Chapter 6 biology 20 12-15

II PU Biology 6.Molecular Basis of Inheritance 2015-16 Maruthi Tutorials KS Town Bangalore-60 Page 1

Chapter 6 Molecular basis of inheritance

DNA and RNA are the two types of nucleic acids found in the living systems. DNA is the

genetic material in most of the organisms. In virus RNA acts as genetic material. RNA also

act as messenger, adapter, structural and catalytic molecule,

6.1 The DNA- DNA is a long polymer of deoxyribonucleotides. The length of DNA is

usually defined as number of nucleotides (or base pairs) present on it. For example a

bacteriophage ϕ x 174 has 5386 nucleotides, bacteriophage lamda has 48502 base pairs (bp),

Escherichia coli has 4.6 x106 bp, and haploid content of human D NA is 3.3 x10

9 bp.

6.11 Structure of polynucleotide chain-

Nucleic acids- It is a macromolecule with acid property and it was isolated from the nucleus

of cells and hence it is named as nucleic acid. It is made up of C, H, O, N and P. Nucleic

acids are found in all organisms such as plants, animals, bacteria and viruses. They are found

in the nucleus as well as in the cytoplasma. It is a long chain polymer. It is composed of

monomeric units, called nucleotides. Each nucleotide consists of a nucleoside and a

phosphate group. Each nucleoside consists of a pentose sugar and a nitrogenous base. The

sugar is ribose in case of RNA and deoxyribose in case of DNA.

The nitrogenous bases are of two types, namely purine and pyrimidine. There are two main

purine bases, adenine and guanine. There are three pyrimidine bases. They are cytosine,

thymine and uracil. Cytosine and thymine are found in DNA. Cytosine and uracil are found

in RNA.

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Nucleosides: A base combined with a sugar molecule is called a nucleoside. In DNA four

nucleosides are present. They are adenosine, guanosine, cytidine and thymidine. In RNA

deoxyribose is replaced by ribose and the base thymine is replaced by uracil.

Nucleotides: A nucleotide is derived from a nucleoside by the addition of a molecule of

phosphoric acid. The DNA contains four different types of nucleotides. They are adenylic

acid, guanylic acid, cytidylic acid and thymidylic acid. The RNA contains uridylic acid

instead of thymidylic acid.

Polynucleotide: a number of nucleotide units linked with one another to form a

polynucleotide chain or nucleic acid.

Nucleic acids are classified into DNA and RNA. The RNA further classified into mRNA,

tRNA and rRNA.and rRNA.

Deoxyribonucleic acid: (DNA): It is the molecule of heredity. It functions as the genes.DNA

is present in all cells except plant virus. In eukaryotic cells, DNA is present in the

chromosomes of nucleus. In addition, the mitochondria and plastids contain DNA. In

eukaryotic nucleus, the DNA is in the form of a double helix. In bacteria, mitochondria and

plastids the DNA molecules are circular. In viruses and bacteriophages they are coiled. DNA

is made up of 3 chemical components, namely 1.Sugar, 2.Phospharic acid and 3. Nitrogenous

bases.

1.Sugar:


The sugar present in the DNA is called deoxyribose. It is a pentose sugar which contains five

carbon atoms (C5H10O4). It contains one O atom less than the ribose sugar. At carbon No.2 of

deoxyribose, is present a H-C-H group. But in ribose sugar the second carbon atom contains

H-C-OH group.

2.Phosphoric acid: (H3PO4)-

Deoxyribose sugar molecule linked with one phosphate group at 5th

position and another

phosphate group is linked with 3rd

position. This forms phosphate diester bond. This bond

links carbon 5’ in one nucleoside with carbon 3’ in the next nucleoside.

3.Nirogenous bases: These are N2 containing organic compounds. They are of two types,

namely purines and pyrimidines.


Purines: Purines are two – ringed N2 compounds. They are of two types, namely adenine and

guanine.

Pyrimidines: These are single ringed N2 compounds. They are two types, namely thymine

and cytosine. Thymine and cytosine are the pyrimidine bases of DNA.

Structure of DNA (Watson and Crick model):

In 1933 Watson and Crik designed the structure of DNA. It is called the Watson and Crick

model of DNA. They were awarded with Nobel Prize in 1962 for this work. According to

them DNA is in the form of double helix. DNA is the deoxy ribonucleic acid. It is a nucleic

acid. It is made up of two chains. Each chain is the polynucleotide chain. Each polynucleotide

is made up of many small units called nucleotides. Each nucleotide is made up of three

chemical components, namely a phosphoric acid, a deoxyribose sugar and a nitrogen base.

The nitrogen bases are adenine, guanine, thymine and cytosine.


The nucleotides of DNA are named according to the type of nitrogen bases present. As there

are four types of nitrogen bases, DNA contain four types of nucleotides, namely

1.AMP- Adenosine monophosphate (adenylic acid)

2.GMP- Guanosine monophosphate (guanylic acid )

3. TMP- Thymidine monophosphate (thymodylic acid)

4. CMP- Cytidine monophosphate (cytidylic acid)

In each nucleotide, the deoxyribose sugar is attached to a phosphoric acid at one side and a

nitrogen base at the other side. The phosphoric acid is linked to the sugar. The nitrogen base

molecule is joined to the sugar by a glycosidic bond. This bond is formed between sugar and

nitrogen base.


Many nucleotides are linked together to form a polynucleotide chain. Two nucleotides are

joined by a phosphodiesterase bond. It is formed between sugar of one nucleotide and

phosphate component of another nucleotide.

The linking between purines and pyrimidines is brought about by hydrogen bonds. There are

two hydrogen bonds between A and T (A=T), and 3 hydrogen bonds between G and C (G=

C). The amount of adenine is equivalent to the amount of thymine and the amount of guanine

is equivalent to the amount of cytosine. The two chains of a DNA are complimentary to each

other. At one end of the polynucleotide chain, the 3rd

carbon atom of the sugar is free and it is

not linked to any nucleotide. This end is called 3 prime (3’) end. At the other end of the 5th

carbon of the sugar is free and this end is called 5prime (5’) end.

DNA strand is antiparallel as they run in opposite direction. The DNA molecule is in the

form of a double helix. The two polynucleotide chains are coiled around each other to form a

double helix. The width (diameter) of DNA is 20A0. The DNA has two external grooves,

namely major groove and minor groove. The major groove is wider and deep. The minor

groove is narrow. The distance between two nucleotides is 3.4 A0.

Properties of DNA;

1. Size of the DNA molecule-The size of the DNA molecule varies from organism to

organism. It depends upon the size of the chromosome and the number of chromosomes


found in each living cell. The size basically depends upon the number of nucleotides present

in each DNA molecule. The size of DNA molecule ranges from 0.7 µm to 40,000mm (4cms).

2. Fragility of DNA molecule: The DNA molecule is highly fragile. Smaller DNA can be

isolated without any damage, but large sized DNA (above 2X 108 deltons) undergoes

breakage during their extraction.

3. Denaturation: Denaturation refers the separation of the two strands of a DNA.

Denaturation is brought about by high temperature, acid, or alkali. During denaturation there

is breakdown of hydrogen bonds between base pairs. Since G-C base pairs have 3 hydrogen

bonds and A-T pairs have 2 hydrogen bonds, G-C base pairs are more stable and it needs

more temperature for denaturation.

4.Renaturation: The denatured single stranded DNA can be made into double stranded DNA

by cooling or by neutralizing the medium. This process is called denaturation.

5.Effect of pH on DNA: The DNA is stable around the neutral pH in the solution. Further

increase in pH (alkali treatment) causes stand separation and finally denaturation occurs.

6.Stability : The DNA is a highly stable molecule. The stability is due to two forces.

a.( Hydrogen bonding between the bases. b.) Hydrophobic interactions between the bases.

Hydrogen bonds


7.Hyper chromic effect: DNA molecule absorbs light energy. This is a property of

individual bases. The intact DNA absorbs less light energy as its bases are packed into a

double helix. A denatured DNA molecule absorbs more light as its bases in single strands are

exposed.

Functions of DNA: It plays an important role in all biosynthetic and heriditory functions of

all living organisms.

1.It acts as the carrier of genetic information from generation to generation.

2. DNA is a very stable macromolecule in almost all living organisms.

3.It controls all developmental processes of an organisms and all life activities.

4.DNA synthesizes RNAs.

5. DNA is the genetic code which is responsible for protein synthesis.

Biological significance of nucleotides:

1. Nucleotides form the main components of nucleic acids.

2. Genetic material: Deoxyribonucleotides of DNA transmit hereditary characters from

parents to offspring.

3. Nucleotides functions as the source of high energy. Eg ATP, UTP, CTP, etc.

4. ATP is involved in oxidative phophorylation.

5. Certain nucleotides function as coenzymes. Eg UDPG, CoA, FMN, FAD.

6. Certain nucleotides function as vitamin B. Eg. FMN, FAD, NAD. Etc.

Types of DNA: DNA is classified into various types:

1.Double stranded DNA: It is also called as double helical DNA. In most of the organisms

except a few viruses, the DNA has a double stranded structure.

2.Single stranded DNA: Eg. Some viruses, E.coli, extra chromosomal satellite.

3. A-DNA: It is a double helical DNA having 11 residues per turn. It has a right handed helix.

It is formed by the dehydration of B.DNA.


A, B and Z DNA

B-DNA: This is the Watson and Crick double helix having 10 residues per turn. It is also

right handed.

Z- DNA: It is the left handed double helix having 12 residues per turn.

Circular DNA: It is circular in shape. It is found in bacteria, virus, mitochondria and

chloroplast. The circular DNA may be single stranded or double stranded. Single stranded

circular DNA is found in some viruses. Double stranded circular DNA is found in bacteria,

viruses, mitochondria, chloroplast, etc.

Relaxed DNA: Circular DNA without any helical coiling is called relaxed DNA.

Supercoiled DNA: It is supercoiled DNA. It can produce negative super coiling and positive

supercoiling. The degree of supercoiling is controlled by topoisomerases and gyrases.

Palindromic DNA: A double helix is formed by two paired strands of nucleotides that run in

opposite directions in the 5I- to 3I sense and the nucleotides always pair in the same way A-T

for DNA , with Uracil (U) for RNA, Cytosine ( C ), a nucleotide said to be palindrome if it is

equal to its reverse complement.


Repetitive DNA or satellite DNA: When very short sequences of base pairs are repeated

many times in DNA, the DNA is called repetitive DNA or satellite DNA. All eukaryotes,

except yeast, contain repetitive DNA. Repetitive DNA is absent in prokaryotes. The

repetitive DNA can replicate but cannot transcribe mRNA for protein synthesis. Repetitive

DNA is therefore inert.

6.1.2 Packaging of DNA Helix

The distance between two consecutive base pairs is 0.34nm.The length of DNA helix in a

typical mammalian cell is calculated by multiplying the total number of bp with distance

between two consecutive bp that is 6.6x109bp x 0.34nm = 2.2 meters. The length of E.coli

DNA is 1.36mm.

In prokaryotes, such as E.coli they do not have a definite nucleus, the DNA is snot scattered

throughout the cell. DNA (-ve charge) is held with some proteins (+ve charge)in a region

termed as ‘ nucleiod’.

In eukaryotes, the DNA is located in the chromatin material. The double helix DNA wraped

twice over bead shaped structure called nucleosome. The part of the DNA between each

nucleosome is the linker DNA. Each nucleosome is made up of 8 proteins known as histone

octamer. This structure is known as beads-on-string. A typical nucleosome contains 200 bp of

DNA helix.


Non-histone chromosomal (NHC) proteins- In a typical nucleus, some region of chromatin

are loosely packed (and stains light), and are referred to as euchromatin. The chromatin that

is more densely packed and stains dark are called as Heterochromatin. Euchromatin is said to

be transcriptionally active chromatin, whereas, heterochromatin is inactive.

6.2 The search for genetic material-

Transforming principle- In 1928, Frederick Griffith observed miraculous transformation

during his experiments on Streptococcus pneumonia. This organism has shown differences in

physical form during the experiment. When Streptococcus pneumonia. Bacteria are grown on

a culture plate, some produce smooth shiny colonies (S) while others produce rough

colonies(R). This is because the S strain bacteria have a mucous (polysaccharide) coat, while

R strain does not. Mice infected with the S strain (virulent) die from pneumonia infection but

mice infected with the R strain do not develop pneumonia.

Griffith was able to kill bacteria by heating them. He observed that heat-killed S strain

bacteria injected into mice did not kill them. When he injected a mixture of heat-killed S and

live R bacteria, the mice died. Moreover, he recovered living S bacteria from the dead mice.

He concluded that R strain bacteria had transformed by the killed S strain bacteria. Some

transforming principle transferred from the heat killed S strain to R strain. This transforming

principle (genetic material) is responsible for the synthesis of a smooth polysaccharide coat

around the R strain bacteria and this becomes virulent and responsible for pneumonia. This

experiment confirms the transformation of genetic material from S strain bacteria to the R

strain bacteria.

Biochemical characterization of transforming principle- Prior to the work of Oswald

Avery, Colin MacLeod, and Maclyn McCarty (1933-44), the genetic material was thought to


be a protein. They worked to determine the biochemical nature of “transforming principle’ in

Griffith’s experiment. They purified biochemicals (proteins, DNA, RNA, etc) from the heat

killed S cells. They discovered that DNA alone from S bacteria caused R bacteria to become

transformed.

They also discovered that protein digesting enzymes (proteases) and RNA digesting enzymes

(RNases) did not affect transformation, so the transforming substance was not a protein or

RNA. Digestion with Dnase did inhibit transformation, suggesting that the DNA caused the

transformation. They concluded that the DNA is the hereditary material.

6.2.1 The genetic material is DNA

In 1952 Alfred Hershey and Martha Chase proved that DNA is the genetic material through

their experiments on viruses that the bacteria known as bacteriophages.


The bacteriophage attaches to the bacteria and its genetic material enters the bacterial cell.

Due to this the bacterial cell produces virus particles. Through this experiment these scientists

discovered whether the genetic material carried by the virus is DNA or protein.

They grew some viruses on a radioactive phosphorus media and some others on a radioactive

sulfur media. Viruses grown in the presence of radioactive phosphorus contained radioactive

DNA (no proteins) as the DNA contains phosphorus. Viruses grown in the presence of

radioactive sulfur media not contained radioactive DNA, but they contain radioactive protein.

This confirms the virus genetic material is DNA and not protein. This indicates that proteins

did not enter the bacteria from the viruses. DNA is therefore the genetic material that is

passed from virus to bacteria.

6.2.2 Properties of Genetic Material (DNA versus RNA)

DNA is major hereditary (genetic) material in most of the organisms. RNA is the genetic

material in tobacco mosaic virus (TMV), ф β bacteriophage etc. RNA mainly performs the

functions of messenger and adapter. This is mainly due to differences between chemical

structure of DNA and RNA.

Required properties of genetic material:

a. Replication: This refers to duplication of its genetic material by faithful replication which

is shown by both DNA and RNA. Proteins and other molecules present in living being do not

posses this property.

b. Stability: Stability of genetic material should exist. It should not change its structure

easily. Even in Griffith’s experiment of ‘transforming principle’, DNA survived in heat killed

bacteria. Both the strands of DNA which are complementary can be separated. RNA is liable

and easily degradable due to presence of 2’—OH group present in each nucleotide. As RNA

is catalytic, it has become reactive. Because DNA is more stable than RNA, it is said to be

better genetic material. Presence of thymine instead of uracil is another reason which leads to

stability of DNA.

c. Mutation:- A mutation is a change in a DNA sequence. Both nucleic acids DNA and RNA

have the capacity to mutate. RNA mutates at a faster rate when compared with DNA. Virus

with RNA genome show mutation and evolution at a faster rate and thus has shorter life span.

d. Genetic expression- RNA expresses easily the characters in the form of proteins. DNA

requires RNA for formation of proteins. DNA being more stable is considered better than

RNA for storage of genetic information.

6.3 RNA world- RNA was the first genetic material. The essential life processes such as

metabolism, translation, splicing, etc are evolved around RNA. RNA also act as a catalyst.


There are some important biochemical reactions in living systems that are catalyzed by RNA

catalysts and not by protein enzymes. RNA being a catalyst is reactive and hence unstable.

Therefore, DNA has evolved from RNA with chemical modifications that make it more

stable. DNA is stable as it resists the changes by a process repair.

Structure of RNA- Ribonucleic acid (RNA): It is a nucleic acid containing ribose sugar. It

is found in large amount in the cytoplasm and at a lesser amount in the nucleus. In the

cytoplasm it is found mainly in the ribosomes and in the nucleus it is mainly found in the

nucleolus. RNA is formed of a single strand. It consists of several units called ribo-

nucleotides. Each nucleotide is formed of different molecules, namely phosphate, ribose

sugar and nitrogen base. The nitrogen bases are purines and pyrimidines. The purine bases

present in the RNA are adenine and guanine. The pyrimidines present in the RNA are

cytosine and uracil. The RNA molecule is normally single stranded, sometimes the stand may

be folded back upon itself and this double strand may be coiled to form a helical structure

like that of DNA. In RNA purines and pyrimidines are not present in equal amount.

Structure of RNA

6.4 Replication-

Replication is the duplication of DNA. By replication DNA produces exact copies of its own

structure. Replication occurs inside the chromosomes. It occurs during interphase. The parent

DNA strands function as templates for the synthesis of new DNA strands. Of the two strands

produced, one strand is the parental strand and the second strand is newly synthesized. This

scheme was termed as semiconservative DNA replication.


Replication starts at a specific point called origins. At this origin, the two strands are

separated. This separation is brought about by the enzyme helicase. At the point where the

two strands are separated, a replication fork is formed. The fork appears in the form of Y. The

duplication of DNA is brought about by the movement of the replication fork.

Leading strand- In one DNA strand, the daughter strand is synthesized as a continuous

strand. This strand is called leading strand because it is synthesized first. The leading strand

is synthesized continuously in the 5’ 3’ direction by DNA polymerase.

RNA primer- A primer is a strand of nucleic acid (10-12 nucleotides) that serves as a

starting point for DNA synthesis.

Lagging strand- In the second DNA strand, the daughter strand begins slightly later. Hence

this daughter strand is called lagging strand. The lagging strand is synthesized in short

fragments called Okazaki fragments. The replication is semi conservative discontinuous

because the DNA is synthesized in short segments. The enzyme DNA ligase joins the

Okazaki fragments into a long polynucleotide chain. DNA ligases catalyze formation of a

phosphodiester bond between the 5’ phosphate of one strand of DNA and the 3’ hydroxyl of

another.


Replication may occur in one direction from the point of origin or in both directions. When

replication occurs in only one direction, it is called unidirectional replication. When

replication occurs in both directions, it is called bidirectional replication.

6.4.1 The experimental Proof- It is now proven that DNA replicates semiconservatively. It

was shown first in Escherichia coli and later in higher organisms.Matthew Meselson and

Franklin Stahl performed the following experiment in 1958-

They grew E.coli in a medium containing 15

NH4Cl (15

N is the heavy isotope of nitrogen) as

the only nitrogen source for many generations. The 15

N was incorporated into the newly

synthesized DNA. This heavy DNA molecule could be distinguished from the normal DNA

by centrifugation in a cesium chloride (CsCl) density gradient. They noticed the heavy

15N

15N fractions of DNA (high density).

Then they transferred the cells into a medium containing 14

NH4Cl and took samples at 20min.

The DNA is separated by centrifugation in a cesium chloride (CsCl) density gradient. They

noticed the heavy 14

N15

N hybrid fractions of DNA (intermediate density).

After 40min (second generation), the extracted DNA is composed of 14

N15

N hybrid fractions

of DNA (intermediate) and 14

N1N fractions of DNA(light).

This experiment confirms that DNA replicates semiconservatively.


6.4.2 The Machinery and the Enzymes- In living cells, such as E.coli, the replication

requires a set of catalysts (enzymes). The main enzyme is the DNA polymerase. It uses a

DNA template to catalyse the polymerization of deoxinucleotides. These enzymes are highly

efficient enzymes, as they have to catalyse polymerization of large number of nucleotides in a

short period of time. E.Coli has only 4.6 x 106

bp (in human diploid cell 6.6 x 109

bp). Hence

it completes the replication within 18 min. The approximate rate of polymerisation is

2000bp/sec. Any mistake during the replication would result in mutation. Deoxynucleoside

triphosphates serve dual purposes. It act as substrate and they provide energy for

polymerization reaction. In addition to DNA-dependent DNA polymerases, many additional

enzymes are required to complete the process of replication with high degree of accuracy.

Replication starts at a specific point called origins. At this origin, the two strands are

separated. This separation is brought about by the enzyme helicase. At the point where the

two strands are separated, a replication fork is formed. The fork appears in the form of Y.

DNA- dependent catalyse polymerisation only in one direction that is 51 -> 3

1. Consequently,

on one strand (the template with polarity 3'5' ), the replication is continuous, while on the

other (the template with polarity 5'3' ), it is discontinuous. The discontinuously synthesised

fragments are later joined by the enzyme DNA ligase. The DNA polymerase on their own

cannot initiate the process of replication. Also the replication does not initiate randomly at

any place in DNA. There is a definite region in E.coli DNA where the replication initiates.

Such regions are termed as origin of replication. During the recombinant DNA procedures, a

vector provides the origin of replication. In eukaryotes, the replication of DNA takes place at

S-phase of the cell-cycle. The replication and cell division should be highly coordinated. A

failure in cell division after DNA replication results into polyploidy (a chromosomal

anomaly).


6.5 Transcription- The process of copying genetic information from one strand of the DNA

into RNA is termed transcription. In this process, the RNAs are required for protein synthesis

are synthesized on DNA strands. Three kinds of RNSs namely mRNA, tRNA and rRNA are

synthesized on different regions of DNA. Here also, the principle of complimentary governs

the process of transcription, except the adenosine now forms base pair with uracil instead of

thymine. In DNA replication, the total DNA of an organism gets duplicated. But in

transcription only a segment of DNA and only one of the strands is copied into RNA.

In DNA double helix, one of the strands serves as a template to produce RNAs. On the

template strand, a special region called promoter region is present. This region contains a set

of nucleotide sequences initiating transcription. This region where the initiator sequences are

present is called initiation site. In this region, the transcription is promoted by the RNA

polymerase.

RNA polymerase attaches to the initiator site with the help of Mn2+

or Mg2+

. The RNA

polymerase attachment makes a local opening in the double helix. From one end of the

unwound DNA strands, the polymerization of new RNA occurs. The new RNA strand grows

in the 5’ 3’ direction as the enzymes moves along the DNA. Proteins factors like rho and

SF help to terminate transcription.

The RNA produced by transcription is inactive and is called pre-RNA or primary transcript or

nascent RNA. It is active after processing.

6.5.1 Transcription unit - The following are the three regions of transcription unit in DNA.


i) A promoter, ii) The structural gene, iii) A terminator

The two strands of DNA have opposite polarity. The DNA-dependent RNA polymerase also

catalyse the polymerisation in only one direction, that is 5’3’ , the strand that has the

polarity 3’5” acts as a template, and is also referred to as template strand. The other strand

which has the polarity 5’3’ and the sequence same as RNA (except thymine at the place of

uracil), is displaced during transcription. This strand is coding strand.

3'-ATGCATGCATGCATGCATGCATGC-5' Template Strand

5'-TACGTACGTACGTACGTACGTACG-3' Coding Strand

The promoter is said to be located towards 5’- end (upstream) of the structural gene. It is a

DNA sequence that provides binding site for RNA polymerase. The terminator is located

towards 3’ end (downstream) of the coding strand and it usually defines the end of the

process of transcription.

6.5.2 Transcription Unit and the Gene- Gene is defined as the functional unit of inheritance

or heredity. Its presence was first proposed by Mendel in 1865. He called it as factor. The

word ‘gene’ was coined by Johannson in 1909. A gene is defined as a segment of DNA

having a particular sequence of nucleotides coding for a specific polypeptide chain. The DNA

sequence coding for tRNA or rRNA molecule also define a gene.

Size of a Gene- An average gene consists of 1500 nucleotide base pairs and has a molecular

weight of 10.6. The haemoglobin has two types of polypeptide chains, namely alpha and beta

chains. The alpha chain is synthesized by a gene containing 423 nucleotides (141x3= 423)

and the beta chain is synthesized by a gene containing 438 nucleotides (146x 3= 438).

Cistron- It is a modern term for gene. This term was coined by Benzer. Cistron is a section of

DNA that contains the genetic code for a single polypeptide chain and functions as a

hereditary unit.

Monocistronic- (mostly in eukaryotes)- Eukaryotic mRNA molecules are monocistronic,

containing the coding sequence for one polypeptide. In most eukaryotic genes, coding regions

(exons) are interrupted by noncoding regions (introns). During transcription, the entire gene

is copied into a pre-mRNA, which includes exons and introns. During the process of RNA

splicing, introns are removed and exons joined to form a continuous coding sequence.


Polycistronic RNA- (mostly in prokaryotes)- Prokaryotic mRNA molecules are polycistronic

containing more than one cistron (gene) and has as many as initiation and termination

codons.

6.5.3 Types of RNA and the process of Transcription-

Types of RNA- There are three types of RNA. They are mRNA, tRNA and rRNA

mRNA: This type of RNA carries genetic information for protein synthesis from the DNA to

the cytoplasm. The mRNA forms about 3 to 5% of the total cellular RNA. The mRNA carries

the message in the form of triplet codes. The life span of mRNA in bacteria is about 2 min. In

eukaryotes it lives for few hours to a few days.

Structure of mRNA: It is a single stranded polynucleotide chain. Each nucleotide is made up

of many nucleotides. Each nucleotide contains a phosphoric acid, a ribose sugar and a

nitrogenous base. The nitrogenous base may be adenine or guanine or cytosine or uracil.

Among RNAs, mRNA is the longest one. Most of the mRNAs contain 900 to 15000

nucleotides. One end of the mRNA is called 5’ end and other end is called 3’ end. At the 5’

end a cap is found in most eukaryotes and animal viruses. The cap is formed by the

condensation of the guanylate residue. The cap helps the mRNA to bind with ribosomes.

mRNA

The cap is followed by a non-coding region. It does not contain code (message) for protein

and hence it cannot translate protein. The non-coding region is followed by the initiation

codon. It is made up of AUG. The initiation codon is followed by the codon region which

contains code for protein. It has an average 1,500 nucleotides. The codon region is followed

by a termination codon. It completes the translation. It is made up of UAA or UAG or UGA

in eukaryotes. The termination codon is followed by non-coding region. At the 3’ end of

mRNA, there is a polyadenylate sequence (poly A). It consists of 200 to 250 adenylate

nucleotides (AAAAA….).


Transfer RNA (tRNA): It is a ribonucleic acid which transfers the activated aminoacids to

the ribosomes to synthesize proteins. It is so small that it remains in the supernatant during

centrifugation. Hence it is also called soluble RNA or supernatant RNA. It serves as an

adaptor molecule to attach amino acids. Hence tRNA is also called adaptor RNA. It

constitutes 10 to 15% of the total weight of RNA of the cell.

Ribosomal RNA (rRNA): It is a ribonucleic acid present in the ribosomes and hence it is

called ribosomal RNA. It is also called insoluble RNA. It constitutes about 80% of the

cellular RNA. It is formed by a single strand. It is a polynucleotide chain. Each strand is

formed of many nucleotide units. Each nucleotide is formed of three components- a

phosphate, a ribose sugar and a nitrogen base. The purine bases present in rRNA are adenine

and guanine and the pyrimidine bases are cytosine and uracil. Each strand has a 5’ end and a

3’ end. In some regions, the single strand is twisted upon itself to form a double helix. In the

helical regions most of the base pairs are complimentary. They are joined by hydrogen bonds.

In the unfolded single stranded regions, the base pairs are not complimentary. In prokaryotes,

the size of a ribosome is 70S, consisting of two subunits: 50S and 30S. The size of a

mammalian ribosome is 80S, comprising a 60S and a 40S subunit.The unit S stand for

Svedberg, which is a measure of the sedimentation rate.

Functions of rRNA; It forms the main bulk of the cytoplasmic RNA. Its function is not

clearly known. However it is believed that rRNA plays the major role in protein synthesis.


There is a single DNA- dependent RNA polymerase that catalyses transcription of all types of

RNA in bacteria. It uses nucleoside triphosphates as substrate. RNA polymerase is the

enzyme that catalyses all the three steps of transcription -initiation, elongation and

termination.

Steps in eukaryotic transcription-

Initiation- The prokaryotic RNA polymerase can bind to a DNA template on its own. But in

eukaryotic RNA polymerase require several other proteins called transcription factors. These

transcription proteins first bind to the promoter region and then help recruit the RNA

polymerase. The most important transcription factors are TFIIA, TFIIB, TFIID, TFIIE,TFIIF

and TFIIH. The synthesis of a polypeptide chain is initiated by a codon called initiation

codon. It is located in the beginning of a cistron (gene). The initiation codon is constant in

most cases. It is AUG and it codes for the amino acid methionine.

In eukaryotes there are three types of RNA polymerases. RNA polymerase I located in the

nucleolus. RNA polymerase II and III located in the nucleus.

Elongation- Transcription elongation occurs in a DNA where the RNA polymerase uses one

strand of DNA as template to catalyze the synthesis of a new RNA stand in the 5’ 3’

direction.

Termination- In bacteria, the polymerase stops transcription at the end of the terminator. The

Rho factor catalyses the termination process. In eukaryotes, the polymerase continues

transcription after the pre-mRNA is cleaved from the growing RNA chain; the polymerase

eventually falls off the DNA.


6.6 Genetic code- It is defined as the sequence of nitrogen bases (nucleotides) in mRNA

molecule which contains the information for the synthesis of protein molecules. A codon is

defined as the sequence nitrogen bases (nucleotides) in mRNA which codes for a single

amino acid. Nirenberg and Mathaei experimentally proved that a single amino acid is

determined by a sequence of three nitrogen bases. The sequence of three nitrogen bases

determining a single amino acid is called a triplet code.

Characteristics of a genetic code-

a.Triplet- A codon is a triplet as it is formed of three bases. 61 codons code for amino acids

and 3 codons do not code for any amino acids, hence they function as stop codons. Some

amino acids are coded by more than one codon, hence the code is degenerate.


b.Universal- Genetic code is universal in nature. One type of code determines the same

amino acid in all organisms including viruses, bacteria, plants and animals.

c.Colinearity- The codons in mRNA and the amino acids in polypeptide chains have a linear

arrangement.

d. Commaless- There is no comma or punctuation between the adjacent codons. That is, each

codon is immediately followed by the next codon without any spaces in between.

e.Non-overlapping- Recent discoveries show that codons are non-overlappng. Hence each

letter is read only once. The codes are consecutive. The codes are read one after another in a

continuous manner, e.g. AUG, CAU, GCA, etc.


f. Polarity- The code has a direction or polarity. It read in only one direction. Usually it is

read from the 5’(prime) end of the mRNA.

g.Initiation codon- The synthesis of a polypeptide chain is initiated by a codon called

initiation codon. It is initiated in the beginning of a cistron (gene). The initiation codon is

constant in most cases. It is AUG and it codes for the amino acid methionine.

h.Termination codon- The synthesis of a polypeptide chain is completed by a codon called

termination codon. It is located at the end of a cistron. The termination codon may be UAA,

UGA or UAG. The termination codon does not code for any amino acid. Hence it is called

non-sense codon.

6.6.1 Mutations and Genetic Code- A gene mutation is defined as an alteration in the

sequence of nucleotides in DNA. Mutation cause changes in the genetic code that lead to

genetic variation and the potential to develop disease. Two types of mutations are point

mutation and base pairs insertions/deletions.

1.Point mutation- In this type mutation changes single nucleotide base pairs.

a.Silent mutation- In this type a change in the DNA sequence occurs, this does not change

the protein. This is because different codons produce same amino acids. If the DNA sequence

AAA is changed to AAG, the amino acid lysine will still be produced.

b. Missense Mutation- In this type a change in the DNA sequence occurs; this alters change

the protein. The altered protein may be beneficial or dangerous. Example- if the codon for

arginine CGC is changed to GGC, the amino acid glycine will be produced instead of

arginine. Another example, in sickle cell anemia due to formation of valine instead of

glutamate.


c.Nonsense mutation- A stop codon is inserted in place of normal codon. This leads to the

formation of a protein which in non functional.

2.Base pairs insertions/deletions (frame-shift) -Mutations can also occur in which

nucleotide base pairs are inserted into or deleted from the original gene sequence. This type

of mutation is dangerous, as it alters the structure of template.

6.6.2 tRNA- the Adapter Molecule-

Transfer RNA (tRNA): It is a ribonucleic acid which transfers the activated amino acids to

to the ribosomes to synthesize proteins. It is so small that it remains in the supernatant during

centrifugation. Hence it is also called soluble RNA or supernatant RNA. It serves as an

adaptor molecule to attach amino acids. Hence tRNA is also called adaptor RNA. It

constitutes 10 to 15% of the total weight of RNA of the cell. It has a molecular weight of

25000 to 30000 and sedimentation co-efficient of 3.8S.


Structure of tRNA: The tRNA is made up of 73 to 95 nucleotide units called

ribonucleotides. Each nucleotides unit is made up of three components, namely a phosphate,

a ribose sugar and a nitrogenous base such as adenine, guanine, cytosine or uracil. The tRNA

is in the form of single polynucleotide chain having 3’ and 5’ ends. The polynucleotide chain

of tRNA is folded on itself and attains the shape of clover leaf. The 3’ and 5’ ends of tRNA

lie side by side as a result of folding. The 3’ end always ends in CCA base sequence. This is

the site for the attachment of activated amino acid. The 5’ end terminates in G or C. The t

RNA has 5 arms.

a.Amino acid acceptor arm, b) D-arm, c) Anticodon arm, d)Variable arm, e) T ΨC arm. Each

arm is made up of a stem and a loop. But the acceptor arm has no loop; the variable arm has

no stem. In the stem, the bases pair with each other (A-U and G-C). There is no base pairing

in the loops.

a) Amino acid acceptor arm: In the amino acid acceptor arm, the stem does not end with

loop. The acceptor has 3’ end of the nucleotide chain. The terminus of the acceptor site has a

constant CCA base sequence. To this base amino acids are attached to form aminoacyl tRNA.

The 5’ end of the arm comes near the 3’ end due to folding. Its terminus is either G or C.

b) D.arm: The D arm has 3 to 4 bases in the stem and 7 to 11 unpaired bases in the loop. The

loop is called dihydrouridine loop (DHU) or D-loop.


c)Anticodon arm: In the anticodon arm, the stem has 5 paired bases and the loop has 7

unpaired bases. The loop is called anticodon loop. Three of the 7 unpaired bases in the loop

(anticodon) determine the pairing of tRNA with the specific codon of mRNA.

d) Variable arm: In variable arm the stem may or may not be formed. The variable arm or

mini arm has a loop with 4-5 bases.

e) T ΨC arm: The T ΨC arm contains a constant T ΨC sequence. Its loop has ribosome

recognition site.

The tRNA molecules are named according to the amino acid to which it gets attached. For

example, tRNA carrying alanine can be called tRNAal. The tRNA molecules are synthesized,

at particular regions of DNA by a process called transcription. About 40 to 80 genes or

cistrons are involved in tRNA transcription.. The hybrid tRNA has base sequences

complimentary to the mother DNA in the beginning. But, after the completion of

transcription, the nitrogenous bases are altered at certain points in the nucleotide chain.

Functions of tRNA: tRNA picks up a specific activated amino acid from the amino acid pool

in the cytoplasm (aminoacyl tRNA). The amino acid is then transferred to the ribosome in the

cytoplasm where the proteins are synthesized. The attachment with ribosome depends upon

the codes in the mRNA and anticodons in the tRNA. Finally it transmits its amino acid to the

new polypeptide chain.

6.7 Translation- Translation is a process by which the base sequence of DNA, transcribed to

the mRNA is interpreted into amino acid sequence of a polypeptide chain. Translation

involves the following steps.

a.Activation of amino acid

b.Attachment of activated amino acid to tRNA.

c.Initiation of polypeptide chain.

d.Elongation of polypeptide chain

d.Termination of polypeptide chain.

a.Activation of amino acid- Amino acids, the building blocks of proteins, are present in the

cytoplasm. They are activated before they are transported by tRNA. The amino acids are

activated by ATP with the help of the enzyme aminoacyl synthetase. The activated amino

acid is called aminoacyl adenylate or aminoacyl AMP.

b.Attachment of activated amino acid to tRNA- The activated amino acid is attached to

the acceptor arm f tRNA. The loading of an amino acid to the tRNA is called aminoacylation


of tRNA. This reaction is catalyzed by an enzyme called aminoacyl tRNA synthetase. The

product formed is called aminoacyl tRNA complex.

c. Initiation of polypeptide chain.-

Protein synthesis is initiated by the selection and transfer of the first amino acid to ribosomes.

This process requires ribosome sub units, amino acyl-tRNA complex, mRNA and initiation

factors (IF). Initiation of polypeptide chain involves the following steps:

1.The 30S ribosomal sub unit attaches to the 5’ end of mRNA to form an mRNA-30S

complex. The attachment is made at the first codon of mRNA.

2.The first codon of mRNA will be always AUG. This codon specifies the amino acid

methionine. So the first amino acid in a polypeptide chain will be always methionine.

3.The tRNA having the anticodon UAC (complimentary to AUG) transports methionine to

the 30S ribosome and attaches itself to the initiation codon on mRNA. The tRNA, mRNA

and 30S subunit forms a complex called 30S pre-initiation complex.

4.The 30S pre-initiation complex joins with 50S ribosomeal subunit to form initiation

complex. The initiation complex is formed of 70S ribosome, mRNA and met-RNA.


5.The 70S ribosome has two slots for the entry of aminoacyl tRNA. They are P site (peptidyl

site) and A- site (aminoacyl site). The first tRNA (met-tRNA) is attached to the P-site of 70S

ribosome. The ribosome also acts as a catalyst for the formation of peptide bond.

d.Elongation of Polypeptide Chain- Elongation refers to addition of amino acids one by one

to the first amino acid methionine, as per the sequences of codon in the mRNA, The second

codon in the mRNA is recognized and corresponding anticodon of the tRNA moves to the

70S ribosome and fits into the A-site. Here the anticodon of tRNA base pairs with the second

codon of mRNA. A peptide bond is formed between the first amino acid of P-site and the

second amino acid of A-site. The peptide bond links the amino acids to form dipeptide. After

the formation of peptide bond, the methionine and tRNA are separated by an enzyme called t

RNA deacylase. The dissociated first tRNA is released from the P-site into the cytoplasm for

further amino acylation. By this way a long polypeptide chain is formed.

e.Termination of Polypeptide Chain- The polypeptide chain is completed when the

ribosome reaches the 3’ end of mRNA. The 3’ end contains a stop codon or termination

codon. It may be UAG, UAAor UGA. The termination is facilitated by proteins like RF-1,

RF-2, RF-3 and CTP. After the release of polypeptide chain, the 70S unit dissociates into 50S

and 30S sub-units. These sub-units are again used in the formation of another initiation

complex.


6.8 Regulation of Gene Expression- Gene expression refers to the manifestation of a

phenotypic character by the activity of gene. A gene expresses its character byproducing a

protein or an enzyme. During gene expression, there is flow of genetic information from

DNA to proteins.

Regulation of Gene Expression in Prokaryotes-In eukaryotes, the regulation could be

exerted at

i) Transcriptional level (formation of primary transcript)

ii) Processing level (regulation of splicing)

iii) Transport of mRNA from nucleus to the cytoplasm

v) Translational level.

The genes in a cell are expressed to perform a particular function or a set of functions. For

example, if an enzyme called beta-galactosidase is synthesized by E.coli, it is used to catalyse

the hydrolysis of a disaccharide, lactose into galactose and glucose; the bacteria use them as a

source of energy. Hence, if the bacteria do not have lactose around them to be utilized for

energy source, the synthesis of beta-galactosidase is not required. Hence the metabolic,

physiological or environment conditions regulate gene expression.

In prokaryotes, control of the rate of transcriptional initiation is the main site for control of

gene expression. In a transcriptional unit, the activity of RNA polymerase at a given promoter

is in turn regulated by interaction with accessory proteins. These regulatory proteins can act

both positively (activators) and negatively (repressors).

6.8.1 The Lac operon-

Operon is a set of closely linked genes regulating metabolic pathway in prokaryotes. A

bacterium contains thousands of genes. When all the genes are functioning at the same time,

the cell will flooded with enzymes and proteins. At any one time the required enzymes alone

are produced. Other enzymes which are not required are not synthesized. The genes for the

required enzymes are switched on and other genes are switched off. This on and off

mechanism was explained by the operon model.

The Lac Operon- It is a set of genes responsible for the metabolism of lactose in E.coli. The

lac operon was discovered by Jacob and Monod in 1961. The lac operon consists of 3

structural genes, namely z, y and a and three 3 control genes, namely a promoter gene (P), a

regulatory gene (I) and an operator gene (O). The structural genes are responsible for the

synthesis of three enzymes- namely β-galactosidase, by the gene z, galactoside permease by

the gene y and thiogalactoside transacetylase by the gene a. The operator gene is closely


linked to the first structural gene z. When the operator gene is active, the structural genes

synthesize enzymes. The activity of operator gene is decided by a repressor (protein)

synthesized by the regulator gene. When the repressor binds to the operator gene, the operator

gene is made non-functional. This state of operon gene is called repressed state and the

phenomenon is called repression. During repression, the enzymes are not synthesized.

When lactose is introduced into medium it diffuses into the cell and binds to the repressor

protein to form an inactive inducer repressor complex. The inactive inducer repressor

complex cannot bind to the operator gene and the operator genes set free to do the function.

This state of the operator gene is called depressed state and the phenomenon is called

derepression. When the operator gene is derepressed, the RNA polymerase binds to the

promoter gene. This initiates the transcription of structural genes. The transcription of

structural genes leads to the synthesis of the 3 enzymes, namely β-galactosidase, galactoside

permease and thiogalactoside transacetylase.

These three enzymes bring about the metabolism of lactose. β-galactosidase splits lactose into

glucose and galactose. Galactoside permease facilitates the entry of lactose into the cell. The

function of galactoside transacetylase is not known. In the lac operon system, the lactose

functions as an inducer for the synthesis of the three enzymes. Hence the lac operon system is

called an inducible system,

Lac operon

9.Human genome project

Education

Chapter 6 biology 20 12-15