determine each specific trait an organism
exhibits. The genes are specific sequences of bases that encode instructions on how to make
proteins. It is estimated that there are 30 40 thousand genes. Oddly, of the 3 billion base
pairs in the genome, the genes comprise only about 2%, or 60 million of the base pairs. The
remaining non-coded regions functions may include structural support and the what-wherewhen and number of proteins made.
DNA and genes seem to get all the publicity, but remember they are the blue prints, they dont
actually do anything other than store information. The proteins that the genes create are
responsible for performing most life functions. The picture below shows an overall view of the
steps from Chromosome to DNA to gene to base pair sequence to amino acid sequence to
protein.

Picture of Human Chromosomes

Components of DNA and RNA:


Bases: heterocyclic rings
purine
C5H4N4
7N

N2

N1
2

N
3

purine
adenine (A)
NH2
N

6
5

5
9

pyrimidine
C4H4N2

purine
guanine (G)
O
N
N

pyrimidine
cytosine (C)
NH2
N

NH
N

pyrimidine
thymine (T)
O

NH2

NH
O

These four bases are found in DNA.

pyrimidine
uracil (U)
O

The uracil base replaces the thymine base in RNA.


The number system for the base and sugar must differ. So, the carbons in
the sugar are given prime superscripts on their numbers, while the positions
in the base are simply given numbers. As you can is in the sugars the
deoxyribose is one oxygen lighter than a normal ribose, and this difference
occurs off the second carbon.
Sugars: two forms of ribose
-D-ribose
HOH2C
4'

5'

3'

N
H

-D-2-deoxyribose
OH

O
H

NH

1'

H
2'

OH
OH
found in RNA

HOH2C
4'

5'

OH

O
H
3'

1'

H
2'

OH

found in DNA

Phosphate, PO4-3:

O-

O P O
ONucleotides:

Upon combining these three structural components, a nucleotide is created. The nucleotide
consists of a base and a phosphate bound to the sugar. To create one nucleotide, the base
binds to the carbon 1 and the phosphate binds to carbon 5. The base-sugar bond is an Nglycosidic bond and the phosphate-sugar bond is a phosphoester bond.
Normally the gylcosidic bond is formed between two carbohydrate monomers and is C-O-C,
which is an particular type of ester. This bond replaces the central oxygen with a nitrogen,
hence the N-glycosidic bond nomenclature. These are both dehydration reactions.
The ester functional group consists of a carbonyl group bonded to an oxygen with R-groups
attached to the ends. The phosphoester bond specifies that one of the R-groups be a
phosphate group.
ester
phosphoester
O
O
O
R'

O C R

O P O C R
O

The DNA nucleotides are drawn below.


NH2
N
-

O P O

5'

CH2

N
N

5'

O P O

CH2

OH

deoxyadenosine monophosphate
dAMP

deoxyguansine monophosphate
dGMP
O

NH2
N

O P O

5'

CH2

OO

1'

H
2'

OH

NH 2

1'
2'

2'

OH

O
H

NH

1'

deoxycytidine monophosphate

O P O

NH
5'

CH2

OH

1'

H
2'

deoxythymidine monophosphate

dCMP

dTMP

The nucleotides for RNA are the same except for the nucleotide containing the thymine, a uracil
has replaced this base and the sugar becomes ribose, instead of deoxyribose.
O
NH
O-

5'

O P O

CH2

OH

O
H

1'

H
2'

OH

OH

uridine monophosphate
UMP
The following table should help with the nomenclature of the nucleotides. The red is for RNA
and the blue is for DNA.
RNA base-ribose
DNA
base
base
base-deoxyribose
adenine adenosine A adenine
deoxyadenosine
dA
guanine guanosine G guanine
deoxyguanosine
dG
cytosine
cytidine
C cytosine
deoxycytidine
dC
uracil

uridine
U thymine
deoxythymidine
dT
The MP in the AMP or UMP stands for monophosphate, obviously meaning one phosphate.
This must be specified as we will find in the topic of metabolism ADP and ATP are very
important sources of cellular energy.
NH2
N
O
-

O
O P O

O P
-

5'

CH2

O
H

1'

NH2
N
O

O P

O P
-

O
O P O

5'

CH2

ADP

O
H

2'

OH

1'

H
2'

OH

OH

ATP

OH

Structure of DNA:
DNA is composed of nucleotides. These nucleotides are linked through a phosphate ester
bond, similar to the one which links the phosphate to the sugar. The same sugar bonds to a
phosphate on the other nucleotide, this time at the 3 carbon. Since this same phosphate now
has two ester style bonds the link is called a phosphodiester bond. A four nucleotide sequence
is depicted below.
NH2
N
OO P O

5'

CH2

H
3'

O
O P O

1'

NH2

5'

CH2

H
3'

O
O P O

H
N

H
5'

CH2

NH

NH2

H
3'

O
O P O

NH

H
5'

CH2

OH

O
H
3'

OH

The DNAs is actually composed to two separate strands and its overall structure is similar to
that of a spiral staircase. The sugar and phosphate are considered the backbone of the
molecule and comprise the hand-rails of the stair case, with the bases forming the stairs. The
two strands are held together by hydrogen bonding formed between the bases. The adenine
and thymine bases form a double hydrogen bond and the cytosine and the guanine form a triple
hydrogen bond. The strands actually are antiparallel. Meaning they are laid out in opposite
directions. From the above 4 nucleotide sequence you can see that one of the ends has a free
3 carbon. The ends are numbered as 3 and 5.
The two below diagrams give a visual picture of the hydrogen bonding.

DNA Replication:
Mitosis is the process that takes place in the nucleus of a dividing cell and results in the
formation of two new nuclei each having the same number of chromosomes as the parent
nucleus. This process must occur prior to a parent cell dividing into two daughter cells.
Replication of DNA occurs at this time.
This process is semiconservative. The each of the parent strands becomes one of the strands
in the new double helix. Using the parent strand as a template maintains a low percentage of
copy error. The semiconservative process was verified by the Meselson and Stahls density
labeling experiment. In the experiment they parent DNA was composed of nucleotides
composed of N-15. The resulting daughter DNA strands where composed of half N-15 and half
N-14. When the next generation was allowed to spawn half of the DNA was composed of N-14,
while the other half was composed of N-15 and N-14. At the time of this experiment a
second theory, the conservative model, rivaled the semiconservative model. The follow picture
depicts the predicted out come for each theory.

Initiation, extension and termination:


Initiation:
Denaturation of helix into single strands occurs at origin sequence
o specific sequence in DNA recognized by enzymes
o bases exposed for synthesis
Untwisting of double helix
Primer synthesis and attachment of first DNA nucleotide
Extension:
DNA replication differs between strands
o leading strand (template 3'-5' direction)
Synthesis towards y junction
Continuous replication
o lagging strand (template 5'-3' direction)
synthesis backwards away from y junction
discontinuous replication
short segments of synthesis: Okazaki fragments
Termination:
Leading strand closes in on lagging strand from other direction
Final bond made by DNA ligase
Each daughter strand is paired with one of the original strands
This process is powered by the nucleotides which are brought to serve as the complementary
bases for the strand being replicated. The nucleotides are brought to the DNA polymerase in
the a triphosphate form. DNA nucleotides are monophosphate, so the other two phosphates are
not needed and are removed. This removal actually provides energy to the replication process.
As ATP provides general cellular energy, dATP, dGTP, dTTP and dCTP all bring their own
energy to be polymerized.

RNA Structure:
There are three main structural differences between DNA and RNA.
RNA normally consists of a single strand
Uracil replaces thymine
the sugar ribose replaces deoxyribose

Types of RNA:

Messenger RNA, mRNA


o carries the gene segment from the DNA in the nucleus to the ribosomes in the cell
cytoplasm
o consist of a chain complementary nucleotides
o short life span, hour or so, before being hydrolyzed back to nucleotides
o size varies, average near 750 nucleotides
Transfer RNA, tRNA
o interprets the mRNA nucleotides, the code is laid out in three nucleotide segments
called codon, each codon corresponds to a specific amino acid
o tRNA anticondons complement the mRNA nucleotides which is the translation of the
genetic code
o the is more than one tRNA for each type of amino acid
o the structure is L-shaped
o end composed of an ACC sequence which binds the proper amino acid to the tRNA
o transport the individual amino acids to the ribosome to be polymerized into a protein
o size varies from 73 to 93 nucleotides
Ribosomal RNA, rRNA
o found in the ribosome
o ribosomes consist of 35% protein and 65% rRNA
o the protein is synthesized in the ribosome by the rRNA

Protein Synthesis:
Transcription and translation are the two steps whereby DNA encodes for the production of
amino acids into proteins. The following figure gives an overview of the entire process of protein
synthesis.

Transcription
Before the synthesis of a protein begins, the corresponding RNA molecule is produced by RNA
transcription. A portion of one strand of the DNA double helix is used as a template, this section
is named a gene. The RNA polymerase synthesizes a mRNA from this gene. This mRNA
migrates from the nucleus to the cytoplasm. The gene contains coded sections, exons and is
sectioned with non-coded portions, introns. The mRNA copies all of this information, including
the intron nucleotides. The mRNA will go through a maturation process called splicing where
the non-coding sequences, introns are eliminated, as depicted below.

Translation:
The coding mRNA sequence can be described as a unit of three nucleotides called a codon.
The ribosome binds to the mRNA at the start codon, AUG that is recognized only by the tRNA.
The ribosome proceeds to the elongation phase of protein synthesis. During this stage,
complexes, composed of an amino acid bound to tRNA, sequentially bind to the appropriate
codon in mRNA by forming complementary base pairs with the tRNA anticodon. The ribosome
moves from codon to codon along the mRNA. This process aligns the amino acids and a
peptide bond is formed between these closely positioned amino acids. Amino acids are added
one by one, translated into polypeptidic sequences dictated by DNA and represented by mRNA.
At the end, a release factor binds to the stop codon, terminating translation and releasing the
complete polypeptide from the ribosome.

The amino acids that correspond to each codon are listed in tables. These tables come in two
formats, each is shown below. Each amino acid corresponds with anywhere from one to six
specific codons. In addition, three codons are used for starting and stopping sequences rather
than specifying amino acids to be added to the sequence. Note that the base sequences
shown here are codons, mRNA rather than anti-codons, tRNA.

A = adenine G = guanine C = cytosine T = thymine U = uracil

Laboratory DNA Synthesis:


PCR, polymerase chain reaction was invented by Kary B. Mullis in 1986, for which he earned
of that years Nobel prize for chemistry in 1993. If you are wondering, Michael Smith won the
other half for his fundamental contributions to the establishment of oligonucleotide-based, sitedirected mutagenesis and its development for protein studies. The Nobel committee could not
decide between these to outstanding discoveries in the field of protein research.
To copy DNA, polymerase requires two other components: a supply of the four nucleotide bases
and something called a primer. DNA polymerases, whether from humans, bacteria, or viruses,
cannot copy a chain of DNA without a short sequence of nucleotides to "prime" the process, or
get it started. So the cell has another enzyme called a primase that actually makes the first few
nucleotides of the copy. This stretch of DNA is called a primer. Once the primer is made, the
polymerase can take over making the rest of the new chain.
A PCR vial contains all the necessary components for DNA duplication: a piece of DNA, large
quantities of the four nucleotides, large quantities of the primer sequence, and DNA polymerase.
The polymerase is the Taq polymerase, named for Thermus aquaticus, from which it was
isolated.
The three parts of the polymerase chain reaction are carried out in the same vial, but at different
temperatures. The first part of the process separates the two DNA chains in the double helix.
This is done simply by heating the vial to 90-75 degrees centigrade (about 165 degrees
Fahrenheit) for 30 seconds.
But the primers cannot bind to the DNA strands at such a high temperature, so the vial is cooled
to 55 degrees C. At this temperature, the primers bind or "anneal" to the ends of the DNA
strands. This takes about 20 seconds.
The final step of the reaction is to make a complete copy of the templates. Since the Taq
polymerase works best at around 75 degrees C, the temperature of the vial is raised.
The Taq polymerase begins adding nucleotides to the primer and eventually makes a
complementary copy of the template. If the template contains an A nucleotide, the enzyme adds
on a T nucleotide to the primer. If the template contains a G, it adds a C to the new chain, and
so on to the end of the DNA strand. This completes one PCR cycle.
The three steps in the polymerase chain reaction - the separation of the strands, annealing the
primer to the template, and the synthesis of new strands - take less than two minutes. Each is
carried out in the same vial. At the end of a cycle, each piece of DNA in the vial has been
duplicated.
But the cycle can be repeated 30 or more times. Each newly synthesized DNA piece can act as
a new template, so after 30 cycles, 1 billion copies of a single piece of DNA can be produced!
Taking into account the time it takes to change the temperature of the reaction vial, 1 million
copies can be ready in about three hours.
Simple, elegant, useful and can be FULLY automated.

Laboratory Protein Engineering:


The synthetic organic chemist faces several problems:
1. The carboxylic acid group (-COOH) in an a-amino acid is not reactive enough to give a
condensation reaction with the amine group (-NH 2) of another amino acid using mild
conditions.
2. As the polypeptide chain is built up unit by unit, care must be taken not to expose it to
conditions that would hydrolyze the peptide links already made.
3. Many a-acids have side chains with functional groups that may react under the conditions
used to make the peptide links.
4. A polypeptide chain has two reactive ends; the free NH 2 and the free -COOH. The synthetic
chemist must make sure that each extra a -amino acid unit adds on to the correct end of the
polypeptide chain being made (see figure on the below).
5. The chain must be elongated by one a -amino acid unit at a time. This means one linking
reaction for each a -amino acid in the chain, followed by separation of the products. Each
reaction must, therefore, have a high yield and each purification step must separate most of
the product.
Chemists solve the problems above as follows:
Problems 1 and 2 by using a coupling reagent that increases the reactivity of the - COOH
group under mild conditions.
Problem 3 by protecting reactive side chains with blocking groups that they can remove
under mild conditions when all the links are complete.
Problem 4 by blocking the a -amino acid and polypeptide groups that they do not want to
join, again using groups they can remove easily later.
Problem 5 by attaching the growing polypeptide chain to an insoluble support made from a
resin. They then wash away soluble by-products and unused reagents before adding the
next a -amino acid to the chain. When the synthesis is complete the polypeptide is detached
from the resin.

The 1984 Nobel Prize for Chemistry was awarded to Professor Merrifield of Rockefeller
University for developing an automated version of this technique capable of producing the
hormone insulin (51 -amino acids). The above diagram summarizes the sequence of steps
involved. The protein is engineered by simply dipping starting material into a series of different
beakers.
Simple, elegant, useful and can be FULLY automated.
The following figure gives the primary structure of the two chains in human insulin.

Biological Protein Engineering:


An organism has genetic material from both parents and so it will inherit characteristics from
both parents. Farmers have been using this idea for thousands of years to help selectively
breed better strains of crops and livestock animals.
Errors can occur when cells divide and genetic information is copied and passed on to offspring.
Scientists refer to these errors as spontaneous mutations. Sometimes the mistakes can be
harmful or even fatal, but occasionally they can give the organism a characteristic that is an
advantage. When the characteristic is an advantage the mutation flourishes in following
generations. This is a brief molecular explanation for the theory of Natural Selection.
Spontaneous mutations are easy to observe and exploit in single celled organisms with short life
cycles. Wine makers and brewers have relied on spontaneous mutations to produce yeasts that
give a wine or a beer its characteristic flavor.
Scientists can increase the frequency of random mutations by exposing cells to ultra violet light.
They then select those cells that have, by chance, mutated to give desirable characteristics.
Scientists, though, have developed the technology that allows them to transfer genes from one
organism to another, even from one species to another. We call this recombinant DNA
technology or genetic modification (engineering) and it provides the opportunity to change the
genetic make-up of an organism quickly, more specifically and with less trial and error than
before.
Genetic modification can alter micro-organisms so that they produce human proteins such as
insulin. Insulin is a protein hormone that helps to regulate the way our bodies metabolize sugar.
Healthy individuals produce it from cells in their pancreas. Diabetics may produce little or no
insulin and need regular injections of it to remain healthy.
It would be good if we could make insulin in a factory by extracting some human pancreas cells
and culturing them in a vessel with a supply of -amino acids.
Unfortunately though, human cells are difficult to culture outside the body. Instead biochemists
have identified the gene that carries the code for insulin, removed it from human cells and joined
it to the genetic material in micro-organisms. Biotechnologists use fermenters to encourage
these micro-organisms to grow and multiply rapidly. The micro-organisms use their chemical
systems for protein synthesis to make human insulin alongside all the other proteins they would
make naturally. Insulin is finally separated and purified.
The following figure gives a general approach showing how genetic modification can produce a
protein biologically.

Biochemists can now use a refinement of the genetic modification technique to redesign
proteins. Once they have isolated the gene for a particular protein they can alter its code so that
a change occurs in the proteins primary structure. They then incorporate the modified gene into
a micro-organism where it is decoded as before, but this time a new protein appears.
Swapping one a-amino acid in a protein can have a large effect on how the protein behaves. As
the primary structure dictates secondary and tertiary structure therefore function. Biochemists

have already used this protein engineering to modify human insulin in a way that makes it
become absorbed more quickly after injection. Multiple changes are needed to humanize
antibodies.
Computer modeling techniques allow protein chemists to make predictions about how proposed
-amino acid changes might change the structure and activity of a protein.
Genetic and protein modification have enormous potential. Besides insulin, genetic modification
can produce human growth hormone and the blood clotting Factor VIII. Genetic engineering
may provide an alternative to conventional plant breeding techniques in farming. In addition it is
already possible to introduce into a plant new genes that enable it to produce its own protein
insecticide or that make it resistant to disease. Hepatitis B vaccine, oil-digesting bacteria and
bacteria that produce biodegradable plastic are all recently developed products of genetic
modification techniques.

DNA Fingerprinting:
In DNA ID "chemical scissors" called restriction enzymes are used to cut the extremely long
DNA molecule into fragments. Each restriction enzyme cuts at each point on the DNA molecule
where specific patterns of nitrogen bases occur. Everyone has these patterns in their DNA, but
not at the same places. Because the patterns occur in different places, the length of the
fragment differs from person to person. In very rare instances two people might have the same
pattern of fragment lengths produced by one restriction enzyme. Therefore, in DNA ID samples
are tested with more than one enzyme. The probability that two or more people will produce the
same pattern when their DNA is cut by two or more restriction enzymes is very small.
Statisticians call this the multiplication rule, because the individual probabilities of a mistaken
identity for each pattern are multiplied together to find the overall probability.

Examples of DNA Fingerprinting Tests