STRUCTURE OF NUCLEAR GENETIC MATERIAL –

DNA, RNA AND GENE

Course: Gp- 603 Genomics in Crop Improvement

Presented by:

M.BHARATH KUMAR

Ph.D 1st YEAR

DEPARTMENT OF GENETICS AND PLANT BREEDING

PROFESSOR JAYASHANKAR TELANGANA

STATE AGRICULTURAL UNIVERSITY(PJTSAU)
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 Contents
 Introduction
 Evidences for DNA as genetic material
 Evidences for RNA as genetic material
 DNA structure
 Components of DNA
 Forms of DNA
 RNA structure
 Types of RNA
 Concept of Gene
 Prokaryotic gene structure
 Eukaryotic gene structure
 Organization of genes

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 Introduction
It is well known fact that transmission of traits takes place from one generation to

other. The offsprings are similar to both the parents in some traits
Gregor Johann Mendel (1866) gave the idea that transmission of traits over
generations take place through Factor or Determiner or Gene which carries
information for expression of trait or phenotype.
Genes are present on the chromosomes which are distributed equally into the two

daughter cells during cell division. The biochemical studies reveal that chromosomes
are composed of proteins (60% ) and DNA (40% )

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 The resemblance of offspring to their parents depends on the precise
transmission of principle component from one generation to the next,
that component is- The Genetic Material

The genetic material must be capable of

• carrying information – Cracking the genetic code
 Must self replicate – DNA replication
 Must govern the expression of the phenotype – Gene function

 Must allow for information to change – Mutation

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 Is the Genetic Material Protein or DNA ?
 Untill 1940 Proteins were considered as genetic material as Proteins are polymer of

20 protein amino acids and present in larger quantity,

encode more and variety of information.
 DNA is polymer of only 4 different deoxyribonucleotides (ATP, CTP, GTP& TTP)

and is present in smaller quantity

 Most geneticists focused on “transmission genetics” and passively accepted proteins

as the genetic material.

 But On the basis of certain experiments conducted from time to time, it was

ultimately demonstrated that DNA carries genetic information and not the proteins
 There are some direct evidences and some indirect evidences which prove DNA as

Genetic Material

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 Direct evidences came from :
 Frederick Griffith’s (1928) experiment on Bacterial Transformation

 Oswald Avery, Colin Macleod and Maclyn McCarty’s (1944 )

experiment on Transformation

 Alfred D. Hershey and Martha Chase (1952 ) experiment on T- Even

(2,4 ) Bacteriophage

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Griffith experiment: Transformation of bacteria
In 1928, Griffith studied the bacterium Streptococcus pneumoniae
Which comes in two strains
S - Smooth (strain IIIS)
 Secretes a polysaccharide capsule (evades immune system)
 Produce smooth colonies on solid media
 Helps make the microorganism virulent, or able to cause disease.
R - Rough (strain IIR)
 Unable to secrete a capsule
 Produce colonies with a rough appearance.
 Does not cause disease as strain lacks the polysaccharide capsule

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 live rough
dead smooth
dead smooth

live smooth

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 When live, avirulent II R and heat-killed virulent III S are mixed, the
heat-killed virulent III S passed on disease-causing information to
the avirulent II R ma ki n g it virulent.
 Transformation is a change in genotype caused when cells take up
foreign material, as bacteria had been Transformed from the rough to
the smooth version
 Principle Component of type III S cells which induced the conversion
of type II R cells into type III S was named transforming principle .

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 Avery, MacLeod and McCarty Experiment
 In1944 repeated Griffith’s experiment of transformation using
purified cell extracts.
 Cell free extract of SIII strain Bacterium was subjected to DNase,
RNase and Protease.
 Each treated extract was mixed with RII and mixture injected to
mouse to see transformation.
 In case of Protease and Rnase transformation was recorded
 In case of DNase no transformation was recorded proving the
transforming material is DNA.

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Avery, MacLeod and McCarty Experiment

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 With the help of experiment they showed that DNA isolated from
SIII strain Bacteria could confer the pathogenic properties to R II
strain Bacteria.
 Avery et al (1944) revealed the chemical nature of the transforming
substance to be DNA
 Two conclusion were derived
1.Active factor is DNA which can cause transformation
2.SIII strain contains the Active factor

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 Hershey and Chase’s Experiments

 1952, American biologists Alfred Hershey and Martha Chase

performed an experiment that settled the controversy
 Proved that DNA carries the genetic material by
experimented with T2 bacteriophages, viruses that attack
bacteria.
 Bacteriophage virus attaches to the surface of bacteria,
injects its genetic material into the bacteria, the viral genes
cause many more viruses to be made inside the bacteria
until the bacteria burst and hundreds of new viruses are
released. 14
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 The results of experiment clearly indicate that only DNA labelled
with 32P entered the bacterial cell, and not the Protein coat (35S) as
it is left outside.
 The DNA entering the host cell carries all the genetic information
for synthesis of new phage particle.
 This certainly proves that DNA is the genetic material in
Bacteriophage and not proteins.

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INDIRECT EVIDENCES FOR DNA AS GENETIC MATERIAL

 Presence of DNA regularly in nuclei of all types of cells.

 Equal amount of DNA is present in all cells of an organism
 Nuclear Division occurs only after DNA duplication during S phase of
Interphase.
 Variation in Diploid amount of DNA amongst different species.
 DNA has same physical and chemical properties in all organisms yet allow
to produce great diversity of organisms
 Indefinite number of combinations are possible with four bases A T G C
 Of all macromolecules DNA is metabolically most stable
 In prokaryotes DNA is not linked with proteins still characters are inherited

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EVIDENCE FOR RNA AS GENETIC MATERIAL

A Gierer and G Schramm ( 1956 ) when inoculated tobacco plants with Purified

RNA isolated from TMV - Leisons appeared on leaves of healthy plant

H Fraenkel Conrat and B Singer (1957) separated RNA from protein of TMV in

first step .
In second step reconstituted virus with protein from mutant strain of TMV

Inoculated Hybrid TMV into healthy tobacco plant Tobacco Mosaic disease

appeared
TMV progeny isolated from diseased plant showed parental RNA only
but not parental proteins
This provided evidence that RNA is the Genetic Material in TMV not but proteins.

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H Fraenkel Conrat and B Singer Experimment in TMV

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EXAMPLES OF PLANT RNA VIRUSES
 Tobbaco Mosaic Virus (TMV ) was crystallized by Stanley (1935)
for the first time
 TMV causes Tobacco mosaic disease
 TMV can also cause disease in Tomato, Pepper, Petunia,
Snapdragon, Delphinium, and Marigold.
 Papaya Ring Spot virus (PRV)
 Potato Leaf Roll Virus (PLRV)
 Holmesrib-grass Virus (HRV)
 Plantago Alfa-alfa Mosaic Virus (AMV)

TM
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Discovery of DNA
 In 1962, James Watson and Francis
Crick were awarded noble prize for
the discovery of double helical
structure of DNA.
 The scientific framework for their
breakthrough was provided primarily
by:
 Rosalind Franklin (X-ray diffraction)
 Erwin Chargaff (chemical
composition)

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Rosalind Franklin and Maurice Wilkins
The data of Wilkin, Franklin and others on X- ray crystallography of
purified DNA revealed following features :
 Multistranded molecule with X –shaped pattern
 Diameter – 22A
 Groups spaced at 3.4A along its length
 A repeating unit occuring at every 34A

The diffraction pattern is interpreted

using mathematical theory which
ultimately provides information
about the structure of the
molecule
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 Watson and Crick Model
 Made of two polynucleotide strands.

 There are two asymmetrical grooves Major groove

and Minor groove where proteins can bind.

 Two strands are oriented anti-parallel i.e., 5'- end of

one strand is located with 3'- end of other strand.

 Anti parallel orientation essential for hydrogen bond

formation between basepairs.

 Composed of many deoxyribonucleotides.

 The two strands of DNA are coiled together in a right

handed helix forming the DNA double helix

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 The DNA double helix is stabilized i.e., the two strands
are held together mainly by hydrophobic forces.
 Diameter of helix -20A
 Pitch of helix- 34A
 Base pairs in one pitch -10bp
 Distance between two base pairs -3.4A
 The base pairs in a DNA are stacked between sugar and
phosphate back bones
 During replication two strands of DNA uncoil. The
unpaired bases in single stranded regions of two strands
pair with complementary bases.
 Sometimes errors in base pairing may occur during
replication resulting in mutations.

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 Hydrogen bonding

 The formation of hydrogen bonds between A and T and between G

and C is called complementary base pairing and these bases are called
complementary bases.

The hyrogen bonds are

crucial for precise
replication and
transcription processes.

A=T G Ξ C

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Chargaff Rules
Chemical analysis of DNA by chargaff and others during
1940s demonstrated following:
 Number of pyrimidine bases = Purine bases
C+T = A+G
• Percent of adenine = percent of thymine (A=T)
• Percent of cytosine = percent of guanine (C=G)
• A+C content of DNA = G+T

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Components of DNA
DNA has three main components
1. deoxyribose (a pentose sugar)
2. base (there are four different ones)
3. phosphate

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Deoxy ribose and Ribose sugars
The oxygen present at the second carbon of ribose is missing in
deoxyribose hence its name 2’- deoxyribose in case of DNA

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 Organic Bases
They are divided into two groups

Purines (made of a 6 member ring,

 fused to a 5 member ring)

Adenine

Guanine

Pyrimidines (made of one 6 member ring)

Thymine and uracil in RNA

Cytosine

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 Nucleoside :

The H – attached to the –N at position 1 of pyrimidines or

H – attached to the -N at position 9 of purines interacts with the –OH at 1’C of pentoses
and forms covalent bond between pentose and organic base resulting in nuceloside.

 Organic base + deoxyribose = Deoxyriboside

 E.g. Deoxyadenosine, Deoxyguanosine

 Nucleotide :

When a phosphate group is attached to either 3’C or the 5’C of the pentose molecule of a

nucleoside then nucleotide is formed.

Organic base + deoxyribose+ Phosphate = Deoxyribotide

Deoxyadenosine + Phosphate = Deoxyadenylic acid

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 Nucleotide Structure
• Nucleotides are covalently linked together by phosphodiester bonds
–A phosphate connects the 5’ carbon of one nucleotide to the 3’ carbon of another
• Therefore the strand has directionality – 5’ to 3’
• The phosphates and sugar molecules form the backbone of the nucleic acid strand

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a

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Alternative Forms of DNA
 The DNA double helix can form different types of secondary structure
 The predominant form found in living cells is B-DNA
 However, under certain in vitro conditions, A-DNA and Z-DNA double helices can form
A-DNA
 Right-handed helix
 11 bp per turn
 Occurs under conditions of low humidity
 Little evidence to suggest that it is biologically important
 DNA.RNA heteroduplexes and RNA double helices occur in vivo in A-form
 Heteroduplex is a double helix in which two strands differ from each other.
Z-DNA
 Left-handed helix
 12 bp per turn
 The sugar –phosphate backbone follows a zig-zag path, which gives the name Z-DNA
 Evidence from yeast suggests that it may play a role in transcription and recombination
C-DNA
• Right-handed helix
• 9.3 basepairs per turn
• It is more tightly wound than the B-form DNA

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 A B Z
Residues/Turn 10 12
11
Clock -wiseTurn Anti Clock-wise Turn

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 RNA structure
 RNA double helices typically right-handed (11-12 base pairs per turn)

 The primary structure of an RNA strand is much like that of a DNA

strand
 RNA strands are typically several hundred to several thousand
nucleotides in length
 In RNA synthesis, only one of the two strands of DNA is used as a
template
 Although usually single-stranded, RNA molecules can form short
double-stranded regions
 This secondary structure is due to complementary base- pairing A to
U and C to G.

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 Types of RNA
Messenger RNA (mRNA) :
 It constitutes about 5-10% of the total cellular RNA.
 a single prokaryotic mRNA molecule codes for more than one polypeptide ; such a
mRNA is known as polycistronic mRNA.
 All eukaryotic mRNAs are monocistronic i.e. coding for a single polypeptide
specified by a single cistron.
 Carry a copy of instructions from nucles to other parts of the cell

Ribosomal RNA (rRNA) :

 It constitutes about 80% of the total cellular RNA.
 The function of rRNA is binding of mRNA and tRNA to ribosomes.

Transfer RNA (tRNA) :

 It is also known as soluble RNA(sRNA). It constitutes about 10-15% of total RNA
of the cell.
 Transfers amino acids(proteins) to the ribosomes to be assembled

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S.No. RNA DNA
1) Single stranded mainly except when Double stranded (Except for certain viral
self complementary sequences are DNA s which are single stranded)
there it forms a double stranded
structure (Hair pin structure)
2) Ribose is the main sugar The sugar moiety is deoxy ribose
3) Pyrimidine components differ. Thymine is always there but uracil is
Thymine is never found(Except never found
tRNA)
4) Being single stranded structure- It It does follow Chargaff's rule. The total
does not follow Chargaff’s rule purine = pyrimidine content.
5) RNA can be easily destroyed by DNA resists alkali action due to the absence
alkalies of OH group at 2’ position
6) RNA is a relatively a labile DNA is a stable molecule. The spontaneous
molecule, undergoes easy and degradation is very 2 slow.
spontaneous degradation

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S.No. RNA DNA
7) Mainly cytoplasmic, but also Mainly found in nucleus, extra nuclear
present in nucleus DNA is found in mitochondria, and
plasmids etc
8) The base content varies from Millions of base pairs are there
100- 5000. The size is variable. depending upon the organism

9) There are various types of RNA – DNA is always of one type and performs
mRNA, r RNA, t RNA. These RNAs the function of storage and transfer of
perform different and specific genetic information.
functions.
10) No variable physiological forms of There are variable forms of DNA (A
RNA are found. The different types to E and Z)
of RNA do not change their forms
11) RNA is synthesized from DNA, it DNA can form DNA by replication, it
can not form DNA(except by the can also form RNA by transcription.
action of reverse transcriptase).

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 Genes
 The genes are the Functional units of Heredity.

 A gene is a specific sequence of DNA containing genetic information

required to make a specific protein

 Modern definition Gene is the Unit of Genetic Information, i.e., the

sequence of DNA that specifies one polypeptide.
 Includes coding as well as non-coding regulatory sequences

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 Gregor Mendel assumed that each trait is determined by a pair of
inherited ‘factors’ which are now called gene.
 Johannsen coined the term ‘GENE’ in 1909
 Each gene is a segment of DNA that give rise to a protein product or
RNA.
 A gene may exist in alternative forms called alleles.
 Prokaryotic gene is uninterrupted.
 In Eukaryotic gene the coding sequences (exon) are separated by non-
coding sequences called introns.
 In complex eukaryotes, introns account for more than 10 times as much
DNA as exons.

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 Gene Theory

 T.H Morgan proposed the gene theory which state that:

i) Chromosomes are bearers of hereditary units and each chromosome

carries hundreds or thousands of genes.

ii) The genes are arranged on the chromosomes in the linear order on the
special regions or locus.

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 Modern concept of gene
S. Benzer (1957) coined different terms for different nature of gene and
genetic material in relation to the chromosome on the basis of genetic
phenomena to which they involve.

• Cistron – Genes as a unit of transmission

 The part of DNA specifying a single polypeptide chain is termed as cistron.
 It transmits characters from one generation to other as unit of transmission.

• Recon - Genes as a unit of Recombination

 The smallest segment of DNA capable of being separated and exchange with
other chromosome is called recon.
 A recon consists of not more than two pairs of nucleotides.

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Muton – Gene as a unit of mutation


Muton is the smallest unit of genetic material which when changed or mutated

produce a phenotypic trait.

muton is delimited to a single nucleotide.

Gene types

Types of gene based on activity

1.House keeping genes ( genes which are always active )

2.Specific genes. (Those genes which are getting active only during some special

condition)

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Types of genes based on behavior.
1.Basic genes: These are the fundamental genes that bring about expression of
particular character.
2. Lethal genes: These bring about the death their possessor.
3.Multiple gene: When two or more pairs of independent genes act together to produce
a single phenotypic trait.
4.Cumulative gene: Some genes have additive effects on the action of other genes.
These are called cumulative genes.
5.Pleiotropic genes: The genes which produce changes in more than one character
is called pleiotropic gene. E.g : Sickle cell anemia causes multiple symptoms,
only one of which is the actual sickle cell condition
6.Modifying gene: The gene which cannot produce a character by itself but
interacts with other to produce a modified effect is called modifier gene.
7. Inhibitory gene: The gene which suppresses or inhibits the expression of another
gene is called inhibitory gene

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Prokaryotic gene structure

5’ 3’

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Prokaryotic gene structure

Prokaryotic Gene is composed of three regions:

1.Promoter region
2.RNA coding sequence
3.Terminator region

Prokaryotic gene lacks introns.

The region 5’ of the promoter sequence is called upstream sequence and
the region 3’ of the terminator sequence is called downstream sequence.

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Promoter region:
 This is situated on upstream of the sequence that codes for RNA.
 This is the site where RNA polymerase interact before RNA synthesis
(Transcription).
 Promoter region provides the location and direction to initiate
transcription.

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 At -10 there is a sequence TATAAT or PRIBNOW BOX. Usually AT rich region
which requires minimum energy for strand separation or melting for initiation .
 At -35 another consensus sequence TTGACA, helps in promoter recognition.
 These two are the most important promoter elements recognized by transcription
factors.

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Type of operon and positin of promoter

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Common Bacterial Promoters used in Research

S.No. Promoter Expression Description

1 T7 Constitutive but requires T7 RNA polymerase Promoter from T7 bacteriophage
2 Sp6 Constitutive but requires Sp6 RNA polymerase Promoter from Sp6 bacteriophage
3 lac Constitutive in the absense of lac repressor (lacI or Promoter from Lac operon
lacIq). Can be induced by IPTG or lactose
4 araBad Inducible by arabinose Promoter of the arabinose
metabolic operon
5 trp Repressible by tryptophan Promoter from E. coli tryptophan
operon
6 Ptac Regulated like the lac promoter Hybrid promoter of lac and trp

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lac Operon

trp Operon

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RNA coding sequence:

 The DNA sequence that will become copied into an RNA molecule (RNA transcript).

Starts with an initiator codon (AUG, GUG, CUG). and ends with termination codon

(Amber-UAG, ochre-UAA, and opal-UGA)

No introns (uninterrupted).

Collinear to its mRNA.

Any nucleotide present on the left is denoted by (-)symbol and the region is called

upstream element. E.g. -10,-20,-35 etc. Any sequence to the right of the start is
downstream elements and numbered as +10,+35 etc.).

Terminator region:

 The region that signal the RNA polymerase to stop transcription from DNA template.

Transcription termination occur through Rho dependent or Rho independent manner.

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 Eukaryotic gene are complex structures compared that prokaryotic gene.
 They are composed of following regions
 Exons
 Introns
 Promoter sequences
 Terminator sequences
 Enhancers and silencers(upstream or downstream)
 Signals (Upstream sequence signal for addition of cap. Downstream
sequences signal for addition of poly A tail.)

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Exons
• Coding sequence, transcribed and translated.
• Coding for amino acids in the polypeptide chain.
• Vary in number ,sequence and length. A gene starts and ends with exons.(5’
to 3’).
• Some exon includes untranslated(UTR)region.
Introns
• Coding sequences are separated by noncoding sequences called introns.
• They are removed when the primary transcript is processed to give the
mature RNA
• All introns share the base sequence GT in the 5’end and AG in the 3’end.
• Introns were 1st discovered in 1977 independently by Phillip Sharp and
Richard Roberts.

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Significance of Introns
• Introns don't specify the synthesis of proteins but have other
important cellular activities.
• Many introns encodes RNA’s that are major regulators of gene
expression.
• Contain regulatory sequences that control trancription and mRNA
processing.
• Introns allow exons to be joined in different combinations(alternative
splicing), resulting in the synthesis of different proteins from the
same gene.
• Important role in evolution by facilitating recombination between exons of
different genes(exon shuffling).

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Promoters
A promoter is a regulatory region of DNA located upstream controlling gene
expression. Responsible for binding of RNA polymerase II which is used for
transcription.

E.g. TA29 promoter has tapetum specific expression

Glutelin (Gt3) promoter in rice has endosperm specific expression.

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Common Eukaryotic Promoters Used in Research

S.No. Promoter Expression Description

1 CMV Constitutive Strong mammalian promoter from human cytomegalovirus

2 EF1a Constituitve Strong mammalian promoter from human elongation factor 1 alpha

3 CAG Constitutive Strong hybrid mammalian promoter

4 PGK Constitutive Mammalian promoter from phospholycerate kinase gene

5 TRE Inducible Tetracycline response element promoter

6 U6 Constitutive Human U6 nuclear promoter for small RNA expression

7 UAS Specific Drosophila promoter containing Gal4 binding sites

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Terminator
Recognized by RNA polymerase as a signal to stop transcription
Enhancer
Enhances the transcription of a gene upto few thousand bp
upstream.
Silencers
Reduce or shut down the expression of a near by gene.

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