INTRODUCTORY

MOLECULAR BIOLOGY

Okunowo O Wahab, Ph.D.

Biochemistry Department
College of Medicine
University of Lagos
 Course outline
• STRUCTURE COMPOSITION OF DNA&RNA

• TYPES OF DNA

• TYPES OF RNA

• DNA REPLICATION

• POST-REPLICATIVE MODIFICATION OF DNA

• CHEMOTHERAPIES TARGETING REPLICATION

• RNA TRANSCRIPTION

• POST-TRANSCRIPTIONAL MODIFICATION OF RNA

• GENETIC CODE

• TRANSLATION.
 DNA structure
Composition of DNA,
deoxyribonucleic acid, and
RNA, ribonucleic acid.

In slide 1, the DNA molecule is

seen as a double stranded helix
or a “ladder” shaped molecule.

The rungs of the ladder are

actually hydrogen bonds (H)
joining two nucleotide bases
together.

The sides of the ladder made

up deoxyribose sugars are
joined together by
phosphodiester bonds.

Slide 1
In slide 2, a nucleotide is seen to be
composed of 3 chemical groups: a
nitrogenous base or nitrogen containing
base, a sugar molecule & a phosphate (PO4) Composition of
group.

There are 2 types of nitrogenous bases

DNA&RNA
found in DNA or RNA known as purines or
pyrimidines; a nucleotide contains either
one.

There are 5 purine or pyrimidine bases found

in DNA or RNA; these are adenine, thymine,
guanine, cytosine, or uracil.

This nitrogen containing base is linked to a 5

carbon sugar – either deoxyribose, which
has a hydroxyl group (OH) at carbon 3’,

or ribose, which has OH groups at carbons 2’

& 3’.

The phosphate group is attached to the

sugar at carbon 5’.

Note that each nucleotide has a direction

– the top part is the 5’-phosphate end &
the bottom is the 3’-OH end
Each nucleotide can be linked together
endlessly to form a nucleic acid. The
phosphate group of the lower nucleotide
links to the –OH group at the 3’ carbon on
the upper nucleotide to form a
phosphodiester bond.

The 5’ end will always will have a free or

unbound phosphate group & the 3’ end
will always have an unbound 3’-OH
group. Direction is an important
concept to remember!

Each side of the DNA helix or ladder is in

the opposite direction of each other.

This is called an anti-parallel structure.

Each side is always anti-parallel.

Also note that the ladder structure is

further stabilized by hydrogen bonds that
join the nitrogenous bases together,
which as are the “rungs of the ladder

Slide 3
The H bonds are very systematic in how they form. Of
the 4 bases in DNA - adenine, guanine, cytosine &
thymine. Adenine always bind to Thymine & Guanine
always bonds with Cytosine.

These are called base pairs or “Watson-Crick base

pairing” after the two scientists who proposed the
structure of DNA in the early 1950’s. This is also called
complementary base pairing – bases only bond with
their complement.

In RNA, 3 of the 4 bases are the same as in DNA –

adenine, guanine, cytosine -- the only difference is uracil
is present in place of thymine. Thus, base pairs in RNA
are Adenine & Uracil and Guanine & Cytosine.
Complementary base pairing is a very important
concept to keep in mind
On the right side is a
segment of DNA, where
H bonds form between A-
T & C-G and nucleotides
are linked together in an
anti-parallel fashion.
Therefore, DNA is a
complementary & anti-
parallel structure.

On the left side of the

slide, RNA tends to be
single-stranded, but it can
loop on itself and when it
does, it is also in a
complementary & anti-
parallel fashion.

Slide 4
 Types of DNA

The double helix of DNA has

been shown to exist in several
different forms, depending upon
sequence content and ionic
conditions of crystal preparation.

The B-form of DNA prevails under

physiological conditions of low
ionic strength and a high degree
of hydration.

Regions of the helix that are rich

in pCpG dinucleotides can exist in
a novel left-handed helical
conformation termed Z-DNA.

This conformation results from a

180 degree change in the
orientation of the bases relative to
that of the more common A- and
B-DNA
 Characteristics of Types of DNA
Parameters A Form B Form Z-Form

Direction of helical rotation Right Right Left

Residues per turn of helix 11 10 12 base pairs

Rotation of helix per residue 33 36 -30

(in degrees)

Base tilt relative to helix axis 20 6 7

(in degrees)

Major groove narrow and deep wide and deep Flat

Minor groove wide and shallow narrow and deep narrow and deep

Orientation of N-glycosidic Anti Anti Anti for Py, Syn for Pu

Bond

Comments   most prevalent within cells occurs in stretches of

alternating purine-pyrimidine
base pairs
 Types of RNA
•RNA is similar to DNA. Like DNA, it is composed of nucleotides joined by 3- to 5-
phosphodiester bonds, the purine bases adenine and guanine, and the pyrimidine
base cytosine. However, its other pyrimidine base is uracil rather than thymine.

• In RNA, the sugar is ribose, which contains a hydroxyl group on the 2´-carbon.

•RNA chains are usually single-stranded and lack the continuous helical structure
of double-stranded DNA. However, RNA still has considerable secondary and
tertiary structure because base pairs can form in regions where the strand loops
back on itself.

•As in DNA, pairing between the bases is complementary and antiparallel. But in
RNA, adenine pairs with uracil rather than thymine. Basepairing in RNA can be
extensive, and the irregular looped structures generated are important for the
binding of molecules, such as enzymes, that interact with specific regions of the
RNA.

•The three major types of RNA (mRNA, rRNA, and tRNA) participate directly in
the process of protein synthesis. Other less abundant RNAs are involved in
replication
 Messenger RNA (mRNA)
It comprises only about 5% of the RNA in the
cell yet is by far the most heterogenous type
of RNA in terms of size.

The mRNA carries the genetic information

from the DNA to the cytosol, where it is used
as the template for protein synthesis.

Each mRNA molecule contains a nucleotide

sequence that is converted into the amino
acid sequence of a polypeptide chain in the
process of translation

Special structural characteristics of eukaryotic

mRNA include a long sequence of adenine
nucleotides (a “poly-A tail”) on the 3’-end of
the RNA chain, plus a “cap” on the 5’-end
consisting of a molecule of 7-
methylguanosine attached “backward”
through a triphosphate linkage.
 Ribosomal RNA (rRNA)

Ribosomes are subcellular ribonucleoprotein

complexes on which protein synthesis occurs.

Different types of ribosomes are found in

prokaryotes and in the cytoplasm and
mitochondria of eukaryotic cells.

rRNA is found in association with a number of

different proteins as components of the
ribosomes.

There are three distinct size species of rRNA

(23S, 16S, and 5S) in prokaryotic cells and
eukaryotic mitochondria.

In the eukaryotic cytosol, there are four rRNA

size species (28S, 18S, 5.8S, and 5S).

rRNA helps to hold mRNA on the ribosome in

order that mRNA can be translated.

{Note: “S” is the Svedberg unit, which is related

to the molecular weight of the compound.}
Together, they make up 80% of the RNA in the
cell.
 Transfer RNAs (tRNA)

Transfer RNAs (tRNA), the smallest of

the three major species of RNA molecules
(4S), have between 74 and 95 nucleotide
residues.

There is at least one specific type of tRNA

molecule for each of the 20 amino acids
commonly found in proteins.

Together they make up about 15% of the

RNA in the cell.

Each tRNA serves as an “adaptor”

molecule that carries its specific amino
acid to the site of protein synthesis. There,
it recognizes the genetic code word that
specifies the addition of its amino acid to
the growing peptide chain.

Typical clover leaf structure of

tRNA
 THE CHEMICAL NATURE OF RNA DIFFERS
FROM THAT OF DNA

RNA is a polymer of purine and pyrimidine ribonucleotides linked together by 3′,5′-

phosphodiester bridges analogous to those in DNA. Although sharing many features with
DNA, RNA possesses several specific differences:

(1)In RNA, the sugar moiety to which the phosphates and purine and pyrimidine bases are
attached is ribose rather than the 2′-deoxyribose of DNA.

(2) The pyrimidine components of RNA differ from those of DNA. Although RNA contains the
ribonucleotides of adenine, guanine, and cytosine, it does not possess thymine. Instead of
thymine, RNA contains the ribonucleotide of uracil.

(3) RNA exists as a single strand, whereas DNA exists as a double-stranded helical molecule.
However, given the proper complementary base sequence with opposite polarity, the single
strand of RNA is capable of folding back on itself like a hairpin and thus acquiring
doublestranded characteristics.

(4) Since the RNA molecule is a single strand complementary to only one of the two strands of
a gene, its guanine content does not necessarily equal its cytosine content, nor does its
adenine content necessarily equal its uracil content.

(5) RNA can be hydrolyzed by alkali to 2′,3′ cyclic diesters of the mononucleotides, compounds
that cannot be formed from alkali-treated DNA because of the absence of a 2′-hydroxyl
group. The alkali lability of RNA is useful both diagnostically and analytically
 Summary

DNA is
• a double stranded helix
• “rungs” joined together by H bonds
• phospodiester linkages between 5’ & 3’ ends of nucleotides; 5’ 3’ direction
• contains deoxyribose sugar
• contains A, T, C, G nucleotides
• found in the nucleus of cells; some is found in the mitochondria

RNA is –
• primarily a single stranded molecule
• linked by the same type of phosphodiester bonds that join DNA together; 5’ 3’
direction
• contains ribose sugar
• contains A, U, C, G nucleotides
• 3 types of RNA – messenger, ribosomal & transfer (mRNA, rRNA, tRNA)
• found in the cytoplasm; however it is manufactured in the nucleus, so some is found
there
 DNA Replication
• The genetic information stored in the nucleotide sequence of DNA serves
two purposes.
• (1) It is the source of information for the synthesis of all protein molecules of
the cell and organism

• (2) It also provides the information inherited by daughter cells or offspring.

• Both of these functions require that the DNA molecule serve as a template
—in the first case for the transcription of the information into RNA

• and in the second case for the replication of the information into daughter
DNA molecules.

• Replication of DNA occurs during the process of normal cell division cycles
• The mechanics of DNA replication was characterized in E. coli.

• 3 distinct enzymes capable of catalyzing the replication of DNA were identified

as DNA polymerase (pol) I, II, and III.

• Pol I is most abundant but has as its primary role to ensure the fidelity of
replication through the repair of damaged and mismatched DNA.

• Replication of the E. coli genome is the job of pol III which is much less
abundant than pol I, however, its activity is nearly 100 times that of pol I.

• There have been 5 distinct eukaryotic DNA polymerases identified, α, β, γ, δ

and ε.

• The ability of DNA polymerases to replicate DNA requires a number of

additional accessory proteins such as:

1. Primase
2. Processivity accessory proteins (dnaA binding protein)
3. Single strand binding proteins (SSB)
4. Helicase
5. DNA ligase
6. Topoisomerases
7. Uracil-DNA N-glycosylase
Steps in DNA Replication
• Steps involved in DNA replication. The figure above describes DNA
replication in an E coli cell, but the general steps are similar in
eukaryotes.

• A specific interaction of a protein (the O protein) to the origin of

replication (ori) results in local unwinding of DNA at an adjacent
A+T-rich region.

• The DNA in this area is maintained in the single-strand conformation

(ssDNA) by single-strand-binding proteins (SSBs). This allows a
variety of proteins, including helicase, primase, and DNA
polymerase, to bind and to initiate DNA synthesis.

• The replication fork proceeds as DNA synthesis occurs continuously

(long arrow) on the leading strand and discontinuously (short
arrows) on the lagging strand.

• The nascent DNA is always synthesized in the 5′ to 3′ direction, as

DNA polymerases can add a nucleotide only to the 3′ end of a DNA
strand.
 DNA Replication is semiconservative

DNA replication is semiconservative. During a

The double-stranded structure of DNA and the template
round of replication, each of the two strands
function of each old strand (dark shading) on which a
of DNA is used as a template for synthesis of
new (light shading) complementary strand is
a new, complementary strand.
synthesized.
 Post-Replicative Modification of
DNA: Methylation
One of the major post-replicative reactions that modifies the DNA is methylation.

The sites of natural methylation (i.e. not chemically induced) of eukaryotic DNA
is always on cytosine residues that are present in CpG dinucleotides.

However, it should be noted that not all CpG dinucleotides are methylated at the
C residue.

The cytidine is methylated at the 5 position of the pyrimidine ring generating 5-

methylcytidine.

Methylation of DNA in prokaryotic cells also occur to prevent degradation of host

DNA from its restriction endonucleases.

This also helps prokaryotic cells to degrade invading viral DNAs. Since the viral
DNAs are not modified by methylation they are degraded by the host restriction
enzymes.
Chemotherapies Targeting Replication
Alkylating Agents

• The class of compounds that have been used the longest as anticancer
drugs are the alkylating agents which includes cyclophosphamide (Cytoxan,
Neosar), ifosphamide, decarbazine, chlorambucil (Leukeran) and
procarbazine (Matulane, Natulan).

• React with and disrupt the structure of DNA (DNA fragmentation).

• Some catalzye the cross-linking of bases in the DNA which prevents the
separation of the two strands during DNA replication.

• Some agents induce mis-pairing of nucleotides resulting in permanent

mutations in the DNA.

• Alkylating agents act upon DNA at all stages of the cell cycle, thus they are
potent anticancer drugs. However, because of their potency, prolonged use
of alkylating agents can lead to secondary cancers, particularly leukemias.
 Topoisomerases Inhibitors
Several classes of anticancer drugs function through interference with
the actions of the topoisomerases.

Two of these classes are the anthracyclines and the camptothecins.

The anthracyclins inhibit the actions of topoisomerase II whose function

is to introduce double-strand breaks in DNA during the process of
replication as a means to relive torsional stresses.

Etoposide (VP-16, Vepesid, Etophos, Eposin) functions through

inhibition of topoisomerase II

Camptothecins inhibit the action of topoisomerase I, an enzyme that

induces single-strand breaks in DNA during replication

Anthracyclines also function by inducing the formation of oxygen free

radicals that cause DNA strand breaks resulting in inhibition of
replication.
 Antimetabolites
• In order for DNA replication to proceed, proliferating cells require a pool of
nucleotides.

• The class of anticancer drugs that has been developed to interfere with
aspects of nucleotide metabolism is known as the antimetabolites.

• There are two major types of antimetabolites used in the treatment of a

broad range of cancers

• componds that inhibit thymidylate synthase and compounds that inhibit

dihydrofolate reductase (DHFR).

• Both of these enzymes are involved in thymidine nucleotide biosynthesis.

• Drugs that inhibit thymidylate synthase include 5-fluorouracil (5-FU, Adrucil,

Efudex) and 5-fluorodeoxyuridine.

• Those that inhibit DHFR are analogs of the vitamin folic acid and include
methotrexate (Trexall, Rheumatrex) and trimethoprim (Proloprim, Trimpex).
RNA transcription
Bacterial RNA transcription is described in
four steps:

(1) Template binding: RNA polymerase

(RNAP) binds to DNA and locates a
promoter (P) melts the two DNA strands
to form a preinitiation complex (PIC).

(2) Chain initiation: RNAP holoenzyme

(core + one of multiple sigma factors)
catalyzes the coupling of the first base
(usually ATP or GTP) to a second
ribonucleoside triphosphate to form a
dinucleotide.

(3) Chain elongation: Successive residues

are added to the 3′-OH terminus of the
nascent RNA molecule.

(4) Chain termination and release: The

completed RNA chain and RNAP are
released from the template. The RNAP
holoenzyme re-forms, finds a promoter,
and the cycle is repeated. The
termination is brought about either by the
action of Rho-protein or hairpin loop
formation of the RNA transcript
• The relationship between the sequences of an RNA transcript and its gene, in which
the coding and template strands are shown with their polarities.

• The RNA transcript with a 5′ to 3′ polarity is complementary to the template strand

with its 3′ to 5′ polarity.

• Note that the sequence in the RNA transcript and its polarity is the same as that in
the coding strand, except that the U of the transcript replaces the T of the gene.
Post transcriptional modification of RNA

• When transcription of bacterial rRNAs and tRNAs is

completed they are immediately ready for use in
translation. No additional processing takes place.

• Translation of bacterial mRNAs can begin even before

transcription is completed due to the lack of the nuclear-
cytoplasmic separation that exists in eukaryotes

• In contrast to bacterial transcripts, eukaryotic RNAs (all 3

classes) undergo significant post-transcriptional
processing.
 Removal of Introns from RNA

• All 3 classes of RNA are

transcribed from genes that
contain introns.

• The sequences encoded by

the intronic DNA must be
removed from the primary
transcript prior to the RNAs
being biologically active.

• The process of intron removal

is called RNA splicing.

• Introns are removed by

nucleophilic attack or
snNRP(small nuclear
ribonucleoprotein)
 5'-Cap of Eukaryotic mRNAs

• The addition of guanosine triphosphate is catalyzed by

Guanyltransferase.

• Methylation is catalyzed by guanine-7-methyl transferase.

• Eukaryotic mRNAs lacking the cap are not translated.

 Polyadenylation of mRNA

• Poly-A tail is not transcribed from DNA, but rather added after transription
by poly-A polymerase

• The tail helps to stabilize the mRNA and facilitate its exit from the nucleus.

• When the poly-A mRNA enters the cytosol the tail is gradually shortened.
 tRNA modification
• Addition of CCA
sequence to 3’-
terminal.

• This is catalyzed by
nucleotidyltransferase
Translation: Synthesis of Proteins

• Proteins are produced by the process of

translation, which occurs on ribosomes
and is directed by mRNA.

• The genetic message encoded in DNA is first

transcribed into mRNA

• The nucleotide sequence in the coding region

of the mRNA is then translated into the amino
acid sequence of the protein.
Genetic Code
 Characteristics of Genetic code
• A. The Code Is Degenerate, But Unambiguous (means that an
amino acid may have more than one codon. However, each codon
specifies only one amino acid, and the genetic code is, thus,
unambiguous.)

• B. The Code Is Almost Universal (All organisms studied so far use

the same genetic code, with some rare exceptions. One exception
occurs in human mitochondrial mRNA, where UGA codes for
tryptophan instead of serving as a stop codon, AUA codes for
methionine instead of isoleucine, and CUA codes for threonine
instead of leucine).

• C. The Code Is Nonoverlapping and without Punctuation

(mRNA does not contain punctuation to separate one codon from
the next and the codons do not overlap).
 Translation of the code
• Translation of the code. The portion of
mRNA that specifies the amino acid
sequence of the protein is read in codons,
which are sets of three nucleotides that
specify individual amino acids.

• The codons on mRNA are read sequentially

in the 5 to 3 direction, starting with the 5-
AUG (or “start” codon) that specifies
methionine and sets the reading frame
and ending with a 3-termination (or “stop”)
codon (UAG, UGA, or UAA).

• The protein is synthesized from its N-

terminus to its C-terminus.

• Each amino acid is carried to the ribosome

by an aminoacyl-tRNA (i.e., a tRNA with
an amino acid covalently attached).

• Base-pairing between the anticodon of

the tRNA and the codon on the mRNA
ensures that each amino acid is inserted
into the growing polypeptide at the
appropriate position.
 Synthesis of the protein
• Initiation involves formation of a complex containing the initial methionyl-
tRNA bound to the AUG “start” codon of the mRNA and to the “P” site of the
ribosome. It requires GTP and proteins known as eukaryotic initiation
factors (eIFs).

• Elongation of the polypeptide involves three steps: (a) binding of an

aminoacyl-tRNA to the “A” site on the ribosome where it base-pairs with
the second codon on the mRNA; (b) formation of a peptide bond between
the first and second amino acids; and (c) translocation, movement of the
mRNA relative to the ribosome, so that the third mRNA codon moves into
the “A” site.

• These three elongation steps are repeated until a termination codon

aligns with the site on the ribosome where the next aminoacyl-tRNA would
normally bind. Release factors bind instead, causing the completed protein
to be released from the ribosome.

• After one ribosome binds and moves along the mRNA, translating the
polypeptide, another ribosome can bind and begin translation. The complex
of a single mRNA with multiple ribosomes is known as a polysome.
 Several Antibiotic and Toxin inhibitors of Translation
Inhibitor Comments

Chloramphenicol inhibits prokaryotic peptidyl transferase

Streptomycin inhibits prokaryotic peptide chain initiation, also induces mRNA misreading

Tetracycline inhibits prokaryotic aminoacyl-tRNA binding to the ribosome small subunit

Neomycin similar in activity to streptomycin

Erythromycin inhibits prokaryotic translocation through the ribosome large subunit

Fusidic acid similar to erythromycin only by preventing EFG from dissociating from the
large subunit
Puromycin resembles an aminoacyl-tRNA, interferes with peptide transfer resulting in
premature termination in both prokaryotes and eukaryotes
Diptheria toxin catalyzes ADP-ribosylation of and inactivation of eEF-2, eEF-2 contains a
modified His residue known as dipthamide, it is this resudue that is the target
of diptheria toxin
Ricin found in castor beans, catalyzes cleavage of the eukaryotic large subunit
rRNA
Cycloheximide inhibits eukaryotic peptidyltransferase
 Protein Synthesis
 DNA passes on genetic
information so that
proteins can be
synthesized in the cell.
 Proteins control many
different chemical
processes in the cell –
such as energy
production, intra &
extracellular
transportation, enzyme
synthesis, hormone
synthesis, and normal
cell maintenance.
 Beside is the summary
of the process from DNA
replication to RNA
synthesis, also known
as transcription, and
translation, also known
as protein synthesis.
 Note that once RNA is
made in the nucleus of
the cell, it leaves and
enters the cytoplasm,
where most cellular
proteins are made.