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CHEM 213 - Nucleic Acids 2

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CHEM 213 - Nucleic Acids 2

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aylarkashi76
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Nucleic Acids

DNA & RNA


Died in 2004
Overview of terms
• The cell’s entire genetic content is its genome, and the study of genomes is
genomics.
• In eukaryotic cells but not in prokaryotes, DNA forms a complex with histone
proteins to form chromatin, the substance of eukaryotic chromosomes.
• A chromosome may contain tens of thousands of genes.
• Many genes contain the information to make protein products. Other genes code for
RNA products. DNA controls all of the cellular activities by turning the genes “on” or
“off.”
• The DNA molecules of eukaryotes never leave the nucleus but instead use an
intermediary to communicate with the rest of the cell. This intermediary is the
messenger RNA (mRNA). Other types of RNA—like rRNA, tRNA, and microRNA—
are involved in protein synthesis and its regulation.
What are nucleotides?

A nucleotide has a
nitrogenous base, a pentose
sugar (ribose or 2′-
deoxyribose), and one or more
phosphate groups
Parts of a Nucleotide
In both DNA and RNA, a nucleotide consists of three parts:
• Nitrogenous base (base): A nitrogenous base (simply called a “base” in
the context of biochemistry) is an organic molecule that contains nitrogen.
Chemically, it’s a base because of the pair of electrons on the nitrogen
atom.
• Pentose sugar: The sugar is called a “pentose sugar” because it contains
five (pent-) carbon atoms. The carbon atoms are numbered. The sugar in
DNA is 2′-deoxyribose, while the sugar in RNA is ribose. The only
difference between the two sugars is that 2′-deoxyribose has one less
oxygen atom attached to the second carbon.
• Phosphate group: A nucleotide has at least one phosphate (PO43-) group.
One oxygen atom of the phosphate connects to the 5′ carbon in the sugar.
When phosphate groups link together (as in ATP or adenosine
triphosphate), the link looks like O-P-O-P-O-P-O. The nucleotide name
refers to the number of phosphate groups it contains
Nucleotides exist in different forms depending on the number of
phosphate groups:

• Monophosphate: AMP (Adenosine Monophosphate), CMP


(Cytidine Monophosphate), etc.
• Diphosphate: ADP (Adenosine Diphosphate), CDP (Cytidine
Diphosphate), etc.
• Triphosphate: ATP (Adenosine Triphosphate), CTP (Cytidine
Triphosphate), etc.
Synthesis of Nucleotides

Nucleotide synthesis in the body occurs through two primary pathways:


• De Novo Pathway: New nucleotides are synthesized from amino acids,
carbon dioxide, and formate.
• Salvage Pathway: Recycled bases and nucleosides are used to create
new nucleotides.
The choice between the pathways depends on the availability of substrates
and the energy cost involved.
Nucleotide Functions in the cell
Beyond being the building blocks of nucleic acids, nucleotides perform various
other functions in cells:
• Energy Currency: ATP serves as the primary energy currency of the cell.
• Enzyme Activity: Nucleotides like NADH and FADH₂ are cofactors in
enzymatic reactions.
• Cell Signaling: cAMP and cGMP serve as second messengers.
• Regulation: Nucleotides like ATP and GTP regulate protein synthesis and
other cellular activities.
What are nucleosides?
A nucleoside is a molecule with a pentose sugar linked
to a nitrogenous base or glycosylamines. A nucleoside
can also be defined as a nucleotide without a phosphate
group attached to it.
The main difference is seen at the second position of
the pentose structure, in the case of 2` – deoxy -ribose
there is an absence of an alcohol group/ oxy group/ -OH
group at the second position, hence the name.
In the case of D – ribose pentose the –OH group is
present at the second position.
In both types, the pentoses are present in their β-
furanose form which is a close five-membered ring
structure.
Pentose (Five-carbon) sugars

• The numbering of the carbon atoms runs


clockwise, following IUPAC chemistry
rules.
• The distinction between the two is the
absence of the hydroxyl (-OH) group on
the 2’ carbon in the deoxy-ribose sugar
in DNA, as compared with the ribose
sugar in RNA.
• The bulk of the extra Oxygen molecule
in Ribose prevents it from forming stable
double-helices.
Nitrogenous bases
The nitrogenous bases are organic molecules and
are so named because they contain carbon and
nitrogen. They are bases because they contain an
amino group that has the potential of binding an
extra hydrogen, and thus decreasing the hydrogen
ion concentration in its environment.
We can divide bases into two categories:
1. Purines have a double ring structure, Adenine
and guanine are purines.
2. Pyrimidines have a single ring, Cytosine,
thymine, and uracil are pyrimidines.
Phosphate group
A phosphate group is just a phosphorus atom
bound to four oxygen atoms, but it has many
important roles. Along with sugars and bases, it
makes up nucleic acids, like DNA and RNA. As
part of energy carriers, like ATP, it provides energy
for moving our muscles..
A phosphate backbone is the portion of the DNA
double helix that provides structural support to the
molecule.
One or more phosphate groups are esterified to
the sugar molecule at the 5′ carbon.
Nucleic Acids and Nucleotides
• Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are
the chemical carriers of genetic information
• Nucleic acids are biopolymers made of nucleotides,
aldopentoses linked to a purine or pyrimidine and a
phosphate
What are nucleic acids?
Nucleic acids are the most important macromolecules for the continuity of life.
They carry the cell’s genetic blueprint and carry instructions for its functioning.
The two main types of nucleic acids are;
1. Deoxyribonucleic acid (DNA): DNA is the genetic material in all living
organisms, ranging from single-celled bacteria to multicellular mammals. It
is in the nucleus of eukaryotes and in chloroplasts and mitochondria. In
prokaryotes, the DNA is not enclosed in a membranous envelope.

2. Ribonucleic acid (RNA). The other type of nucleic acid, RNA, is mostly
involved in protein synthesis.
Historical commentary on nucleic acids
• In 1944: Avery, MacLeod & McCarty presented Strong evidence that DNA is
genetic material
• In 1950: Chargaff reported careful analysis of DNA from a wide variety of
organisms. Content of A,T, C & G varied widely according to the organism,
however: A=T and C=G (Chargaff’ Rule)
• In 1953: Watson & Crick - structure of DNA (1962 Nobel Prize with M.
Wilkens, 1962)
DNA Double-Helix Structure
The sugar and phosphate lie on the outside of the helix,
forming the DNA’s backbone. The nitrogenous bases are
stacked in the interior, like a pair of staircase steps.
Hydrogen bonds bind the pairs to each other. Every base
pair in the double helix is separated from the next base pair
by 0.34 nm.
The helix’s two strands run in opposite directions, meaning
that the 5′ carbon end of one strand will face the 3′ carbon
end of its matching strand.
Each cell contains about two meters of DNA. DNA is
“packaged” by coiling around a core of proteins known as
histones. The DNA-histone assembly is called a
nucleosome. Histones are rich is lysine and arginine
residues.
Rules of base pairing
Because of the structure of the bases, A can only
form hydrogen bonds with T, and G can only form
hydrogen bonds with C (remember Chargaff’s
Rules).
• Each strand is therefore said to be
complementary to the other, and so each
strand also contains enough information to act
as a template for the synthesis of the other.
• This complementary redundancy is important
in DNA replication and repair. If the sequence
of one strand is AATTGGCC, the
complementary strand would have the
sequence TTAACCGG.
• During DNA replication, each strand is copied,
resulting in a daughter DNA double helix
containing one parental DNA strand and a
newly synthesized strand.
DNA

DNA stands for deoxyribose nucleic acid

This chemical substance is present in the nucleus


of all cells in all living organisms
DNA controls all the chemical changes which
take place in cells
The kind of cell which is formed, (muscle, blood,
nerve etc) is controlled by DNA
DNA
molecule
DNA is a very large molecule made up of a long
chain of sub-units
The sub-units are called nucleotides

Each nucleotide is made up of


a sugar called deoxyribose
a phosphate group -PO4 and
an organic base
Heterocycles in DNA and RNA
• Adenine, guanine, cytosine and thymine are in DNA
• RNA contains uracil rather than thymine
Hydrogen Bonding Interactions

• Two bases can hydrogen bond to form a base pair


• For monomers, large number of base pairs is
possible
• In polynucleotide, only few possibilities exist
• Watson-Crick base pairs predominate in double-
stranded DNA
• A pairs with T
• C pairs with G
• Purine pairs with pyrimidine
Functions of
Nucleotides and Nucleic Acids
• Nucleotide Functions:
• Energy for metabolism (ATP)
• Enzyme cofactors (NAD+)
• Signal transduction (cAMP)

• Nucleic Acid Functions:


• Storage of genetic info (DNA)
• Transmission of genetic info (mRNA)
• Processing of genetic information (ribozymes)
• Protein synthesis (tRNA and rRNA)
Base Pairing in DNA: The Watson–Crick
Model
• In 1953 Watson and Crick noted that DNA consists
of two polynucleotide strands, running in opposite
directions and coiled around each other in a double
helix
• Strands are held together by hydrogen bonds
between specific pairs of bases
• Adenine (A) and thymine (T) form strong hydrogen
bonds to each other but not to C or G
• (G) and cytosine (C) form strong hydrogen bonds to
each other but not to A or T
The Difference in the Strands
•The strands of DNA are
complementary because of H-
bonding
•Whenever a G occurs in one strand,
a C occurs opposite it in the other
strand
•When an A occurs in one strand, a T
occurs in the other
Primary Structure of Nucleic Acids
• The primary structure of a nucleic acid is the nucleotide sequence
• The nucleotides in nucleic acids are joined by phosphodiester bonds
• The 3’-OH group of the sugar in one nucleotide forms an ester bond
to the phosphate group on the 5’-carbon of the sugar of the next
nucleotide
Generalized Structure of DNA

20
Reading Primary Structure

• A nucleic acid polymer has a free 5’-


phosphate group at one end and a
free 3’-OH group at the other end
• The sequence is read from the free
5’-end using the letters of the bases
• This example reads
5’—A—C—G—T—3’
Example of DNA Primary Structure
• In DNA, A, C, G, and T are linked by 3’-5’ ester bonds
between deoxyribose and phosphate
Nucleic Acid Structure
Polymerization

Nucleotide
Sugar Phosphate
“backbone”
Describing a Sequence

•Chain is described from 5′ end,


identifying the bases in order of
occurrence, using the abbreviations A
for adenosine, G for guanosine, C for
cytidine, and T for thymine (or U for
uracil in RNA)
• A typical sequence is written as
TAGGCT
Properties of a DNA double
helix

The strands of DNA are antiparallel

The strands are complimentary

There are Hydrogen bond forces

There are base stacking interactions

There are 10 base pairs per turn


• An overview of DNA can be seen
at http://www.youtube.com/watch?v=_-vZ_g7K6P0
The central dogma of molecular biology
The Central Dogma (F. Crick):

• Replication: is the process by which DNA is copied with very high fidelity.
• Transcription: process by which the DNA genetic code is read and transferred to
messenger RNA (mRNA). This is an intermediate step in protein expression.
• Translation: The process by which the genetic code is converted to a protein, the
end product of gene expression. The DNA sequence codes for the mRNA
sequence, which codes for the protein sequenc
Enzymes in DNA replication

DNA is replicated by the coordinated efforts of a number of proteins and enzymes. For
replication, DNA must be unknotted, uncoiled and the double helix unwound.
1. Topoisomerase: Enzyme that unknots and uncoils DNA.
2. Helicase: Protein that unwinds the DNA double helix.
3. DNA polymerase: Enzyme that replicates DNA using each strand as a template
for the newly synthesized strand.
4. DNA ligase: enzyme that catalyzes the formation of the phosphodiester bond
between pieces of DNA.
DNA replication is semi-conservative: Each new strand of DNA contains one parental
(old, template) strand and one daughter (newly synthesized) strand
Interesting Links
• https://youtu.be/Qqe4thU-os8?si=6JNOc5W5z5dSSKWN (DNA Replication)

• https://youtu.be/a5jmdh9AnS4?si=vZlTV4YnT4JpzeYg (PCR in DNA)

• https://youtu.be/YeHtjO7vlyg?si=vNH1CIL46mPcasIw (Sanger Sequencing)


DNA Polymerase: the new strand is replicated from the 5’ → 3’
(start from the 3’-end of the template) DNA polymerases are Mg2+
ion dependent.
The deoxynucleotide 5’-triphosphate (dNTP) is the reagent for
nucleotide incorporation. 3’-hydroxyl group of the growing DNA
strand acts as a nucleophile and attacks the α-phosphorus atom of
the dNTP.
Replication of the leading strand occurs continuously in the 5’ → 3’
direction of the new strand. Replication of the lagging strand occurs
discontinuously. Short DNA fragments are initially synthesized and
then ligated together.
DNA ligase catalyzes the formation of the phosphodiester bond
between pieces of DNA.
DNA replication occurs with very high fidelity: Most DNA polymerases
have high intrinsic fidelity Many DNA polymerases have “proof-
reading” (exonuclease) activity Mismatch repair proteins seek out
and repair base-pair mismatches due to unfaithful replication
Ribonucleic Acid (RNA)
RNA contains ribose rather than 2-deoxyribose and uracil rather than thymine.
RNA usually exist as a single strand.
There are three major kinds of RNA
1. messenger RNA (mRNA):
2. ribosomal RNA (rRNA)
3. transfer RNA (tRNA)
DNA is found in the cell nucleus and mitochondria; RNA is more disperse in the
cell.
During Transcription only one of the DNA strands is copied (coding or
antisense strand). An RNA polymerase replicates the DNA sequence into a
complementary sequence of mRNA (template or sense strand).
Messenger RNA (mRNAs): are transported from the nucleus to the cytoplasm,
where they acts as the template for protein biosynthesis (translation). A three
base segment of mRNA (codon) codes for an amino acid.The reading frame of
the codons is defined by the start and stop codons.The mRNA is positioned in
the ribosome through complementary pairing of the 5’-untranslated region of
mRNA with a rRNA.
Transfer RNA (tRNA): t-RNAs carries an amino acid on the 3’-terminal hydroxyl
(A) (aminoacyl t-RNA) and the ribosome catalyzes amide bond formation
• In the ribosome, large assembly of proteins and rRNAs that catalyzes protein
and peptide biosynthesis using specific, complementary, anti-parallel pairing
interactions between mRNA and the anti-codon loop of specific tRNA’s.
• Although single-stranded, there are complementary sequences within tRNA
that give it a defined conformation. The three base codon sequence of mRNA
are complementary to the “anti-codon” loops of the appropriate tRNA. The
base pairing between the mRNA and the tRNA positions the tRNAs for amino
acid transfer to the growing peptide chain.
Ribosomal protein synthesis

Protein synthesis, as the name implies, is the


process by which every cell produces specific
proteins in its ribosome. In this process,
polypeptide chains are formed from varying
amounts of 20 different amino acids. It is one of
the fundamental biological processes in both
prokaryotes and eukaryotes.
DNA sequencing
DNA sequencing is the process
of determining the sequence of
nucleotides within a DNA
molecule. Every organism’s DNA
consists of a unique sequence of
nucleotides. Determining the
sequence can help scientists
compare DNA between organisms,
which can help show how the
organisms are related.
DNA sequencing methods
• Sanger sequencing
This method relies on a primer that binds to a denatured DNA molecule and
initiates the synthesis of a single-stranded polynucleotide in the presence of a
DNA polymerase enzyme, using the denatured DNA as a template. In most
circumstances, the enzyme catalyzes the addition of a nucleotide. A covalent
bond, therefore, forms between the 3′ carbon atom of the deoxyribose sugar
molecule in one nucleotide and the 5′ carbon atom of the next.

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