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SELF INSTRUCTIONAL MATERIAL
M.Sc. PREVIOUS (BOTANY)
PAPER –I CELL & MOLECULAR
BIOLOGY OF PLANTS
BLOCK -2
UNIT III – Nucleus, Ribosomes
MADHYA PRADESH BHOJ ( OPEN)
UNIVERSITY, BHOPAL
Editor: Dr. (Smt.) Renu Mishra
HOD, Botany & Microbiology
Sri Sathya Sai College for Women,
Bhopal
Writer: Smt. Shikha Mandloi
Asst. Prof. Microbiology
Sri Sathya Sai College for Women,
Bhopal
PAPER-I
M.SC. PREV.
CELL AND MOLECULAR BIOLOGY OF PLANTS
UNIT-III
NUCLEUS
STRUCTURE
3.1
INTRODUCTION
3.2
OBJECTIVE
3.3
NUCLEUS
3.4
NUCLEOLUS & NUCLEAR PORE
3.5
NUCLEOSOME ORGANISATION
3.6
DNA STRUCTURE
3.7
FORMS OF DNA
3.8
REPLICATION
3.9
DAMAGE AND REPAIR
3.10 TRANSCRIPTION
3.11 RNA SPLICING
3.12 RIBOSOME STRUCTURE, r-RNA & BIOSYNTHESIS
3.13 MECHANISM OF TRANSLATION
3.14 SUM UP
3.15 CHECK YOUR PROGRESS KEY
3.16 ASSIGNMENT
3.17 REFERENCES
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3.1 INTRODUCTION
Since plant cell is a eukaryotic cell, the DNA of the cell remains organized in the
form of nucleosome. It remains surrounded by double membrane structure called as
nuclear membrane.
This organizational complex form nucleus in plants
The various functions of nucleus are
1. A site of DNA replication, transcription, translation etc.
2. Participates in ribosomal biosynthesis
3. Participates in biosynthesis of nucleotides
4. Exchange of materials with cell through nuclear pore.
During the process of growth of cell, replication of DNA occurs before cell
division. All the machinery required for "duplication" of DNA is located in nucleus.
Further, the flow of genetic information follows the pathway: DNA
RNA
transcription
Protein
translation
The Information coded in DNA cannot act directly as a template for protein
synthesis but must be first transcribed into messenger RNA. This RNA is synthesized by
RNA Polymerase Enzyme. The translation of the four base codes into a 20-amino acid
protein sequence involves many cellular components like nRNA, 4 RNAs, 70 protein
molecules assembled together in the nucleolus.
Apart from this, other process like ribosomal biosynthesis, nucleotide synthesis
is associated with nucleus. Thus in this unit, you will be learning in sequence a detailed
study of eukaryotic nucleus.
3.2 OBJECTIVE
After studying this unit you are expected to
1. Understand the structure and functioning of nucleus.
2. Know the structural and conformational details of DNA and RNAs.
3. Understand the process of Protein synthesis.
4. Fulfill the basic requirement to move a step towards molecular and genetic studies.
Nucleus : In Greek language the term nucleus means Karyon.
Robert Brown for the first time in 1833 discovered and coined the term Nucleus.
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On the basis of the absence of well-defined nucleus, living organisms were earlier
classified into two groups by molecular biologist viz.
Prokaryotes: Cells not having well defined nucleus as in bacteria and blue green algae.
Eukaryotes: Included remaining type of organisms having well-organized nucleus.
A nucleus may be described as having three important parts, viz
Nuclear membrane or nuclear envelope
Nucleolus
Chromosomes
The fluid in which nucleolus and chromosomes are present and which is enclosed in
nuclear membrane is called nucleoplasm.
3.3 NUCLEUS
Nucleus was first discovered by Robert Brown (1831) in orchid cells. It is the
most important part of the cell which directs and controls all the cellular functions. That’s
why nucleus is very often regarded as ‘director of the cell’. Presence of true nucleus
with nuclear membrane and linear chromosomes is the characteristic of all the eukaryotic
cells. However, there are some exceptions viz., mature mammalian RBCs, sieve tubes of
phloem, tracheids and vessels of xylem.
As far the number of nucleus in a cell is concerned, most of eukaryotic cells have
single nucleus within them. However, the number may vary in some cells.
Depending on the number of nuclei cells may be of following types :
Anucleate (without nucleus) :
Mammalian RBCs.
Uninuclate
:
Most of Eukaryotic Cells.
Binucleate
:
Basidiomycetes, Paramoecium
Multinucleate
:
Phycomycetes like Mucor, Rhizopus etc., Red
Algae.
The true Nucleus may be defined as: ‘The cellular structure limited externally by a
nuclear membrane surrounded by cytoplasm which contains linear
nucleoproteinous chromosomes and carry genetic information’s from generation to
generation’. The carrier of genetic information nature of nuclei was established by
Hammerling (1953) who worked on the macroscopic unicellular alga, Acetabularia and
concluded that the morphology of the plant is solely determined by the type of nucleus
contained in the plant body.
STRUCTURE OF NUCLEUS
Study of the cell cycle has revealed that each cell has two phases in its cycle:
Interphase and
Phase of cell division.
In fact, interphase is the phase between two cell divisions. This is much longer
than the phase of cell division, structure of nucleus is studied in this interphase only.
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The electron microscopic studies of interphase nucleus have revealed that the nucleus
may consists of following four parts:
1. Nuclear Membrane: It limits the nucleus externally and also known as karyothica. It
is bilayedred, lipoproteineous and trilaminar in nature. Outer membrane is called
ectokaryotheca and the inner is endokaryotheca. The outer membrane is studded with
ribosomes while the inner is free of that. The two membranes have a thickness of 75-90
Å
And are apart from each other by a distance of 100-300Å. This space is called
perinuclear space.
The nuclear membrane has many pores. Its number may vary from 1000-10000 in
a nucleus. Each pore is about 400-1000 Å in diameter. The number and size of pores may
depend on the needs of the cell. Each nuclear pore is fitted with a cylindrical structure
called annulus. The pore and the annulus both collectively form the pore complex or pore
basket.
Figure: nucleus
2. Nucleoplasm: It is transparent semi fluid, homogenous, colloidal ground substance
inside the nuclear membrane. It is also called nuclear sap, karyolymph or karyoplasm.
Nuclear chromatin and nucleolus are embedded within nucleoplasm, chemically, it is
formed of water, sugars, minerals (Mn2+, Mg2+, etc.), Nucleotides, ribosomes, enzymes,
DNA and RNA polymerases, mRNA, tRNA molecules etc. It is alkaline in nature (pH =
7.4 ).
Functions :
Nucleoplasm forms the skeleton of nuclei and helps in maintaining their shape.
The process of transcription takes place in the nucleoplasm in which different
molecules of RNA are formed.
It supports nuclear chromatin and the nucleolus.
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Ribosomal subunits are synthesized in the nucleoplasm.
3. Chromatin Net or thread : Electron microscopic studies of well stained eukaryotic
nuclei have revealed that presence of darkly stained network of long, fine and interwoven
threads which is called chromatin net or thread. It is also known as nuclear reticulatum. It
was first reported by Fleming in 1882. During the phase of cell division, the chromatin
net is transformed into chromosomes due to high condensation of DNA molecules. These
chromosomes are rod like and have definite shape and size chracteristic of an organism.
The chromatin is chemically nucleoprotein and formed of nucleic acid (DNA) and base
proteins i.e., histones . It may be classified in to two categories:
1. Heterochromatin : It is made of comparatively thick regions which is darkly
stained. DNA strands in this chromatin are more condensed. Transcriptionally, it
is inactive and late replicative. It does not contain active genes.
Euchromatin : It is true chromatin and is formed of thick and less darkly stained
areas. It has loose, less condensed DNA which is trancriptionally, inactive and
early replicating.
4. Nucleolus : Within each nucleus, there is a darkly stained, granular, naked and large
organelle without limiting membrane. It was discovered by Fontana in 1781. The term
nucleolus was coined by Bowman (1840). The size of nucleolus is comparatively larger
in those cells which have rapid rates of protein biosynthesis.
The position of nucleolus is generally definite within nucleus. It is associated with
nucleolar organizer region (NOR) of nuclear chromosome. It is absent in muscle fibres ,
RBC, Yeast, sperm and prokaryotes. In general, each nucleus has one or two nucleoli. Its
number depends on the number of chromosomes in the species. For each haploid set of
chromosomes in the nucleus, there is a single nucleolus. However, a pair of nucleoli may
be found in haploid nuclei. In human beings, two pairs of nucleoli are found in each
diploid nuclei. In human beings, two pairs of nucleoli are found in each diploid nucleus
Xenopus oocytes may contain upto 1000 nucleoli in the nucleoli in the nucleus.
Ultrastructure : The ultrastructure of nucleolus was studied by Borysko and Bang in
1951 and again by Berhard in 1952. On the basis of electron microscopic studies of the
structure of nucleolus, de Robertis et al., (1971) described it to be made up of four parts:
i.
Fibrillar regions: This part is made up of ribonucleoprotein fibres. It is also
called nucleolemma. Each fibre has a length of around 50-80 Å.
ii.
Granular regions : This part has many granules each having the diameter of 150200 Å. These are derived from nucleolar fibres, chemically, these granules are
also ribonucleoproteins.
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iii.
Protein region: This proteinous part is also called parsamorpha. This is the
fluid part of nucleolus in which other parts are found.
iv.
Chromatin part: It is made up of chromatin fibres containing DNA. These DNA
molecules function as template for RNA synthesis. The chromatin part may be
differentiated into two parts
a) Perinucleolar Chromatin: it forms a covering or envelope around nucleolus. It
may have ingrowths at certain places inside the nucleolus, which are called
trabeculae.
b) Intranucleolar chromatin: These chromatin fibres are found in internal protein
region. These form many septa like structures.
Each nucleolus has dense fibrillar region due to presence of which it is associated
with nuclear organizer region of chromosomes. These region have been reported to
contain many copies of DNA responsible for synthesis of ribosomal RNA. These rRNA
molecules are rapidly synthesized in this region. The protein of ribosomes are
synthesized in the cytoplasm which is transported to nucleus and finally to nucleolus. The
rRNA and protein molecules combine to form complete ribosome molecules. These
newly synthesized ribosomes are associated with thin fibrils of RNA and look like
beaded string. This structure is called nucleonema. On the basis of the presence and
structure of nucleonema, following three types of nucleoli may be recognized:
Nucleolus with nucleonema which is more common is all types of cells.
Nucleolus without nucleonema which is commonly found in salivary gland cells.
Ring shaped nucleolus containing ribonucleoprotein granules and RNA fibrils.
This is common in endothelial cells and muscle cells.
Functions:
Nucleoli are the site of rRNA biosysthesis.
It stores rRNA.
It helps in the biogenesis of ribosomes.
It helps in the formation of spindle fibres.
It plays important role in mitosis.
Functions of Nucleus
It controls all the cellular functions.
It controls the synthesis of all the structural and enzymatic proteins.
Synthesis of all the 3 types of RNA (mRNA, tRNA and rRNA) takes place in the
nucleus.
It plays important role in cell division.
Cell growth is controlled by nucleus
Nucleus controls cellular differentiation by regulating differential gene expression
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It induces genetic variation and thus helps in organic evolution.
Sexual reproduction happens due to fusion of two nuclei gametes of opposite sex.
Due to presence of all these organelles and other structures, a cell functions as
self-regulatory systems and provides a definite set of characteristics to different
organisms.
3.4 NUCLEOLUS & NUCLEAR PORE
Nucleolus can be seen as a very conspicuous structure in the interphase nucleus. It
disapperar during mitosis and reappears at the next interphase. The process by which the
nucleolus is formed, is described as nucleologenesis. During prometaphase to early
telophase, when the nucleolus remains disappeared, a number of non-ribosomal nucleolar
proteins as well as U3 s- RNA are found in (i) the peripheral regions of chromosomes and
in the (ii) nucleolus derived foci (NDF) found as cytoplasmic particles 1-
Fig.UltraStructure of Nucleolus
CHROMOSOMES
Chromosomes are rod like or filamentous bodies, which are typically, present in
nucleus and become visible during the stage of cell division. Presence of true
chromosomes is the characteristic of eukaryotic cells.
Literally, the term chromosomes have been derived from two Greek words;
Chroma and soma meaning by ‘colored body’. This is named because they appear as
darkly stained bodies during cell division when stained with a suitable dye and viewed
under compound microscope.
Chromosomes can well be defined in following way “Chromosomes are
individual protoplasmic entities found in the nuclei of almost all eukaryotic cells
which multiply themselves through sequential cell divisions and provide
physiological and morphological stability to protoplasm and so to a particular
individual”
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Some important points to remember about chromosomes.
1. Chromosomes were first observed by Straburger (1875) in mitotically dividing
cells and the name ‘chromosome’ was proposed by Waldeyer in 1888.
2. Each species has a definite and constant number of chromosomes in their cells.
The chromosome number found in somatic cells of the species is called somatic
chromosome number and is usually represented by ‘2n’. This is because,
ordinarily, somatic cells contain two copies of each chromosome which are
morphologically identical and also have same gene content and gene location.
They are known as homologous chromosomes.
3. Chromosomes can darkly be stained by treating the dividing cells by
acetocarmine, acetoarcine, feulgan and some other basic dyes.
4. In plant kingdom, lowest number of chromosome is found in Haplopappus
gracilis and highest in Ophioglossum reticulatum. In animal kingdom, Ascaris
mega-locaphala has been found to have lowest number of chromosome.
5. Chromosomes are chemically nucleio protein consisting of DNA and proteins.
The bear genes therefore, regarded as ‘bearer of hearedity.’
6. Each chromosome is made up of two longitudinally held chromatids which are
visible during mitotic metaphase.
7. The two chromatids of a chromosome are joined at centromere the main function
of which is the formation of spindle fibres during cell division.
8. Nucleolus within nucleus is associated with secondary constriction of
chromosomes. Therefore, the later is called ‘nucleolar organiser.
3.5 NUCLEOSOME ORGANIZATION
This model was proposed by Kornberg and Thomas in 1974 to explain the
structure of chromatin fibres. This has been widely accepted all over the world.
According to this model, chromatin is composed of a repeating unit called nucleosome.
Important points of this model are as follows:
Chromatin fibres of a chromosome are made up of DNA and histone proteins.
The repeating unit of chromatin is called nucleosome. It is a disc like structure 11nm in
diameter and 6nm in height. The core of a nucleosome is made up of an octamer of
proteins having two molecules each of H2A, H2B, H3 and H4 histones.
Around this octamer, a DNA segment having the length of 200 base pairs is
wound round making one 3/4 turns. This segment of DNA in chromatin fibre is nuclease
resistant. The structure of nucleosome is invariable in all the eukaryotes.
P. Oudet et al. (1975) worked extensively on the structure of nucleosome and
proposed that the length of DNA segment in the core of nucleosome is 146 base pairs.
Two nucleosome units are joined with a segment of DNA, which is called linker. It
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consists of 50-70 base pairs. H1 histone is associated with this linker DNA which makes
a connection between two adjacent nucleosomes.
The nucleosome, model explains the ‘string of beads’ concept of chromatin. This
is just opposite, to the concept of ‘beads on string’ explaining the interrelationship of
genes and chromosomes. Aron Clug (1977-80) made further electron microscopic
studies of chromosomes and chromatin and proposed ‘Solenoid model of nucleosome’.
This model describes the dense compaction of DNA in chromosomal chromatids. It
further illustrates that chromatin fibres tightly coil in a chromosome and form lump like
structure. The average diameter of this chromatin lump is 300 Å in which several
nucleosomes of 100 Å diameter are found. As has been mentioned earlier each
nucleosome is made up of protein octamer around which DNA segment of 200 base pairs
was found forming one3/4 turn. Through the process of super coiling, such nucleosomes
with the help of linker DNA easily form the solenoid like structure.
SOLENOID MODEL:
It was also shown that 11nm wide fibre of nucleosomes gets coiled upon itself to
form – 30nm wide helix with five or six nucleosomes per helix. In this helix successive
nucleosome units came close together, so that their centre to centre distance was about 10
nm. This 30nm structure was called a solenoid. Formation of solenoid from nucleosomes
can be compared with winding of a cable on a spool and then folding of wrapped spools.
It was also proved that H1 protein helped in folding of 110 A wide fibre in to 300
armstrong wide solenoid, It has been shown that H1 molecules aggregate by cross linking
to form polymers and may thus control the formation of solenoid. The above account
gives patterns of coiling and packing of DNA. Since 60 nm along DNA is coiled in a
nucleosome, only 6nm long, and then nucleosomes are coiled in 30nm wide solenoid
fibres, it gives DNA a packing ratio of 1:50. However, in highly condensed
chromosomes, the packing ratio is actually 1:5000, which is 100 times greater than
provided by solenoid, would take place by further coiling and folding of solenoid.
Ubiquitination, acetylation, methylation and phosphorylation of histones in
the nucleosome.
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The histone proteins, which are integral parts of nucleosome undergo a variety of
modifications to bring about decondensation of chromatin, to allow access of DNA
replication or transcription machinery to naked DNA. These modifications include
ubiquitination, acetylation, methylation and phosphorylation of some specific amino acid
residues of histones.
Acetylation and methylation occur on the free amino groups of lysines residues.
Methylation also occurs on arginine and histidine. Similarly, phosphorylation occurs on
the hydroxyl group of serine and histidine. Methylation and acetylation remove the
positive charge on NH3+, while phosphorylation introduces a negative charge in the form
of phosphate group.
3.6 DNA STRUCTURE
THE WATSON AND CRICK’S MODEL OF DNA DOUBLE HELIX
In 1953, James Watson and Francis Crick deduced the three dimensional structure
of DNA and immediately inferred its mechanism of replication, Watson and Crick
analyzed X-ray diffraction photographs of DNA fibres taken by Rosalind Franklin and
Maurice Klilkins and derived a structural model that has proved to be essentially correct.
The salient features of their model are:1. Two helical polynucleotide chains are coiled around common axis, the chains run
in the opposite directions.
2. The purine and pyrimidine bases are on the inside of the helix, where as the
phosphate and deoxyribose units are on the outside the planes of the bases are
perpendicular to the helix axis. The planes of the sugars are nearly at right angles
to those of the bases.
3. The diameter of the helix is 20 A0, adjacent bases are separated by 34 A0 along
the helix and related by a rotation of 360, hence the helical structure repeats after
in residues on each chain, i.e., at interval of 34A0.
4. The two chains are held together by hydrogen bonds between the pairs of bases
adenine is always paired with thymine guanine is always paired width cytosine.
5. The sequence of bases along a polynucleotide chain is not restricted in any way,
the precise sequence of bases carries the genetic information.
6. The ratio of A+G/C+T always equals to one
7. In every organism, the sequence of nucleotides in constant. The ratio of
A=T/G=C is also specific to organisms.
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8. Each pitch of DNA has two major and two minor groves.
Fig. DNA Structure
The most important, aspect of the DNA double helix is the specificity of the
pairing of the bases. Watson and Crick deduced that adenine must pair with thymine and
guanine with cytosine.
The steric and hydrogen bonding factors restriction is imposed by the regular
helical nature of the sugar-phosphate backbone of each polynucleotide chain the
glycosidic bonds that are attached to a bonded pair of bases are always 10.85 A apart a
pair of pyrimidine base pair fits perfectly in this shape, in contrast, there is insufficient
room for two purines. There is more than enough space for two pyrimidine but they
would be too far apart to form hydrogen bonds Hence, one member of a base pair in a
DNA helix must always be a purine the other a pyrimidine, because of stric factors. The
base pairing is further restricted by hydrogen bonding requirements. The hydrogen atoms
in the purine and pyrimidine bases have well defined positions. Adenine can not pair with
cytosine because there would be two hydrogen near one of the bonding positions and
none at the other like wise guanine can’t pair with thymine, where as guanine forms three
bonds with cytosine. The orientation and distance of those hydrogen bonds are optimal
for achieving strong interaction between the bases. The base pairing scheme was strongly
supported by the base compositions of DNA’s from different types. In 1950, Erwin
chargaff found that the ratios of adenine to thymine and guanine to cytosine were nearly
1 in all the samples studied.
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3.7 FORMS OF DNA
CONFORMATIONAL FLEXIBILITY OF DNA MOLECULES:The vast majority of DNA molecules present in the aqueous protoplast of living
cells almost certainly exist in the living cells almost certainly exist in the Watson Crick
double helix model.
Another model content with the X-ray diffraction was constructed in which the
two anti-parallel DNA strands remain associated via. Complementary base pairing, but
lie “side by side” instead of being wound in to a continuous double helix. But the
existence of as double helix has been confirmed by experiments to measure directly the
number of base pairs per turn. This proves to be 10.4 instead of 10 bases pair by the
classic B-model. The change requires a slight adjustment in the angle of rotation between
adjacent base pairs along to helix, to 34.60 So that it takes slightly longer to accomplish
the full 3600 turns. Changes in the condition or in the particular base sequence could lead
to lighter or looser helical structures in Particular regions. Indeed the structure of a
particular 12 base pair molecule has been shown by X-ray crystallography to have 10.1
base pairs per turn, achieved by a slight twist of each base pair that improves base
stacking relative to original model.
Because of this variation, the idea that there is a single structure for the DNA
double helix has been replaced width a view that there are families of structures each of a
characteristics type, but shows difference in the no. of nucleotides per turn and the
distance between adjacent repeating units the variation is achieved by changes in the
rotation of groups about bonds with rotational freedom. Thus the DNA molecule exhibit a
considerable amount of conformational flexibility, Till today 6 forms of DNA molecule
are KN but most of these have been found only under rigidly controlled experimental
conditions., these forms are distinguished by:
1. The no. of base pairs that occupy each turn of helix.
2. The pitch or angle between each base pair
3. The helical diameter of molecule
4. The bounded length of double helix.
Some of these forms inter convert if salt and hydration condition are manipulated.
B-FORM OF DNA: This is the dominant form of DNA under physiologic condition has pitch of 3.4
nm per turns, 10 base pairs exist each planer base being stacked to resemble 2 binding
stacks of coin sides by side. The 2 stacks are held together by hydrogen bonding at each
level between the 2 coins on opposite stacks and by 2 ribbons would in a right handed
turn about the 2 stacks and representing the phosphodiaster back bone.
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A- FORM OF DNA:It is found in fibres at 75% relative humidity and requires the presence of Na+, K+
or Cesium as the counter ion. Instead of lying flat the bases are tilted with regard to the
helical axis and there are more base pairs per turn. The A form is biologically interesting
because its is probably very close to the conformation adopted by DNA-RNA hybrids or
by RNA-RNA double stranded regions, the reason is that the presence of 2 hydroxyl
groups prevents RNA from lying in the B form. In this A form II nucleotides per turn are
present.
C-FORM OF DNA: This occurs where DNA fibers are maintained in 66% relative humidity in the
lithium. It has fewer base pairs per turn than B-DNA. It has 91/3 no. of nucleotides base
pairs per turn. Some further forms have been found that appear to represent option open
only to DNA molecules with particular quick’s in their base composition.
D-FORM AND E-FORM OF DNA:These are actually possibly extreme variants of the same form. They have the fewest
base pairs per turn and are taken up only by certain DNA molecules that lack guanine.
Z-FORM OF DNA: They are left handed double helix in which the phosphodiaster backbone zigzags
along the molecule hence, the name Z-DNA. It is least twisted and it has only one grove.
Z-DNA occurs in repeated sequence of alternating purines and pyrimidine
deoxynucleotides but also requires one or more stabilizing influences. These stabilizing
influences include
1. The presence of high salt or specifications such as spermine or spermidine
2. A high degree of negative supercoiling of DNA.
3. The bending of Z-DNA specific protein.
4. The methylation of 5-carbon of come of the deoxycytidine nucleotides in the
alternating sequence.
Z-DNA could exert regulatory effects both proximal and distal to the site of is
existence. For instance, some proteins that bind in the major or minor groove of B form
DNA could probably not bind to the Z-form . In addition, the reversion of Z-form to a Bform of DNA, an event that might occur as a consequence of low of CH3 group from 5methydeoxycytidine, would likely reveal in torsional difference of DNA actual to the
actual site of the Z-DNA.
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The existence of Z-DNA in Drosophila chromosomes has been demonstrated utilizing
antibodies that recognize and bind specifically to Z-DNA human DNA contain potential
Z-DNA forming regions dispersed through out the genome, and stabilizing influences
may also exist.
Helix Type
A
B
C
Z
D
Base
Per Turn
11
10
9
12
8
E
7
Pair
Rotation per
Helical
base pair
Diameter
+ 32.70
23
+ 36.00
19
0
+ 38.6
19
- 30.00
18
Denaturatio
n
Denaturatio
n
Vertical rise
per base pair
2.56
3.38
3.32
3.71
Check Your Progress – (1)
Note: 1. Write your answer in the space given below.
2. Check your answer with the one at the end of the unit.
1.
2.
3.
4.
5.
The Main function of Nucleolus is the synthesis of ___________.
Protein synthesis is performed by __________.
Nucleosome model of chromatin structure was proposed by ___________.
The Unit of DNA measurement is _______________.
Highly condensed chromatin is called _______________.
3.8 REPLICATION
Semi-conservative replication of Chromosomes in eukaryotes:
Autoradiography experiment in Vicia faba, by J.H. Taylor and his co-workers for
the study of duplicating chromosomes in the root tip cells were first published in 1957.
They reported that
DNA in all the organisms has the inherent capacity of self-replication. The
mechanism of DNA replication is so precise that all the cells derived from a zygote
contain exactly similar DNA both in terms of quality and quantity. The replication takes
place in interphase after every cell division.
Theoretically, there may be following three possible modes of DNA replication:
Dispersive Method
Conservative Method
Semiconservative Method
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Semiconservative mode ist the most accepted of all.
Semiconservative Method: - During replication, the two strands of the DNA molecule
uncoil with the help of some proteins and enzymes. The unpaired bases in the single
stranded regions of the two strands binds with their complementary bases in the single
stranded regions of the two stands bind with their complementary bases present in the
cytoplasm in the form of nucleotides. These nucleotides become joined by
phosphodiester linkages generating complementary strands on the old ones. This provides
for an almost error free, high fidelity replication of DNA.
Fig. Semiconservative replication.
Detailed mechanism of semiconservative mode of DNA replication was given by
Kornberg. He proposed that following enzymes are important for functional replication of
DNA.
1.
2.
3.
4.
5.
6.
Nucleases
Unwinding Proteins
DNA Polymerases
DNA Ligases
RNA primer
Primases or RNA polymerases.
Out of the above six enzymes/proteins, the three; nucleases, RNA polymerases and DNA
polymerases are known as “Kornberg Enzyme”.
1. NUCLEASES
These are the enzymes which digest or breakdown nucleic acid molecules. These attack
on phosphodiester bonds of the nucleic acid backbone and release nucleotides through
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hydrolysis. On the basis of their mode of function, nucleases may be classified into
following two:
(i) Exonucleases
(ii) Endonucleases.
Exonucleases function on phosphodiester bonds of the DNA at both the terminus. The
endonucleases, on the other hand attack the phosphodiester bonds at intercalary regions
of the DNA breaking it into as many parts as the site of function.
2. UNWINDING PROTEINS.
Unwinding proteins are those proteins or enzymes, which uncoil the DNA helix and
separate the two DNA strands by breaking hydrogen bonds between them. Due to this
function these are known as unwinding proteins.
Due to separation, the two strands form a ‘Y’ or fork like structure, which is
known as replication fork. Usually two types of unwinding proteins have been
recognized:
(i) DNA helicases : These enzymes or proteins uncoil the helix of DNA which may now
appear as ladder.
(ii) DNA gyrases: These enzymes breakdown the hydrogen bonds (A=T, C=G) between
two strands of a DNA molecule. E.g., DNA topoisomerase.
3. DNA Polymerases or Replicase Enzyme
DNA polymerases are the first enzymes suggested to be implicated in DNA replication. It
mainly function in the polymerazation of nucleotides on the DNA template producing
thereby the polynucleotide chain.
In eukaryotic cells, these three enzymes have been name as DNA polymerase, βDNA polymerase and -DNA polymerase. Due to their role in DNA replication, DNA
polymerases are also known as DNA replicases.
(1) DNA POLYMERASE-I: This enzyme was first discovered by Arthur Kornberg in
E.coli. After the name of the scienctist, DNA polymerase-I is also known as ‘Kornberg
enzyme’ or ‘Kornberg polymerase’. It is the most extensively studied DNA polymerase
studied in DNA replication machinery of E.coli. Nowadays, it is believed that this
enzyme is not responsible for DNA replication and it mainly function in DNA repair.
Structurally, a molecule of DNA polymerase-I consists of a single polypeptide chain
having the molecular wt. 109,000. An atom of Zn is associated with each molecule of this
enzyme.
Following sites have been reported to be present on its surface:
(a) Template Site: it is occupied the DNA strand.
(b) Primer Site: The site at which the growth of DNA chain occurs.
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(c) Nucleotide triphosphate site: The site where the incoming nucleotide triphosphate
is received.
(d) Primer terminus site: This site, which is used for removing any mismatched
nucleotide at the end of growing chain.
(e) Site for 5’ 3’ cleavage: The site which is used for removing any strand coming in
the path of growing primer. DNA polymerase-I function in following activities within
cells.
(i) 5’ 3’ polymerization activity: Attachment of nucleotides with each other by the
activity of DNA polymerase forming a polynucleotide chain is called polymerization.
The rate of polymerization in E.coli at 370 C has been noted to be 1000 nucleotides per
minute. It occurs in 5’ 3’ direction. It forms small DNA segments through
polymerization, which is used in repair mechanism.
(ii) 3’ 5’ Exonuclease Activity: The activity of DNA polymerase-I is performed to
remove any nucleotide which mispair during elongation of growing strand.
(iii) 5’ 3’ Exonuclease Activity: This activity is performed by this enzyme to remove
any DNA segment which comes as an obstruction in the way of growing DNA Strand.
(iv) Removal of Thymine Dimmers: DNA polymerase-I function in the removal of
thymine dimers from the DNA strand. Such thymine dimmers are produced to UV
irradiation. After removal of diers, it also fills the gap, formed dut to excision.
(2) DNA Polymerase-II : This enzyme has also been isolated from E.coli and has the
molecular wt. 90,000. Polymerization rate DNA polymerase-II is much slower than
polymerase-I. It is only 50 nucleotides per minute in E.coli, it has 3’ 5’ exonuclease
activity but lacks 5’3’ exonuclease activity unlike polymerase-I.
(3) DNA- Polymerase-III : This is the main DNA polymerase involved in DNA
replication. This polymerase enzyme was originally discovered in a lethal mutant of
E.coli having mutation at dnaE locus. The enzyme has higher affinity for nucleotides than
polymerase-I and II have. The rate of polymerization by polymerase-III is approximately
10-15 times higher than the polymerase-I.
Structurally, DNA polymerase molecule consists of two polypeptide chains each
having the molecular wt. of 90,000. This dimeric enzyme does not function unless it
associates with two more chains of copolymerase-III each having the molecular wt. of
77,000. The holoenzyme may be represented as α2β2 where α2 polymerase-III and β2
copolymerase-III. ATP is also needed for the growth tof polynucleotide chain.
17
Fig.- Enzyme polymerase.
The enzyme DNA polymerase-III has 3’5’ exonuclease activity and 5’3’ polymerase
activity.
4. DNA LIGASES
Through polymerase activity by DNA polymerase-III, polynucleotide chains is
formed in the form of small fragments which are known as Okazaki fragments. In order
to form a complete chain complementary to that of the template, ligation of okazaki
fragments is essential. The ligation reaction is performed by RNA ligases.
5. RNA PRIMER
DNA replication really starts with the formation of a RNA fragment known as
RNA primer. It is formed at the point of origin. The primer is formed throught
polymerization activity by RNA polymerase. Due to this reason, RNA polymerase is also
needed for functional replication of DNA.
6. PRIMASES
This is the group of enzymes, which are involved in RNA primer synthesis. RNA
polymerase is an example of primases.
Detailed molecular biological studies of the process of DNA replication now have
revealed that the process is much complex and requires a multi-enzyme complex. About
two dozens of enzymes are involved in this complex. The complex is known as
‘replisome’
STEPS OF DNA REPLICATION
Before studying the mechanism of DNA replication, we must be familiar with
following terms:
1. Replicon: A replicon is the unit of DNA in which individual act of replication takes
place. It has capacity of DNA replication independent of other segments. Therefore, each
replicon has its own origin and terminus, at which DNA replication stops.
2. Origin: This is the sequence of a replicon which supports initiation of DNA
replication and also regulates the frequency of replication initiation. A general feature of
origin is that it is A=T rich. An origin in E.coli, oric has been identified to 250 base pairs
long.
3. Terminus: In most of prokaryotes, replicons has a specific site at the extreme
downstream of the strands. This site stops replication fork movement and thereby
terminates DNA replication.
18
In order to understand the exact mechanism of DNA replication. The process
must be studied in stepwise manner.
The overall process is completed in following steps:
(1) Before the start of DNA replication and formation of origin point, the enzyme DNA
helicase associates with the site of DNA. Its molecules unwind the two strands of the
DNA. Another enzyme, DNA gyrase or DNA topoisomerase breaks the hydrogen bonds
between the two strands and separate them from each other forming a ‘Y’ shaped
replication fork.
(2) The two strands of a DNA molecule separated in the way explained in the first step
function as template. It should be noted here that template is the single strand of DNA on
which polymerization of nucleotides forming a new strands takes place.
In eukaryotes, evidences for bi-directional DNA replication are available.
DNA replication starts at many points each of which start as a loop and can be seen as
expanding bubbles or eyes in electron micrograph.
The number of eyes or bubbles indicates the number of replicons.
Figure- DNA Replication
19
(2) Formation of RNA Primer: Before the actual replication of DNA starts at origin, a
short fragment of RNA is synthesized with the help of RNA polymerase. This RNA
fragment is called RNA primer. It is believed that it provides safety to the new DNA
strands, which is synthesized extending the RNA primer itself.
(3) Synthesis of Complementary Strand of DNA: DNA replication or synthesis of a
new DNA strand complementary to the template is catalysed by the enzyme, DNA
polymerase-III. It starts at the end RNA primer in 5’3’ direction. The nucleotide
sequence in the new strand is always complementary to the sequence of nucleotides in the
template.
Some features of DNA replication are as follows:
(i) DNA Replication is Bi-Directional: Johan Cairsn on the basis of his experiments on
atoradigraphy concluded that DNA synthesis starts at a fixed point on the chromosome
and proceeds in one direction only. Subsequently, it was realized that Cairns results could
be interpreted in terms of bi-directional replication also. On the basis of many other
experiments it has convincingly been demonstrated that DNA replication begin at a
unique site at origin and proceeds in both the direction on a strand. It takes place in the
form of pieces called ‘Okazaki fragment which are joined with each other with the help
of DNA ligases.
(ii) The two strands of the parent DNA at the point of replication fork or origin replicate
together with each other.
(iii) Replication of 3’5’ strand of DNA molecule is continuous and the new strand
grows in 5’3’direction. Replication in the second strand of the DNA molecule is
discontinuous. Replication of this strand starts somewhat later than that of strand.
Consequently, a given segment of 53’ strand always replicates i.e., the 3’5’ strand.
Therefore, the 3’5’ strand of the parent DNA molecule is known as the leading strand
while the 5’3’ strand is termed as the lagging strand.
(iv) Formation of RNA primer takes place in the beginning of each and every okazaki
fragments.
4. Termination of DNA Replication: The termination of DNA replication so signaled
by specific sequences, the ter-elements. E.coli, the ter-element of R6 K plasmid has a 23
base pair sequence. This site functions as the binding site of Tus, a 36 K dal protein
necessary for termination. This stops the replication fork movement and thereby stops
DNA replication.
5. Removal of RNA Primer: After the whole DNA molecule is replicated on both the
strands, the RNA primer on all the segments are removed or degraded with the help of
DNA polymerase-I through its nuclease activity.
20
6. Synthesis of DNA Strand at the place of RNA Primer: in order to have replication
of a complete DNA molecule, replication of the segment at the place of RNA primer is
necessary. This process is performed with the help of DNA polymerase-I instead of
polymerase-III through polymerization of deoxyribonucleotides. After DNA replication
at the place of RNA primer, the replication process is completed.
7. Proof Reading and Repair Mechanism: The complementary base pairing during
DNA replication is much accurate and precise, however, there are chances of error in this
process of base pairing. It is mainly due to various physical and chemical forces involved
in rate of error in E.coli are 5 x 10-8 to 5 x 10-10.
8. The proof reading of newly synthesized DNA strand is done by DNA polymerase-III
through correction of mismatched base pairing. It deletes the wrong base and replaces it
by a correct base. The process of proof reading also takes place in 5’3’ direction. In
E.coli, mutants defective in proof reading show an increase in mutation frequency by
over 1000 times.
3.9 DAMAGE AND REPAIR.
Both in prokaryotic and eukaryotic cells, there are repair enzyme systems to deal
with DNA damage, which is caused rather frequently. The changes in DNA leading to
damage are broadly divided into two general classes: (i) Single base changes may be
caused by conversion of one base to another. These are corrected throught DNA
replication leading to changes in DNA sequence. (ii) Structural distortions may results
from a single strand nick, removal of a base or introduction of a covalent link between
bases of same or different strands eg. formation of thymine dimer.
A cell may have several systems to deal with DNA damage. These systems include the
following: (i) Direct repair involves reversal of the damage; for instance, photo
reactivation of pyrimidine dimer involves reversal of covalent bonds giving the original
structure. (ii) Excision repair involves recognition of damaged or altered base followed
by excision of a sequence including damaged or altered base(s) followed by excision of a
sequence including damaged bases. This is achieved by an endonuclease, or an
exonuclease. A new stretch of DNA is then synthesized to replace the excised material.
(iii) Mismatch Repair involves correction of mismatches or pairing between bases
which are not compulsory. Mismatches may arise either (a) during replication or (b) due
to base conversion and are corrected by a process described as error correction during
DNA replication.
Direct Repair
As mentioned above, direct repair involves reversal of DNA damage. Abnormal
chemical bonds between bases or between a nucleotide and an abnormal substituent are
broken down, thus restoring the original DNA structure. The enzymes currently known to
catalyze this DNA repair include the following: (i) DNA photolyase repairs
cyclobutane
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Pyrimidine dimers, induced by Ultra-violet The enzyme splits the cyclobutane
ring, only on activation by light. (ii) 6-4 photoproduct photolyase deals with the DNA
damage involving the formation of ‘6-4 photoproduct’ due to UV. (iii) Spores
photoproduct lyase repairs the lesion caused by UV in B. subtilis spores, where a spore
photoproduct is produced instead of cyclobutane dimers on UV irradiation. (iv) O6methyl guanine DNA methyl transferase (MGMT) is a common enzyme found in all
species tested. The enzyme transfers the methyl group of O6- methylguanine to a cystine
residue on the enzyme.
Excision Repair system in E.coli
Excision repair has been classified into the two types on the basis of the nature of
excised products. Which can either be free bases as in base excision repair, or one or
more nucleotides, as in nucleotide excision repair?
Base excision repair. BER is always initiated by a DNA glycosylase. A number
of these glycosylases are known, depending upon the substrate used by each of them.
These glycosylases hydrolyse N-glycosyl bonds linking bases to the sugar-phosphate
backbone of the DNA, leaving sites that are abasic.Sites are then acted upon by an
apurinic/aprrimidinic endonuclease and an AP lyase, so that sugar-phosphate group is
removed by hydrolysis of 5’phosphodiester bond.
Nucleotide excision repair. NER is initiated by endonucleolytic excisions, either
only on one side of the lesion or on each of the two sides of the lesion. In the
endonuclease-exonuclease repair, an endonuclease first makes an incision at either the 5’
or the 3’ end of the lesion. The exonuclease then digests segment of the strand involving
the lesion. The gap is later filled by DNA synthesis and ligation.
Relationship between NER and transcription
In prokaryotes, as well as in eukaryotes, it has been shown that the nucleotide
excision repair of template strand of the transcriptionally active DNA is faster than that of
its complementary strand.
Recombination repair (dimmer tolerance) involves DNA replication in E. Coli
Dimers, whenever formed in DNA, are reversed either by photo reactivation, due
to Photolyase or removed by UvrABC catalyzed excision repair. However, some of the
dimmers still remain and are tolerated by the replication machinery.
An SOS repair system in E.coli.
In E. coli, many treatments causing DNA damage or inhibition of DNA
replication induce a complex series of phenotypic changes described as SOS response.
The SOS response is initiated by interaction of RecA protein with LexA repressor.
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3.10 TRANSCRIPTION
The production of RNA copies from a DNA template is known as transcription. It
is catalysed by a specific enzyme RNA polymerase or transcriptase. During this process,
only one strand of DNA duplex is known as template strand or antisence strand. This
results into the production of m-RNA molecule having base sequence complementary to
the template DNA strand. It should be noted here that the sense. strand or coding strand
of DNA is now copied and has the same base sequence as the RNA produced by the
antisense strand.
The RNA polymerase is a complex enzyme and usually consists of a larger protein part
(apoenzyme), which is known as core enzyme and a cofactor, which is known as sigma
factor. The two combines to produce the complete enzyme of holoenzyme. Unless and
until the two parts of RNA polymerase do not combine with each other, it is not
functional. As far as the nature of RNA polymerase in prokaryotes and eukaryotes is
concerned, it shows much diversity. While in prokaryotes like E.coli a single species of
this enzyme is found, at least three distinct RNA polymerases have been reported in
nuclei of most of eukaryotes. These have been named as : 1. RNA polymerase-I or A, 2.
RNA polymerase-II or B and 3. RNA Polymerase-III or C. They have different functions
as:
RNA polymerase-I or A: It is located in the nucleolus and responsible for the
synthesis of rRNA.
RNA polymerase-II or B: It is found in the nucleoplasm and is responsibe for the
synthesis of hnRNA which is a precursor of mRNA.
RNA polymerase-III or C: It is also found in the nucleoplasm. It is responsible for
the production of 5s rRNA and tRNA.
1.a Promoters for RNA PolymeraseI. Promoters for RNA polymerase I have atleast
two elements:
A GC-rich upstream (-180 to -107) control element
A core region that overlaps the transcription start site (-45 to +20).
Protein coding structural genes in higher eukaryotes are transcribed in the
nucleus, but the primary RNA transcripts in the nucleus differ from mRNAs used in the
cytoplasm for translation. The RNA transcripts in the nucleus are collectively described
as heterogeneous nuclear RNA or pre-mRNA molecules each of which is generally much
larger than its corresponding mRNA. The hnRNA molecules, which are destined to
produce mRNA, undergo RNA processing which includes the following events: (i)
Modification of 5’ end by capping and modification of 3’ end by a tail after enzymatic
cleavage; (ii) Splicing out of intron sequences from RNA transcripts of interrupted genes.
Cleavage and polyadenylation usually preceed RNA splicing.
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Promoter, enhancer and silencer sites for initiation of transcription in eukaryotes
In eukaryotes there are three RNA polymerases: RNA polymerase I or RNAPI for
synthesis of pre-rRNA; RNA polymerase II or RNAPII for synthesis of re-mRNA or
hnRNA and several snRNAs, and RNA polymerase III or RNAPIII for synthesis of 5S
RNA, tRNA. Different promoter sequences have been identified for different RNA
polymerases.
MECHANISM OF TRANSCRIPTION: The overall process of transcription is
completed in following steps:
1. Formation of holoenzyme: The core enzyme of RNA polymerase cannot start
the polymerization process producing RNA. It first combines with the sigma
factor and produce the holoenzyme, It is assumed that the sigma factor helps the
enzyme in recognition of the initiation site on the DNA template.
2. Attachment of holoenzyme on DNA duplex: The holoenzyme first binds at the
promoter site of DNA forming the closed promotor complex or ‘closed binary
complex’. In this stage the DNA still remains in the form of double helical.
3. Unwinding of DNA: It includes strand separation in the DNA duplex in a stretch
of the DNA bound with RNA polymerase; It extends just beyond the start point so
that the template becomes available for transcription initiation. The open DNA
strands form the ‘open binary complex’
4. Synthesis of RNA: After the open binary complex is formed on DNA, synthesis
of RNA starts. Once the template or antisense strand of DNA becomes available,
the enzyme begins to incorporate RNA nucleotides beginning at the start points.
The polymerization of these nucleotides takes place in 5’ 3’ direction. As the
enzyme molecule move ahead in this direction, phosphodiester linkage or bond is
formed between two adjacent nucleotides.
The process of elongation of RNA synthesis take place when the holoenzyme
leave the promoter region and move ahead in 5’3’ direction. Together with the
movement of the holoenzyme, the trancription bubble also moves in the same direction.
The transcription bubble represents the region of the DNA duplex in which the two
strands are separated from each other. The length of the bubble ranges from 12 to 20 base
pairs. The bubble movement and sequential adding of correct nucleotides on RNA chain
take place simultaneously. The 5’ end of the newly synthesized RNA progressively
separate from the DNA template DNA. In the back of the bubble, the two DNA strands
reassociate to form DNA duplex.
5. Termination of RNA formation: Specially in prokaryotes, termination of
transcription or RNA formation is brought about by certain termination signals on
DNA The termination may be of two types:
24
Rho Independent Terminations: This types of RNA synthesis termination is due to
specific sequences on DNA. A typical hairpin like structure is formed on DNA
template due to which the movement of RNA polymerase on the template is
obstructed. The hairpin structure is formed due to inverted repeat sequences on
DNA. The hairpin or stem-loop is followed by a run of adenine residues in DNA
and U residues in mRNA in the downstream.
Rho Dependent Termination: This type of termination is due to presence of special
factor, which is called Rho factor. It has a mol. Wt. Of 60,000 and is not a part of
RNA polymerase. After the synthesis of mRNA on template DNA is completed, it
attaches with the template. The site ofr its attachment is characterized by 5’CAATCAA-3’. The actual and precise mechanism of the function of factor is not
known.
In eukaryotes, the termination process is more completed. The termination sites
similar to prokaryotes are also operative in eukaryotes but these sites are believed to be
present away up to 1 kb from the site of the 3’end of the mRNA. AAUAAA sequence on
mRNA and ‘snurp’ are assumed to play important role in termination of the process in
eukaryotes.
Maturation of mRNA from hnRNA in eukaryotes: The mature mRNA
molecules very often have much lower molecular wt. And base sequence length in
comparison to the DNA segment from which it is transcribed. The primary RNA
transcript of a structural gene is called pre-mRNA. It is also known as the heterogeneous
RNA, high molecular wt. RNA. It is much bigger in size than mRNA.
The later is formed by splicing of hnRNA followed by some other modifications.
The heteronuclear mRNA undergoes following modifications: Addition of Cap (m7G) and Tail (Poly A) for mRNA in Eukaryotes
1. Addition of methylated cap at the 5’ end
The initial RNA transcript, derived from genes coding for proteins, gets modified
so that its 5’ end gains a methylated guanine and its 3’ end is polyadenylated. Capping at
5’ end occurs rapidly after the start of transcription and much before completion of
transcription. Transcription starts with a nucleoside triphosphate, and a 5’ triphosphate
group is retained at this first position. The initial sequence at 5’ end of hnRNA is
therefore 5’ pppApNpNp…3’. To the 5’ end is added a terminal G with the help of an
enzyme guanyl transferase as follow:
5’Gppp+5’pppApNpNp 5’Gppp5’ApNpNp+pp+p
The new G residue is in the reverse orientation with respect to all other
nucleotides and undergoes methylation at its 7th position. The cap with a single methyl
group at this terminal guanine residue is found in unicellular eukaryotes and described as
cap0, but in most eukaryotes, methyl group may also be present on the penultimate base
25
at 2’ position of sugar moiety, so that nucleotides, it is now described as cap1. Removal
of cap leads to loss of translation activity due to loss of the formation of mRNA-ribosome
complex. It suggests that the ‘cap’ helps in recognition of ribosome. Only in some
eukaryotic nRNAs, caps may be absent and may not be required for translation.
2.
Polyadenylation and the generation of 3’ end in eukaryotes
The 3’end of n RNA is generated in two steps (i) Nuclease activity cuts the transcript at
an appropriate location. (ii) Poly (A) is added to the newly generated end by an enzyme,
poly (A) polymerase (PAP), utilizing ATP as a substrate. Ordinarily 30% of hnRNA and
70% of mRNA are polyadenylated. In addition to AAUAAA, there are following
consensus sequences, that are involved in polyadenylation: (i) a G-U rich element is
present downstream to the site of cleavage, and is important for efficient processing for
polyadenylation: (ii) a G-A sequence immediately5’ of the cleavage site;(iii) consensus
upstream element situated 5’ of a poly A signal or AAUAAA.
3. Splicing of RNA parts coded by introns: Self splicing is a very common
phenomenon found in hnRNA. In this process generally those parts of the RNA
are removed or spliced out, which have been transcribed from intron regions of
the template DNA. These regions have short consensus sequences which pair to
formstem-loop like secondary structure. These are helpful in self or autosplicing.
Stem-loop like structures were observed for the first time in the hnRNA of
Tetrahymena thermophila.
4. Editing of RNA: Theoretically, the base sequence of a mRNA is just
complementary to the base sequence of the segment of the template DNA from
which it is transcribed. However, in many cases, the base sequence of mRNA has
been found to be changed after transcription at the level of RNA. This process of
change in the base sequence of mRNA is known as RNA editing. It may be
confined to a single base or may affect the entire mRNA.
3.11 RNA SPLICING.
In Eukaryotes, the RNA transcribed from DNA almost invariably undergoes RNA
splicing to yield mature RNA sequences. It involves removal of sequences mainly
corresponding to introns in split genes. The mechanisms available for this purpose
include the following:
I. Self splicing of fungal mitochondrial and other group I introns;
II. Splicing of higher eukaryotic nuclear introns, through the formation of
spliceosomes at intron-exon junction;
III. Self splicing of mitochondrial group II introns through lariat formation without
assistance from any proteins or spliceosomes;
26
IV. Splicing of yeast tRNA precursor molecules by cleavage due to endonuclease
followed by fusion due endonuclease followed by fusion due to ligase.
Self-splicing of RNA molecules involving group I introns, found in rRNA genes
of Tetrahymena and Physarum nuclei, in fungal mitochondria and in phage T4, takes
place through two transesterification relations. Group I introns are characterized by (i) the
absence of conserved sequences at splicing junction, and by (ii) the presence of short
conserved consensus sequences internally. In the first transesterification, the 5’ splice site
is cleaved. In the second transesterification step, the 3’ splice site is cleaved. The excised
IVS or introns can form a circle by cyclization reaction and these circles can again
regenerate linear molecules due to autocatalysis.
In case of viroids and virusoids= satellite RNA, a consensus sequence forms a
‘hammerhead’
SPLICING OF hnRNA OF HIGHER EUKARYOTES THROUGH SPLICEOSOMES.
Splicing of major class of GU-AG introns. Splicing of introns sequences of
eukaryotic hnRNA involves a well defined multi-step pathway. Small nuclear
ribonucleoprotein particles and about 50 protein factors are essential for the formation of
an active spliceosomes, in which introns excision proceeds in two successive
transesterification reactons. Each step of the splicing reaction is mediated by a number of
snRNA associated proteins and non-snRNP splicing factors. The major components of
the splicing machinery in mammals, which may differ in details worked out in budding
yeast. (i)snRNPs consisting of snRNAs and common Sm proteins, (ii) SR family of
splicing proteins; (iii) polypyrimidine tract-binding proteins; (iv)branh-site binding
proteins; (v)hnRNP proteins; (vi) snRNP associated non-snRNP proteins; (vii) Some
other non-snRNP splicing factors.
THE SPLISOSOME ASSEMBLY: The splicing reaction involves the formation of
spliceosome. For spliceosome assembly. The splicing reation involves the formation of
spliceosome. For spliceosome associate with pre-nRNA using large number of essential
protein factor.
1) SR proteins: They are called SR proteins serine, abbreviated as S and arginine
abbreviated as R; the N-terminal region consists of an RNA recognition motif or
RRm. SR proteins are among the first components that interact with pre-nRNA,
thus committing pre-nRNA to the splicing pathway.
2) Polypyrimidine tractbinding proteins; They bind a polypyrimidine tract and
play an important role in spliceosome assembly. Following steps are involved in
spliceosome assembly (i) The first specific stage of spliceosome assembly is the
binding of U1 sn RNP, which requires interaction of pre-mRNA with SR.
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Splicing of UA rich introns in plants. Plant introns have UA-rich elements spread
throughout their length. These UA-rich elements help in recognition of 5’ and 3’ splice
sites.
Following interactions are involved in plant intron recognition:
(i)
U1 snRNP binds to 5’ splice site,
(ii)
U-tract binding factors bind the U-rich sequence preceeding the 3’
splice site,
(iii) UA-island binding proteins bind to UA-rich elements of intron and
(iv)
exon sequence element binding proteins associate with AG elements in
the adjacent exon. In plants, splice site selection is primarily defined
by UA- rich sequences within the intron. Consequently UA richness in
plant introns is essential for efficient splicing and for 5’ and 3’ splice
site recognition. However, the monocot splicing machinery is more
permissive than the dicot recognition machinery.
Formation of lariat during splicing. Nuclear splicing, where spliceosome is formed,
involves formation of a lariat structure. This occurs in two stages: (i) In the first stage, a
cut is made at the left end of the intron, releasing a separate RNA molecule with left exon
and a right RNA molecule with intron and the right exons.
The left RNA molecule is linear, but the right intron-exon molecule is not linerar The 5’
terminus at the left end of intron-exon molecule gets liked by a 5’-2’ bond to the A of the
sequence CUGAC located-30 bases upstream of the right end of the intron. This linkage
generates a lariat. (ii) In the second stage, cutting at the right splicing junction releases a
free intron in lariat form, and the left exon is ligated to the right exon. The lariat is
debranched to give a linear excised intron, which is rapidly degraded.
Splicing of group II introns
The group II introns resemble introns of nuclear pre-mRNA or hnRNA of higher
eukaryotes and are excised as lariats like those produced in nuclear pre-mRNA introns of
higher eukaryotes. They have consensus sequences at the splicing junctions, GT and AP y
and a branch sequence resembling TACTAAC box. In group II introns, higherly
conserved sequence elements juxtapose splice sites and branch sites by intramolecular
base pairing interactions, in the nuclear pre-mRNA introns, the same functions are
compensated by intermolecular interactions between the RNA substrate and the RNA
moieties of snRNPs. This self splicing reaction of group II introns may be regarded as an
intermediate step between RNA mediated self splicing in group I introns and protein
dependent RNA splicing of the introns of nuclear pre-mRNA of higher eukaryotes.
Yeast tRNA splicing by cutting and rejoining
About 40 genes of appro. 400 genes for yeast nuclear rRNAs are interrupted, each with a
single intron, located one nucleotide away from 3’ end of anticodon. The splicing
requires following two steps (i)phosphodiester bond cleavage by an endonuclease; this
does not require ATP; and (ii) ligation reaction, which requires ATP and involves bond
formation with the help of RNA ligase;
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Constitutive vs alternative splicing
In the earlier sections of this chapter, we have discussed the various mechanisms
involved in RNA splicing. Generally, the pre-mRNAs undergo processing, so that the
non-coding intervening sequences, i.e. introns, are excised, and exons correctly ligated.
When this splicing takes place in all cells, without any variation, it is described as
‘constitutive splicing’ in contrast to ‘alternative splicing’, which differs in time and
space. Thus ‘alternative splicing’ is an adjunct to the regulation by promoter activity,
gene rearrangements and by the occurrence of multigene families. In alternative splicing,
splice sites that are selected for splicing in some circumstances are completely by-passed
by the splicing machinery under other circumstances. The latter may lead to (i) insertion
of a peptide segment or, (ii) functionally different product or (iii) no functional product.
Ribozymes (RNA splicing, DNA Cleavage and RNA Amplification)
RNA molecules can cut, splice and assemble themselves without any outside help, thus
extending the range of chemistry of enzymes. These RNA molecules working as
enzymes, were called ribozymes. It has also been shown that enzymes can also be
synthesized chemically, and are then described as chemzymes.
3.12 RIBOSOME STRUCTURE, r-RNA AND BIOSYNTHESIS
Ribosomes are round, granular and membraneless cell organelle which are
chemically nucleoprotein and found enormously in all the prokaryotic and eukaryotic
cells. These were discovered first by Claude in 1943 and were named as ‘microsomes’.
Robinson and Brown isolated ribosomes from root cells of broad bean. Palade coined the
term ‘ribosomes’ and isolated it from animal cells. After his name ribosomes are laso
called ‘Palade granules.’ Ribosomes may be defined as “The smallest known electron
microscopic, ribonucleoprotein particles attached the on RER or floating freely in the
cytoplasm and are the sites of protein biosynthesis”.
OCCURRENCE: Ribosomes are generally found in all known prokaryotic and
eukaryotic organisms except mature RBCs. In prokaryotes these are found only in free
form in the cytoplasm while in eukaryotes these are found both in the cytoplasm and on
the surface of RER. The former is called cytoplasmic and later is called bound form of
ribosomes. These may also be found on the surface of nuclear membrane. Some
organelles like mitochondria and chloroplast contain ribosomes in the matrix. These are
called organellar ribosomes and reffered as ‘mitoribosomes’ and ‘plastidoribosomes’.
The ribosomes found on the surface of RER is bound with the membrane with special
proteins called ribo-phorines.
Number: The number of ribosomes in a cell depends on the content of RNA. These are
more in number in metabolically active cells like plasma cells, livercells, nissl’s granules
of nerve cells, meristemati c cells, cancer cells, endocrine cells etc. In a cell of E.coli, the
number of ribosomes vary from 10,000-20,000.
29
Structure: Ribosomes are globular structures having the diameter of 150-250 Å. Each
ribosome is made up of two subunits one is smaller and another is larger in size. The later
in dome shaped and is covered by cap like smaller unit. In 70S type of ribosome the
larger and smaller units are 50S and 30S type. On the other hand, in 80S type, these are
of 60S and 40S type, respectively. The two subunits of ribosomes are freely distributed in
the cytoplasm. The two subunits unite to form a complete ribosome. Likewise, the two
subunits dissociate with each other when the concentration of Mg++ ion decreases in the
cytoplasm.
During protein synthesis many ribosomes become attached with mRNA forming a
peculiar structure called polyribosome.
Ultrastructure of Ribosomes
The last point about the ultra structure of ribosomes has not been said till date.
The credit of giving the present knowledge of the ultrastructure of ribosomes goes to
Nauninga. According to him, the size of larger (50S) subunit of 70S type of ribosome is
160 to 180 Å which is pentagonal in shape. This unit has a groove of 40-60 Å size in
which the smaller subunit is attached during association. The smaller subunit has a
platform, cleft, head, base and also a binding site for nRNA. The smaller unit of 70S and
80S type of ribosomes does not have a definite shape. Florendo in 1968reported a pore
like transparent area on the larger unit of 50S of 70S ribosomes.
In between two subunits of ribosomes, mRNA is found. t-RNA molecule is found
in the side of nRNA. The new-formed polypeptide chain being synthesized on the
ribosome mRNA complex has been seen passing through the transparent pore on the
larger unit.
It also has a protuberance, a ridge and stalk. Two binding sites, peptidyl and
amino acyl sites are found on the larger units.
The 50S and 30S subunits have been reported to have the molecular weight of 1.8
X 10 Daltons and 0.9 X 106 Daltson, respectively. It must be noted here that size and
type of ribosomes and their subunit are determined on the basis of their sedimentation
coefficient.
6
Types of Ribosomes
On the basis of their sedimentation coefficient, ribosomes have been classified
into two main types:
70S ribosomes:
These are found in prokaryotes and mitochondria and plastids of eukaryotes. Each 70S
ribosome is about 200-290 Å in size and 2.7 X 106 Daltons in its mol. Weight. It
consists of two subunits of 50S and 30S size. Both of these units are made up of
ribosomal RNA and ribosomal proteins. The 50S subunits again consist of 23S
30
and 5S rRNA and 30 types of proteins. Similarly, the smaller unit is made up of
16S type of rRNA and 20 types of proteins.
80S ribosomes:
These are the characteristic of eukaryotic cells and found. In their cytoplasm. It consists
of two subunits. The size of larger subunits is 60S and that of smaller subunit is
40S. It is also made up rRNA and proteins. The 60S subunits consists of 28S
rRNA, 5.8 S rRNA and 5S rRNA and about 50 types of proteins. The smaller
subunits is similarly made up of 18S rRNA and 30 different proteins.
Polyribosomes or Polysomes: When many ribosomes are attached to same mRNA
strand, it is called polyribosomes or polysome.
It is formed when a simple protein is required in high quantity. The number of
ribosomes in a polysome depends on the length of mRNA. The distance between two
adjacent ribosome is about 90 nucleotids.
Origin of ribosomes:
We have studied that ribosomes are solely made up of rRNA and proteins. The former is
formed inside the nucleus and the later is produced in the cytoplasm. Therefore, these are
partly nuclear and partly cytoplasmic is nature. However, in prokaryotes, since there in
no nucleus, ribosomes are totally cytoplasmic in nature.
Functions of Ribosomes.
I. Ribomes are called factories of proteins or engineers of the cell because these are
the side of protein synthesis.
II. Sometimes rRNA of ribosomes has been found to function as enzymes controlling
the cellular functions. These are called ribozymes.
III. The process of translation of genetic language into the language of enzymes or
protein take place at ribosomes. It takes place with the help of nRNA, which, is
produced during transcription of nuclear DNA.
IV. In general the ribosomes bound on RER synthesise enzymes for extracellular use
e.g., pancreatic cells, chief cells of gastric glands, liver cells etc.
V. Ribosomes temporarily store proteins.
VI. Ribosomes keep the mRNA molecules functionally alive.
31
RIBOSOMAL RNA or rRNA
The RNA, which is found in ribosomes, is called ribosomal RNA. Ribosomes are
chemically ribonucleoprotein as they consist of RNA and proteins. It is known as soluble
RNA. Its quantity in a cell is much higher than that of mRNA and tRNA. It constitutes
about 80% of total RNA.
On the basis of their sedimentation coefficient or rate of sedimentation, rRNA
molecules may be classified into following categories:
1) 28S-rRNA: It has molecular weight more than 10,00000. Sedimentation
coefficient is between 21S and 29S. It is found in 60S subunit of eukaryotic
ribosomes.
2) 18s-rRNA: It molecular weight is less than a millions. Sedimentaion varies
between 12S to 18S. It is found in 40S subunit of ribosomes.
3) 5S-rRNA: It has much lower molecular weight and is found in 30S unit of
ribosomes.
Structure of rRNA
Ribosomal RNA molecules are single stranded but in the solution of high ionic
concentration, irregular spiral coiling of rRNA is formed. As the ionic concentration of
the solution increases, the degree of irregular coiling of rRNA also increases. In this
coiling the intramolecular bases show base pairing. The pairing is normal as A pairs with
U and C pairs with G.
Function of rRNA
The main function of rRNA may be summarized as below:
In many viruses specially in plant viruses, RNA function as genetic material and carry
genetic information from generation to generation.
Different RNAs function as structural component of a cells
mRNA are the site of protein synthesis where polymerization of amino acids takes
place through peptide bond formation between amino acid molecules during
translation process.
A tRNA molecule has anticodon site and has the capacity of attachment with the
complementary condon on mRNA. tRNAs further carry activated amino acids to
the mRNA and catalyse peptide bond formation between two amino acid
molecules.
Ribosomal RNA(rRNA) is the constituent unit of ribosomes.
32
Check Your Progress – (2)
Note: 1. write your answers in the space given.
2. Compare your answers with that given at the end of the unit.
I Write Short Notes on:
1. Nucleosome Model
2. Watson & Cricks Model of DNA.
3. Transcription
4. RNA Polymerase Enzyme.
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------II. Name these:
a. Initiation Condon -------------------------------------------------------------------b. Termination Codon ------------------------------------------------------------------
3.13 MECHANISM OF TRANSLATION
33
The synthesis of protein from mRNA involves translation of the language of nucleic
acids into language of proteins. For initiation and elongation of a polypeptide, the
formation of aminoacyl transfer RNAs is a prerequisite,
Formation of Aminoacyl rRNA
a) Activation of amino acid
This reaction is brought about by the binding about by the binding of an amino
acid with ATP and is mediated by specific activating enzymes known as amino acyl
tRNA syntehtases or aaRs. As a result of this reaction between amino acid and
adenosine triphosphate, mediated by specific enzyme, a complex (amino acyl-AMPenzyme complex) is formed. Amino acyl-RNA synthetases are specific with respect to
amino acids. For different amino acids, different enzymes would be required.
(Enzl)
Aa1 + ATP
(aa1-AMP) Enz1 + PP
b) The transfer of amino acid to rRNA
The amino acyl-AMP-enzyme complex, formed during the step outlined above,
reacts with a particular tRNA and transfers the amino acid to the tRNA. A particular
amino acid would require a particular enzyme and a particular species of tRNA. This
would mean that for 20 amino acids, at least 20 different enzymes and also atleast 20
different t-RNA species would be required.
(aa1-AMP) Enz1 + t-RNA1
aa1- t-RNA1 + AMP + Enz1
Initiation of Polypeptide
The initiation of polypeptide chain is always brought about by the amino acid
methionine, which is regularly coded by the condon AUG,
In eukaryotes, formylation of initiating methionine is not brought about due to the
absence of tRNAfmet in plants and animals. Initiation in higher organisms will therefore,
take place without formylation.
Initiation in eukaryotes
Initiation of polypeptide chain in eukaryotes is similar to that is prokaryotes,
except the following minor differences. (i) In eukaryotes there are more initiation factors.
They are named by putting a prefix ‘e’ to signify their eukaryotic origin. These factors
are eIF1, eIF2, eIF3, eIF4A, eIF4B, eIF4C, eIF4D, eIF4F, eIF5 and eIF6. (ii) In
eukaryotes, formylation of methionine does not take place. (iii) In eukaryotes, smaller
subunit associates with initiator tRNAimet, without the help of mRNA, while in
prokaryotes, generally the 30S-mRNA complex is first formed which then associates with
f-met-tRNAfmet.
Kozak’s ribosome scanning hypothesis for translation in eukaryotes
In 1983, Marilyn Kozak proposed a hypothesis for initiation of translation by
eukaryotic ribosome. According to this hypothesis, 40S smaller subunit of a eukaryotic
ribosome with its associated met-tRNA moves down the mRNA from 5’ end, until it
encounters the first AUG. At this point, the 60S subunits join and the translation begins.
34
The 80S ribosome, after reaching termination, releases protein and dissociates in two
subunits.
Elognation of Polypeptide
The following three steps are important in the elongation process.
Binding of AA-rRNA at site ‘A’of ribosome (classical vs hybrid state models for
translation)
In earlier classical model, each ribosome had two cavities, in which tRNA could
be inserted. These were ‘P’ site and ‘A’ site. However, later a third cavity was suggested.
F-met-tRNAfmef comes on ‘E’ site, to make ‘A’ site available for the next amino acyl
tRNA (AA-rRNA).
35
Various steps of protein synthesis: (A-B) Attachment of tRNA-fmet-mRNA
and smaller unit of ribosome, (C) Union of subunits of ribosomes, (D) Union of second
Another site called ‘R’ sites located on smaller subunit of ribosome, was
proposed ‘R’ site plays a role in the improvement of accuracy of translation. The
aminoacyl rRNA first binds to R site involving codon-anticodon pairing.
Later aminoacyl rRNA is flipped to ‘A’ site using energy from GTP molecule.
During this flipping, tRNA is held only by condon-anticodon pairing. After formation of
70S initiation complex, the next amino acyl tRNA enters ‘A’ site. Elongation factors EFTu and EF-Ts participate. The elongation factor EF-Tu first combines with GTP and
changes to an active binary complex, which binds with aa-tRNA, to form a ternary
complex.
EF-Tu-Ts+GTP
EF+Tu.GTP+EF-Ts
Binary Complex
EF-Tu.GTP+aa-tRNA
EF-Tu..GTP.aa-tRNA
(Ternary Complex)
(Hybrid states Models), it has been shown that above ternary complex actually
binds in an A/P hybrid state, the anticodon binding to the A-site of the 30S subunit and
the CCA end binding to the P-site of the 50S subunit as well as to the 30S subunit. The
‘P’ site is already occupied by f-met. tRNAfmet or by a peptidyl tRNA. Following the
GTP hydrolysis, EF-Tu.GDP+P are released from the ternary complex, permitting
movement of CCA end of aa-tRNA into A site of the large 50S subunit. EF-Ts now
displaces GDP in the EF-Tu.GDP binary complex and associates with EF-Tu, so that
GTP can again associate with EF-Tu to start another cycle for the binding of aa-tRNA.
Formation of peptide bond
This is a catalytic reaction during which a peptide bond is formed between the
free carboxyl group of the peptidyl tRNA at the ‘P’ site and the free amino group present
with amino acyl tRNA, which is available at the A site. The 50S rRNA have peptidyl
transferase activity, sothat the ribosome is described as a ribozyme.
According to this displacement model, peotidyl chain remains in a constant
position relative to ribosome, while the tRNA moves during the peptide reaction. After
the formation of peptide bond, the tRNA at ‘P’ site is deacylated and the tRNA at ‘A’ site
now carries the polypeptide.
36
Translocation of peptidyl tRNA
From ‘A’ to ‘P’ site.
The peptidyl tRNA present at ‘A’ site is now Translocated to ‘P’ site. For
translocation of peptidyl tRNA from ‘A’ site to P site, there are two models available: (i)
According to two sites model, deacylated tRNA is liberated from ‘P’ site, and with the
help of one GTP molecule and an elongation factor EF-G, the peptidyl tRNA is
translocated from ‘A’ to ’P’ site. Thus according to this model, tRNA is either entirely in
the A site or entirely in the P Site.
Various stages of protein synthesis: (E Formation of peptide bond and (F)
Treansference peptidyle tRNA from A-site to P-site , (G) Attachment of third amino
acid (serine) at A- site (H) Union of RF factor at A-site after the completion of
formation of peoptide bonds in between amino acids.
37
The requirement of EF-G and GTP for translocation was revealed by the use of
antibiotic. The elongation factor EF-G binds to ribosome and is released on hydrolysis of
GTP, which is a ribosomal function. EF-G and EF-Tu cannot bind to ribosome
simultaneously, so that the entry of a fresh aa-tRNA on ‘A’ site and the translocation of
peptidyl tRNA from ‘A’ to ‘P’ site has to follow each other and cannot occur
simultaneously.
In eukaryotes, the elongation factor needed for translocation is called eEF-2, for
the formation of one peptide bond. One ATP molecule and two GTP molecules (one for
transfer of aa-tRNA to ‘A’ site and the other for translocation of peptidyl tRNA from ‘A’
to ‘P’ site) are required.
Termination of Polypeptide
Terminations in mRNA with stop condon
Termination of the polypeptide chain is brought about by the presence of any one
of the three combination condons, namely UAA,UAG and UGA. These termination
condons are recognized by one of the two release factors RF1 and RF2. The release
factors to act on ‘A’ site, since suppressor rRNA capable of recognizing by entry at ‘A’
site. A third release factor RF3 stimulate the action of RF1 and RF2 in a GTP-dependent
and condon independent manner GTP molecule is hydrolysed during release of a
polypeptide, when RF3 stimulates RF1 amd RF2. For release reaction, the polypeptidyl
tRNA must be present on ‘P’ site and the release factors help in splitting of the carboxyl
group between the polypeptide and the last tRNA carrying this chain. Polypeptide is thus
released and the ribosome dissociates into two subunits with the help of ribosome release
factor or RRF.
It has been shown that the translation apparatus in chloroplasts and mitochondria
differs from that in cytoplasm in eukaryotes in the following respects. (i) Ribosomes in
these organelles are smaller in size than these in cytoplasm. (ii) The tRNAs are specific
and differ, the number of tRNAs in mitochondria being 22 as against 55 in cytoplasm.
(iii) Initiation of translation takes place by formyl-methionyl tRNA both in chloroplasts
and mitochondria, although no formylation takes place in cytoplasm. (iv) Translation in
chloroplasts and mitochondria can be inhibited by chloramphenicol.
38
Modification of Folding of Released Polypeptide
Modification of released polypeptide
After translation, the released polypeptide is modified in various ways.
Due to the action of certain other enzymes, exo-amino-peptidases, amino acids
may be removed from either the N-terminal end or the C-terminal end or
both.
The polypeptide chain singly or in association with other chains also folds into a
tertiary structure. This problem of protein folding is sometimes described
as ‘Second Half of the Genetic Code’.
Check Your Progress – (3)
Note: 1. Write your answer in the space given below.
2. Check your answer with the one at the end of the unit.
Eassy Type Question
Q.1 What do you mean by Transcription and Translation?
Q.2 Describe the role of ribosomes in protein synthesis?
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
3.14. SUMMARY
The functional significance of nucleus can be understood through the process ofReplication, Damage and repair ,Transcription factors splicing ,m-RNA transport
process, And mechanism of translation
Nuclear Pore: The nuclear pore is a large complex structure, 30 times large than eukaryotic ribosome .
The four separate elements are
1. The scaffold
2. The transporter or hub.
3. Thick filaments
4. Basket
39



The scaffold allows diffusion of proteins and metabolise between the nucleus and
the cytoplasm.
Transporter helps in passing of RNA from the nucleus to the cutoplasm.
Basket plays an important role in RNA export
Nucleolus:  It is a conspicuous structure during interphase nucleus.
 It is associated with nucleolous organizing region of chromosome (NOR)
 It consist of RNA, acidic dyes, basic dyes, phospholipids and alkaline
phosphatase
 The small nucleolar RNA, U3, U7, U8, U14 etc. are found in nucleolus.
Nuleosome Organization:The length of DNA exceeds the dimensions of the nucleus, which contains it.
Therefore, it must be compressed lightly to fit into the space available.
The density of DNA is about 100 mg/ml in eukaryotic Nucleus.
This packaging is facilated by histone proteins known as H, H2A, H2B, H3 and H4.
A variations of H, as H5 has also been described. RNA polymerase is non-histone
protein. The diameter of nucleosome is 11 nm and height is 6 nm. The length of DNA is
around 70 nm which is equivalent to 200 base pairs. Variation- in fungus it is 150 bpl in
seaurchin sperm it is 260 bp. The core is formed by 8 histone protein H3, H2B, H3 ,2H4
in pairs.it actually contains 165 pairs of DNA and copy per H1 is binder protein. It is
DNA which is wrapped around a solid core/nucleosome.(H3)2 (H4)2 tetramer makes a
central ‘Kernel.’
Solenoid Model
 11nm wide fibre of nucleosomes gets coiled upon itself to form – 30 nm wide
helix with five/six nucleosome per helix.
 The center to center distance remains 10 nm.
 This 30 nm structure is called a solenoid that means a wire coiled on a central
axis.
 It is the H1 molecules which aggregates by cross linking to form polymers. And
thus control the formation of solenoid
DNA Structure & Forms.







The Watson & Cricks model of DNA, actually describes B form of DNA.
Two Polynucleolide chains run antiparallely.
Purines and Pyremedines are on inside while phosphate and deoxyribose units are
on outside.
The diameter of Helix is 20 A0, Adjacent basee are separted by 3.4 A0 along the
helix and are related by a rotation of 360.
The helical structure repeats after ten residues at interval of 34 A0.
G = C & A = T are hydrogen bonds.
G always pairs with C & A with T
40
Replication of DNA: -Basic Rules of Replication
 Replication is a semi conservative process.
 Replication has direction. It could be unidirectional or bi-directional fork.
 Unidirectional –
 Bidirectional - In E.coli & Eukaryotic Chromosome.
 Replication starts at a unique point on bacterial and viral chromosome.
 Replication of both strands proceed by the addition of nucleotide monomers in the
5’ 3’directional
 Replication starts in short discontinuous pulses.
 Replication at the level of short fragments in initiated by the production of a short
segment of RNA to serve as a primer for DNA polymerase.
Replication of viral DNA is circular but progeny has linear DNA,
Replication time is 30 Minutes in E.Coli.
Enzymes of Replication
 RNA Polymerase or Primerases
 DNA Polymerase
 Nucleases
(Endonucleases & Exonucleases)
 DNA Ligases
 Restriction Enzymes
 Swivelases
 Unwinding enzymes and proteins
Steps: Binding of Unwinding proteins
Initiation of chain
Elongation
Termination
Binding or joining by ligases.
Damage and Repair.
 The changes in DNA leading to damage are broadly divided into two general
classes
1. Single Base Changes
2. Structural Distortions
 Systems of damage repair are
1. Direct Repair
2. Excision Repair
3. Mismatch Repair
 Direct Repair- Involves reversal of DNA Damage
 The enzymes involved are
DNA Photolyase Reparis cyclobutane pyrimidine divers (CPD) included by U.V.
6-4 photoproduct photolyase deals with the DNA damage involving formation of
6-4 photoproduct due to U.V
Spore photporduct lyase repairs the lesion caused by U.V in B. Subtilis
41
O6- methylguanine DNA methyl transferase MAMT is a common enzymes found
in all species tested.
Excision Repair
 It is of two types on the basis of the nature of excised product
1. Base excision repairs (BER) when free bases are excised- enzyme is DNA
glycosylase
OR
Nucleotide excision repair (NER) When one or more nucleatide are excised –
Pudonuclease repair

Involves correction of mismatches or pairing between bases which ae not
complementary.
 Mismatches may arise either
During replication of
Due to base conversion and are corrected by a process described as error
correction during DNA replication.
Transcription:  Transcription is the synthesis of mRNA and stable RNA molecules from a
DNA Template by RNA Polymerase, using ribonucleotide triphosphates as
precursor.
 Bacterial cells contain only on RNA Polymerase where as eukaryotes have at
least three viz. RNA Poly. I, II & III.
 The three stages of transcription are
Initiation
Elongation
Termination
 Promoters consist of conserved seq. necessary for the initiation of
transcription.
 There are many different types of promoters.
 The promoter sites for RNA polymerase I and II are located before the start
site for transcription.
 RNA Polymerase III recognizes sites within the gene itself.
In eukaryotes the promoters sites for RNA Polymerases I and II are located at the
5’ end of the gene but the polymerase III promoter lies with in the gene.
RNA Polymerase II promoters have TATA box with consensus seq. of TATAAA.
Main features of eukaryotic Transcription
The template for transcription is a completes of DNA and Protein that has beaded
appearance.
Eukaryotic RNA synthesis starts at precise promoter seq. As a result, genes that is
very actively transcribed and show “fern leaf” or “Christmas Tree”
configuration in tRNA and lampbrush chromosome.
42
At any one time, only a very small fraction of the total chromatin is transcribed.
The nascent RNA gets associated with protein as it is being transcribed,
producing ribonucleus protein particles (RNP).
In eukaryotes the nuclear envelope introduces as barrier between transcription and
protein synthesis
Eukaryotic mRNA
Eukaryotic mRNA is metabolically stable as compared to mRNA of pro. As they
have comparatively longer half-life.
It is monocistronic.
The 5’ end of blocked by 7-methyl –G.]
The 3’ end of euk. mRNA ends with A poly A segment./
Eukaryotic genes. Frequently contain insertions of non-coding DNA.
Heterogeneous nuclear RNAs are mRNA precursors containing intervening
sequences.
Eukaryotic mRNA are associated with proteins
RNA Splicing.








The mRNA are transcribed in nucleus in eukaryotes and is known as
heterogeneous (hnRNA) nuclear RNA.
These are destined to produce mRNA by undergoing processing as : Modification of 5’ end by capping.
Modification of 3’ end by tailing.
Splicing out of interrupted genes.
Cleavage and polyadenylation usually preceedes RNA splicing.
Splicing : The mechanism of splicing includes the following
1. Self splicing by Gr I introns.
2. Splicing of hnRNA and higher eukaryotes through splicesomes
3. Splicing of group II introns.
4. Yeast tRNA splicing by cutting and rejoining.
Translation: Main steps of translation are: 1. Activation of amino acids
2. Transfer to t-RNA.
3. Initiation
4. Elongation
5. Termination
I. Activation of amino acids
It includes screening of amino acids
There activation at carboxyl gr.
Enzyme is amino acyl tRNA synthetase
43
II. Transfer of a.a to t-RNA
t-RNA are specific and named after amino acids
Ester bonds are formed between amino acids & t-RNA
III Initiation
 In 1975 Anderson reported IF- MP, IF –M1, IF-M2A, IF-M3.
 Formylation of methionine does not occur in rule.
 Reaction is catalyzed by transformylase enzymes
 Initiation complex is formed by mRNA + 40S ribonucleus S.U + tRNA +
GTP + 3 Initiation factors.
 Here, nmet-tRNA binds first to 40s sub unit. met RNA.
 The P and A sites are located on 70s. subunit. Met RNA binds to the P site.
 AU other tRNA as first build to A site, then shift to P site.
 60s& 40s sub units join to form 80s subunit elongation of polypeptide chain.
 EF1& EF2 are required for elongation of polypeptide chair.
 In the presence of elongation factor, amino acyl tRNA bends to A site (by
using GTP) on ribosome. Thus ternary complex is formed.
Peptide bond formation:  First amino acid is now united by peptide bond formation with second amino
acids.
 Peptide bond formation does not require external energy source.
 This reaction is catalyzed by peptide transferase complex located in large
subunit
 Alpha Amino group of one amino acid is bonded to the alpha- carboxyl of
other with elimination of H2O.
Translocation : The movement of ribosome relative to mRNA is called translocation.
It occurs in 53’ direction.
Termination.
 RNA polymerase recognizes termination signal UAA,UAG & UGA.
 The termination condons provides signals to the ribosomes for attachment of
release factors. RF-1, RF-2, RF-3.
3.15.
CHECK YOUR PROGRESS –
KEY (1)
1. rRNA
2. Ribosomes
3. Kornberg and Thomas
4. Picogram
5. Heterochromatin.
44
3.16. ASSIGNMENTS/ ACTIVITY:
Q.1 Prepare a model of Eukaryotic Nucleus.
Q.2 Prepare a Solenoid & Nucleosome model of chromosome?
Q.3 Prepare a model of DNA, given by Watson & Crick?
Q.4 Explain the process of Replication in Eukaryotes?
Q.5 Write Short Notes On:a. DNA Damage and Repair.
b. Heteronuclear m-RNA Splicing.
c. Transcription.
3.17. REFERENCE:-
1. E.D.P De Roberties & E.M.F. De Robertis Jr. Cell and Molecular Biology.
2. David Friefielder
-
Molecular Biology.
3. P.K. Gupta
-
Cell and Molecular Biology.
4. Stanier
-
Microbiology
5. Benjamin Lewin
-
Genes.
6. C.B. Powar
-
Cell Biology.
45