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.
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• 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 Crick’s model
 Watson and Crick’s 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
NH2 O
Adenine Thymine
N H3C
N NH

NH N NH O

Guanine O NH2
Cytosine
N
NH N

NH N NH2
NH O
DNA: The Sugar

HO OH
O

OH

Deoxyribose
 DNA: The Nucleotides
NH2 NH2

O A N
N
O C N

HO P O N N N O
HO P O
O O
OH OH

OH OH

T
O O

O G N

N
NH
O
H3C
NH

HO P O N NH2
O HO P O N O
OH 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
Species Base Pairs
(estimated)
Yeast 6,000 12 million
(Saccharomyces cerevisiae)
Round Worm 19,000 99 million
(Caenorhabditis elegans)
Mouse 40,000 3 billion
(Mus musculus)
Fruit Fly 13,600 165 million
(Drosophila melanogaster)
Human 40,000 3 billion
(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 RNA
 Double-stranded  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 -8–10-11 errors

per base inserted.

 Polymerase can detect mismatch through
incorrect hydrogen bonding
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.