Introduction to

Molecular Biology I
Cell Biology
TBB 1013
Sem1 - 2009/2010

Objectives:
1 DNA and RNA structure and function
1.1 DNA replication
2 Gene Expression
2.1 Central Dogma
2.2 Transcription
2.3 Translation
2.4 Mutations
3 DNA Technology
3.1 Recombinant DNA Technology
Polymerase Chain Reaction
Genetic engineering applications
4 Gene Regulation
4.1 Control of gene expression in prokaryotes and
eukaryotes
Signalling between cells in eukaryotes

Introduction To Molecular Biology:

Key terms
Genome = complete set of genes.
sequences in the genetic material of an organ.

Chromosomes = Discrete unit of the genome carrying

many genes. composed of 2 Macromolecules:
i.
Protein.
Company
Name
ii. Nucleic acids
\ DNA(d) and RNA(s)

Gene = Segment of DNA that codes for a protein.

It codes for a RNA

Protein.

DNA(Deoxyribonucleic Acid)=The genetic material of

all cellular organisms and most viruses; which is used
to encode genetic information for all life on Earth.

Introduction To Molecular Biology:

Key terms

Viruses contain either RNA or DNA genomes

Can be linear or circular
Can be single or double stranded

Plasmids: replicate separately from chromosome

Company
Great
majorityName
are double stranded
Circular (most)
Generally beneficial for the cell (i.e., antibiotic
resistance)
NOT extracellular, unlike viruses

Plasmid : is a genetic element that is expendable and rarely contains genes

needed for growth under all conditions.

Kinds of Genetic Elements

Company Name

Molecular Biology

Molecular biology; the study of gene structure and

functions at the molecular level to understand the
molecular basis of hereditary, genetic variation, and
the expression patterns of genes.

The Molecular biology field overlaps with other

areas, particularly genetics and biochemistry.

The genome of an organism is the totality of

genetic information and is encoded in the DNA
(or, for some viruses, RNA).

The DNA

Deoxyribonucleic Acid (DNA), the genetic material of all

cellular organisms and most viruses, the gigantic molecule
which is used to encode genetic information for all life on
Earth.

The Chromosome, the storage place for all genetic

information, the number of chromosomes varies from one
species to another.

In normal human cell DNA contained in the nucleus, arranged

in 23 pairs of chromosomes .

The gene, the basic units of inheritance; it is a segment

within a very long strand of DNA with specific instruction for
the production of one specific protein. Genes located on
chromosome on it's place or locus.

Within cells, DNA is organized into structures called

chromosomes.
Eukaryotic organisms:
store their DNA inside the cell nucleus
Prokaryotic organisms :
it is found in the cell's cytoplasm.
In normal human cell DNA contained in the nucleus, arranged in
23 pairs of chromosomes.

What is a DNA?

A nucleic acid that carries the genetic information

in the cell and is capable of self-replication and
synthesis of RNA.

DNA consists of two long chains of nucleotides

twisted into a double helix and joined by hydrogen
bonds.

The sequence of nucleotides determines individual

hereditary characteristics.

Functions of DNA

Storage of genetic information

Transmission of genetic information

General Structure of Nucleic Acid

DNA & RNA are long chain polymers of small compound

called nucleotides.

Each nucleotide is composed of a nitrogen-containing

base; pentose sugar (5 carbon) ribose in RNA or
deoxyribose in DNA) and a phosphate group.

The phosphate joins the sugars in a DNA or RNA chain

through their 5 and 3 hydroxyl group by phosphodiester
bonds.

Phosphates connect 3- carbon of one sugar to 5 of the

adjacent sugar

General Structure of Nucleic Acid

There are four different types of nucleotides found in DNA,

differing only in the nitrogenous base: A is for adenine; G is for
guanine; C is for cytosine and T is for thymine.

These bases are classified based on their chemical structures

into two groups:

adenine and guanine are double ringed structure termed purine

thymine and cytosine are single ring structures termed pyrimidine

The bases pair in a specific way: Adenine A with thymine T

(two hydrogen bonds) and guanine G with cytosine C (three
hydrogen bonds).

Within the structure of DNA, the number of thymine is always

equal to the number of adenine and the number of cytosine is
always equal to guanine.

General Structure of Nucleic Acid

The structure of DNA was described by British Scientists

(Watson and Crick) as long double helix shaped, with its sugar
phosphate backbone on the outside and its nitrogenous bases
on inside;

The two strands of the helix run in opposite direction and are
anti-parallel to each other. The DNA double helix is stabilized by
hydrogen bonds between the bases.

This structure explains how genes engage in replication,

carrying information and acquiring mutation.

The G+C content of a natural DNA can vary from 22-73% and
this can have a strong effect on the physical properties of DNA,
particularly its melting temperature.

DNA: Watson and Cricks model

Watson and Cricks model

Double Helix
Sugar-Phosphate backbone =
sugar is deoxyribose
4 types of nucleotide base =
A, C, G, T
2 complementary strands
where A = T, C = G

"complementary = fitting
together of 2 molecules
with hydrogen bonds

DNA has 4 Bases

The Double Helix of DNA

DNA: Four DNA Bases

Adenine

NH2

N
NH

Guanine

H3C

N
N

NH
NH

N
NH

Thymine

Cytosine

NH2

NH
N

N
NH2

NH

DNA: The Sugar

HO

OH
O

OH

Deoxyribose

DNA: The Nucleotides

O
HO

NH2

HO

OH

OH

OH

HO

OH

NH2

G
O

OH

N
N

NH
N

NH2

O
H3C

NH

O
HO

N
O

OH
OH

OH

Adenine pairs with Thymine

Hydrogen
Bonding

Guanine pairs with Cytosine

Hydrogen
Bonding

DNA
A) Sugar ;
Deoxyribose.
B) Nitrogen base;
(Adenine, Guanine,
Cytosine, Thymine)
C) A phosphate group

Genes and Base Pairs:

A Comparison
Genes
(estimated)

Base Pairs

6,000

12 million

Round Worm

19,000

99 million

Mouse

40,000

3 billion

Fruit Fly

13,600

165 million

Human

40,000

3 billion

Species
Yeast

(Saccharomyces cerevisiae)
(Caenorhabditis elegans)
(Mus musculus)
(Drosophila melanogaster)
(Homo sapiens)

RNA

In contrast to DNA;

RNA is a single stranded

the pyrimidine base
Uracil (U) replaces
thymine
Ribose sugar replaces
deoxyribose.

DNA vs. RNA

DNA
Double-stranded

RNA
Single-stranded

Deoxyribose sugar

Ribose sugar

Thymine (T) base

Uracil (U) base

The RNA

Three major classes of RNA:

messenger (mRNA) : encodes polypeptides

transfer (tRNA) : plays role in protein synthesis

ribosomal (rRNA) : plays role in protein synthesis

The RNA

mRNA molecules represent transcripts of structural

genes that encode all the information necessary for the
synthesis of a single type polypeptide of protein.

mRNA; intermediate carrier of genetic information;

deliver genetic information to the cytoplasm where
protein synthesis take place.

The mRNA also contains regions that are not translated:

in eukaryotes this includes the 5' untranslated region, 3'
untranslated region, 5' capand and poly-A tail.

The RNA

All tRNAs share a common secondary structure represented

by a coverleaf.

They have four- base paired stems defining three stem loops
(the D loop, anticodon loop, and T loop) and the acceptor
stem to which amino acids are added in the charging step.

RNA molecules that carry amino acids to the growing

polypeptide.

The RNA

Ribosomal RNA (rRNA) is the central component of the

ribosome, the function of the rRNA is to provide a mechanism
for decoding mRNA into amino acids and to interact with the
tRNAs during translation by providing peptidyl transferase
activity.

The concentration of purine and pyrimidine bases do not

necessarily equal one another in RNA because RNA is single
stranded.

However, the single strand of RNA is capable of folding back

on itself like a hairpin and acquiring double strand structure.

The RNA

mRNA
tRNA

Chromosomes
and DNA

DNA: General Rules

Two nucleotide chains

Hydrogen Bonds
Nitrogenous Bases are located together
Bases pair specifically (A-T, G-C)
Forms Double Helix
DNA is wrapped around histones to form chromosomes

Genomic DNA Organization

Eukaryotic genes: DNA molecules complexed with other proteins

especially basic proteins called histones to form a substance
known as chromatin.
A human cell contains about 2 meters of DNA. DNA in body
could stretch to the sun and back almost 100 times. So it is
tightly packed.
Eukaryotic chromatin is folded in several ways.

The first order of folding involves structures called nucleosomes,

which have a core of histones, around which the DNA winds ( four
pairs of core histones H2A, H2B, H3 and H4 in a wedge shaped
disc).
The second order of chromatin folding is the 30 nm fiber.
The third order of chromatin condensation appears to involve
formation of a radial loop structure in eukaryotic chromosomes.

Genomic DNA Organization

Chromosomes

Chromosomes are comprised of:

- 2 Chromatids (2C)
- Centromere

Ecology Physiology Molecular

Biology

DNA Replication

DNA Replication:

all gene duplication.

the transfer the genetic information from a parent to a

daughter cell.

the DNA base sequence are precisely copied.

DNA Replication

Semiconservative replication would produce two copies that

each contained one of the original strands and one entirely
new strand.

Conservative replication would leave the two original template

DNA strands together in a double helix and would produce a
copy composed of two new strands containing all of the new
DNA base pairs.

Dispersive replication would produce two copies of the DNA,

both containing distinct regions of DNA composed of either
both original strands or both new strands.

Possible Modes of DNA

Replication

Replication: 5 to 3 Direction ONLY

Continuous replication
on one strand

Discontinuous replication
on one strand

Semiconservative DNA Replication

DNA Replication is
Catalyzed by Enzymes

* Okazaki fragments are on lagging

strand.

Templates and Enzymes

DNA polymerases

catalyze the addition of

nucleotides.
DNA polymerases require a primer

Primer made from RNA by an enzyme Primase

Events at the DNA Replication Fork

Proofreading
DNA replication is extremely accurate

Proofreading helps to ensure high fidelity

Mutation rates in cells are 10 -810-11 errors

per base inserted.

Polymerase can detect mismatch

incorrect hydrogen bonding

through

DNA Polymerase Adds Complementary Bases

DNA Polymerase Sees Errors

DNA Polymerase Repairs Errors

Gene Expression

Gene expression process by which a gene product

(an RNA or polypeptide) is made.

In transcription steps, RNA polymerase make a copy

of information in the gene (complementary RNA)

(mRNA) complementary to one strands of DNA

In translation step, ribosomes read a messenger

RNA and make protein according to its instruction.
Thus any change in gene sequence may lead to
change in the protein product.

Gene Expression

Transcription is the process by which mRNA is made

from DNA template to copy the information in the
gene and pass it to mRNA (in the cytoplasm).
- Transcription = mRNA from DNA (in the nucleus)

Translation is the process by which protein is made

from an mRNA template.
- Translation = Protein from mRNA (in the cytoplasm)

Types of control in eukaryotes:

Transcriptional,
prevent transcription, prevent mRNA from being synthesized.
Posttranscriptional,
control mRNA after it has been produced.
Translational,
prevent translation; involve protein factors needed for
translation.
Posttranslational,
after the protein has been produced.

Overview of Transcription

Transcription (DNA to RNA) is carried out by

RNA polymerase.

RNA polymerase uses DNA as template.

RNA precursors are ATP, GTP, CTP, and UTP.

Chain growth is 5 to 3 just like DNA replication.

Overview of Transcription

Only one of the two strands of DNA are

transcribed by RNA polymerase for any gene.

Genes are present on both strands of DNA, but

at different locations

RNA polymerase has five different subunits

RNA polymerase recognizes DNA sites called

promoters

Overview of Transcription

Promoters: site of initiation of transcription

Promoters are recognized by sigma factor of RNA

polymerase.

Transcription stops at specific sites called

transcription terminators .

Unlike DNA replication, transcription involves smaller

units of DNA

Often as small as a single gene.

Allows cell to transcribe different genes at different rates.

Transcription: Steps in RNA Synthesis

Termination of Transcription

Termination of RNA synthesis is governed by a

specific DNA sequence.

Unit of transcription: unit of chromosome bounded

by sites where transcription of DNA to RNA is
initiated and terminated

mRNA have short half-lives (a few minutes)

The Genetic Code

The purine and pyrmidine bases of the DNA molecule are

the letters or alphabet of the genetic code.
All information contained in DNA represented by four letters:
A,T,C,G.
Three nucleotides of DNA (1st, 2nd and 3rd) form triplet
codons.
there are 64 possible codons,
most amino acids have more than one possible codon.
Out of the 64 possible 3-base codons, 61 specify amino
acids; the other three are stop signals (UAG, UAA, or UGA).
The sequence of codons in the mRNA defines the primary
structure of the final protein.

The Genetic Code

Translation: the synthesis of proteins from RNA.

Genetic code: a triplet of nucleic acid bases (codon)

encodes a single amino acid.

Specific codons for starting and stopping translation.

Degenerate code: multiple codons encode a single

amino acid.

Anti-codon on tRNA recognizes codon.

Transfer RNA

Transfer RNA: at least one

tRNA per amino acid.

Specific for both a codon

and its particular amino
acid.

tRNA is cloverleaf in shape

Anti-codon: three bases

of tRNA that recognize
three complementary
bases on mRNA.

The Genetic Code

Codons

There are twenty amino acids, so a three-base

code is the minimum required.

The Genetic Code

Series of codons in part of a mRNA molecule. Each codon
consists of three nucleotides, representing a single amino acid.

The Genetic Code as Expressed by

Triplet Bases of mRNA

Synthesis of the Informational

Macromolecules

The
Central Dogma

Macromolecules and Genetic

Information

Central dogma of molecular biology

DNA to RNA to protein

Eukaryotes: each gene is transcribed individually

Prokaryotes: multiple genes may be transcribed

together

Transcription in Prokaryotes

Central Dogma of Molecular Biology

Ribosomes

Ribosomes:
Factory for protein synthesis; thay are composed of
ribosomal RNA and ribosomal proteins (known as a
Ribonucleoprotein or RNP).

They translate messenger RNA (mRNA) to build

polypeptide chains using amino acids delivered by
transfer RNA (tRNA).

Thousands of ribosomes per cell

Combination of rRNA and protein

Ribosomes

Eukaryotic ribosomes are larger than prokaryotic

ribosomes. They consist of two subunits:

60S subunit holds (three rRNAs 5S, 5.8S, 28S and about
40 proteins).
40S subunit contains (an 18S rRNA and about 30
proteins), which come together to form an 80S particle
compared with prokaryotic 70S ribosome.

Most mRNA are translated by more than one

ribosome at a time; the result, a structure in which
many ribosomes translate an mRNA in tandem , is
called a polysomes.

Ribosomes

Polysomes

Four levels of protein structure

Translation: The Process of

Protein Synthesis
Translation is broken down into three main steps:
Translation is broken down into three main steps:

1) Initiation: two ribosomal subunits assemble with mRNA

Begins at an AUG start codon.
2) Elongation: amino acids are brought to the ribosome and are
added to the growing polypeptide.
Occurs in the A and P sites of ribosome.
Translocation: movement of the tRNA holding the
polypeptide from the A to the P site.
Polysomes: a complex formed by ribosomes simultaneously
translating mRNA

Translation: The Process of

Protein Synthesis
3) Termination: occurs when ribosome reaches a stop codon.
Release factors (RF): recognize stop codon and cleave
polypeptide from tRNA.
Ribosome subunits then dissociate.
Subunits free to form new initiation complex and repeat
process.

Translation: Initiation

Translation: Elongation

Translation: Termination

Mutation

Mutation include both gross alteration of chromosome and

more subtle alteration to specific gene sequence.

Gross chromosomal aberrations include:

large deletions
addition
translocation (reciprocal and nonreciprocal).

Mutation in a gene's DNA sequence can alter the amino acid

sequence of the protein encoded by the gene.

Point mutations are the result of the substitution of a single

base.
Frame-shift mutations occur when the reading frame of the
gene is shifted by addition or deletion of one or more bases.

Mutation

Mutations can have harmful, beneficial, neutral, or uncertain

effects on health.

Mutations may be inherited as autosomal dominant, autosomal

recessive, X-linked traits.

Mutations that cause serious disability early in life are usually

rare because of their adverse effect on life expectancy and
reproduction.