Sample Theory - CSIR NET LS (Post Transcription Modification) (UNIT-3) PDF

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Life Sciences
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Life Sciences-LS (Sample Theory)
1. GENE REGULATION
Gene regulation is a label for the cellular processes that control the rate and manner of gene
expression. A complex set of interactions between genes, RNA molecules, proteins (including transcription
factors) and other components of the expression system determine when and where specific genes are
activated and the amount of protein or RNA product produced.
Some genes are expressed continuously, as they produce proteins involved in basic metabolic
functions; some genes are expressed as part of the process of cell differentiation; and some genes are
expressed as a result of cell differentiation.
Mechanisms of Gene Regulation Include
 Regulating the rate of transcription. This is the most economical method of regulation.
 Regulating the processing of RNA molecules, including alternative splicing to produce
more than one protein product from a single gene.
 Regulating the stability of mRNA molecules.
 Regulating the rate of translation.
OPERON MODEL
The basic concept about how gene regulate occurs at the level of transcription in bacteria was
provided by the classical model called operon model (formulated by Jacob and Monod in 1961). Operon,
genetic regulatory system found in bacteria and their viruses in which genes coding for functionally
related proteins are clustered along the DNA. This feature allows protein synthesis to be controlled
coordinately in response to the needs of the cell. By providing the means to produce proteins only when
and where they are required, the operon allows the cell to conserve energy (which is an important part
of an organism's life strategy). A typical operon consists of a group of structural genes that code for
enzymes involved in a metabolic pathway, such as the biosynthesis of an amino acid. To achieve this
aspect, some bacterial genes are located near together, but there is a specific promoter for each of
them; this is called gene clustering.
Many of the genes in E. coli are expressed constitutively; that is, they are always turned "on".
Others, however, are active only when their products are needed by the cell, so their expression must
be regulated.
Two Examples
 If the amino acid tryptophan (Trp) is added to the culture, the bacteria soon stop producing
the five enzymes previously needed to synthesize Trp from intermediates produced during
the respiration of glucose. In this case, the presence of the products of enzyme action
represses enzyme synthesis.
 Adding a new substrate to the culture medium may induce the formation of new enzymes
capable of metabolizing that substrate. If we take a culture of E. coli that is feeding on
glucose and transfer some of the cells to a medium contain lactose instead, a revealing
sequence of events takes place.
The three enzymes are :
 A permease that transports lactose across the plasma membrane from the culture medium
into the interior of the cell
 Beta-galactosidase which converts lactose into the intermediate allolactose and then
hydrolyzes this into glucose and galactose. Once in the presence of lactose, the quantity
of beta-galactosi dase in the cells rises from a tiny amount to almost 2% of the weight
of the cell.
 A transacetylase whose function is still uncertain.
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1. The lac Operon - an inducible system

The first control system for enzyme production worked out at the molecular level described the
control of enzymes that are produced in response to the presence of the sugar lactose in E. coli cell.
The following is the pathway that leads to the production of glucose and galactose.
Lactose 
â-galactosidase
 Glucose +Galactose
Several proteins involved in lactose metabolism in the E. coli cell. They are:
 -galactosidase - converts lactose into glucose and galactose
 -galactoside permease - transports lactose into the cell
 -galactoside transacetylase - function unknown
Structure of lac operon:
I P O || Z | Y | A |
_________________________________________________________
Controlling || Structural genes
Region
The lac operon consists of three structural genes, and a promoter, a terminator, regulator, and
an operator. The three structural genes are: lacZ, lacY, and lacA. Only lacZ and lacY appear to be
necessary for lactose catabolism.
 lacZ encodes -galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide
lactose into glucose and galactose.
 lacY encodes lactose permease (LacY), a transmembrane symporter that pumps -
galactosides into the cell using a proton gradient in the same direction.
 lacA encodes galactoside O-acetyltransferase (LacA), an enzyme that transfers an acetyl
group from acetyl-CoA to -galactosides.
 I : Gene for repressor protein
 P: promotor
 O: operator
Effector molecule - a molecule that interacts with the repressor and affects the affinity of the
repressor for the operator
Mutant lacgene Mutant Phenotype
I- constitutive expression because the operator is never closed
-
O constitutive expression because the repressor protein can not bind
-
P no expression of the operon because RNA polymerase cannot bind
-
lac Z no glucose or galactose production from lactose
lac Y- no induction because lactose will not be taken into the cell
Gene transcription begins at a particular nucleotide shown in the figure as "+1". RNA polymerase
actually binds to a site "upstream" (i.e., on the 5' side) of this site and opens the double helix so that
transcription of one strand can begin.
The binding site for RNA polymerase is called the promoter. In bacteria, two features of the
promoter appear to be important:
 a sequence of TATAAT (or something similar) centered 10 nucleotides upstream of the +1
site and
 another sequence (TTGACA or something quite close to it) centered 35 nucleotides
upstream.

The lac Operon : In the absence of lactose, the repressor protein encoded by the I gene binds
to the lac operator and stops transcription. Binding of allolactose to the repressor causes it to leave the
operator. This enables RNA polymerase to transcribe the three genes of the operon. The single mRNA
molecule that results is then translated into the three proteins. In fig. lactose present, repressor inactive,
operon on.
The lac repressor binds to a specific sequence of two dozen nucleotides called the operator.
Most of the operator is downstream of the promoter. When the repressor is bound to the operator, RNA
polymerase is unable to proceed downstream with its task of gene transcription.
 Lactose is not the preferred carbohydrate source for E. coli. If lactose and glucose are
present, the cell will use all of the glucose before the lac operon is turned on. This type
of control is termed catabolite repression.
 To prevent lactose metabolism, a second level of control of gene expression exists. The
promoter of the lac operon has two binding sites. One site is the location where RNA
polymerase binds. The second location is the binding site for a complex between the
catabolite activator protein (CAP) and cyclic AMP (cAMP).
 The binding of the CAP-cAMP complex to the promoter site is required for transcription
of the lac operon. The presence of this complex is closely associated with the presence
of glucose in the cell. As the concentration of glucose increases the amount of cAMP
decreases. As the cAMP decreases, the amount of complex decreases.

 This decrease in the complex inactivates the promoter, and the lac operon is turned off.
Be cause the CAP-cAMP complex is needed for transcription, the complex exerts a
positive control over the expression of the lac operon.
Positive Control of Transcription : CAP
 Absence of the lac repressor is essential but not sufficient for effective transcription of the
lac operon. The activity of RNA polymerase also depends on the presence of another
DNA-binding protein called catabolite activator protein or CAP. Like the lac repressor,
CAP has two types of binding sites:
 One binds the nucleotide cyclic AMP; the other binds a sequence of 16 base pairs
upstream of the promoter. Yet, CAP can bind to DNA only when cAMP is bound to CAP.
So when cAMP levels in the cell are low, CAP fails to bind DNA and thus RNA polymerase
cannot begin its work, even in the absence of the repressor. So the lac operon is under
both negative (the repressor) and positive (CAP) control.
2. Riboswitches
Protein repressors and corepressors are not the only way in which bacteria control gene
transcription. It turns out that the regulation of the level of certain metabolites can also be controlled by
riboswitches. A riboswitch is section of the 5'-untranslated region (5'-UTR) in a molecule of messenger
RNA (mRNA) which has a specific binding site for the metabolite (or a close relative).
Some of the metabolites that bind to riboswitches:
 The purines adenine and guanine
 The amino acids glycine and lysine
 Flavin mononucleotide (the prosthetic group of NADH dehydrogenase)
 S-adenosyl methionine (that donates methyl groups to many molecules) including
 DNA
 the cap at the 5' end of messenger RNA
 tRNAs: When these are bound to their amino acid (aminoacyl-tRNA), they bind to the
riboswitch in the mRNA that encodes the enzyme (an aminoacyl-tRNA synthetase)
responsible for loading the amino acid onto the tRNA. This causes transcription of the
mRNA to terminate prematurely. tRNAs with no amino acid attached also bind to the
riboswitch but in such a way that transcription of the mRNA continues. Its translation (in
bacteria, translation begins while transcription is still going on) produces the aminoacyl-
tRNA synthetase used to load the amino acid onto the tRNA. Thus these riboswitches
regulate the level of aminoacyl-tRNAs producing more when needed, less when not (a
kind of feedback inhibition). In each case, the riboswitch regulates transcription of genes
involved in the metabolism of that molecule. The metabolite binds to the growing mRNA
and induces an allosteric change that for some genes causes further synthesis of the
mRNA to terminate before forming a functional product and For other genes, enhances
completion of synthesis of the mRNA. In both cases, one result is to control the level of
that metabolite.
3. Tryptophan Operon System
The trp operon of E. coli controls the biosynthesis of tryptophan in the cell from the initial
precursor chorismic acid. This operon contains genes for the production of five proteins which are used
to produce three enzymes. The products of the E and D genes form a multimeric protein comprised of
two copies of each protein to produce the enzyme anthranilate synthetase. This enzyme catalyzes the
first two reactions in the tryptophan pathway. The next enzyme, which is responsible for catalyzing the
next two steps in the pathway is indole glycerolphosphate synthetase and it is the product of the C locus.
The final step in the reaction is the pathway produces tryptophan from indole-glycerol phosphate and
serine. This single step is catalyzed by tryptophan synthetase, an enzyme that is a multimer of two
proteins that are the product of the B and A genes. Control Circuit for the trp Operon
trp Operon Gene Gene Function
P/O Promoter; operator sequence is found in the promoter
trp L Leader sequence; attenuator (A) sequence is found in the leader
trp E Gene for anthranilate synthetase subunit
trp D Gene for anthranilate synthetase subunit
trp C Gene for glycerolphosphate synthetase
trp B Gene for tryptophan synthetase subunit
trp A Gene for tryptophan synthetase subunit

Fig. : Tryptophan Operon System

The trp operon is a repressible system. The primary difference between repressible and inducible
systems is the result that occurs when the effector molecule binds to the repressor. With inducible
systems, the binding of the effector molecule to the repressor greatly reduces the affinity of the repressor
for the operator, the repressor is released and transcription proceeds. The lac operon is an example of
an inducible system. With repressible systems, the binding of the effector molecule to the repressor
greatly increases the affinity of repressor for the operator and the repressor binds and stops transcription.
Thus, for the trp operon, the addition of tryptophan (the effector molecule) to the E. coli environment
shuts off the system because the repressors binds at the operator.
a. Attenuation of The trp Operon : One element of the trp operon is the leader sequence
(L) that in immediately 5' of the trpE gene. This sequence about 160 bp is size also
controls the expression of the operon through a process called attentuation. This sequence
has four domains (1-4). Domain 3 (nucleotides 108-121) of the mRNA can base pair with
either domain 2 (nucleotides 74-94) or domain 4 (nucleotides 126-134). If domain 3 pairs

with domain 4, a stem and loop structure forms on the mRNA and transcription stops.
This structure forms when the level of tryptophan is high in the cell. If domain 3 pairs with
domain 2, then the stem and loop structure does not form and transcription continues
through the operon, and all of the enzymes required for tryptophan biosynthesis are
produce. These events occur when tryptophan is low in the cell.
If domain 4 is deleted, the stem and loop structure cannot form and transcription of the
remainder of the operon will occur even in the presence of tryptophan. Domain 4 is called
the attenuator because its presence is required to reduce (attenuate) mRNA transcription
in the presence of high levels of tryptophan.
Domain 1 is also an important component of the attenuation process. The section of the
leader sequence encodes a 14 amino acid peptide that has two tryptophan residues.
b. trp Operon Transcription Under High Levels of Tryptophan : When the cellular levels
of tryptophan are high, the levels of the tryptophan tRNA are also high. After transcription,
the mRNA moves quickly through the ribosome complex and the small peptide is translated.
Translation is quick because of the high levels of tryptophan tRNA. Because of the quick
translation, domain 2 becomes associated with the ribosome complex. Then domain 3
binds with domain 4, and transcription is attenuated because of the stem and loop formation.
c. trp Operon Transcription Under Low Levels of Tryptophan : Under low cellular levels
of tryptophan, the translation of the short peptide on domain 1 is slow. Because of the
slow translation, domain 2 does not become associated with the ribosome. Rather domain
2 associates with domain 3. This structure permits the continued transcription of the
operon. Then the trpE-A genes are translated, and the biosynthesis of tryptophan occurs.
Fig. : Attenuation of The trp Operon mRNA

4. Eukaryotic Gene Regulation
Many features of gene regulation are common to both bacterial and eukaryotic cells. For example,
in both types of cells, DNA-binding proteins influence the ability of RNA polymerase to initiate transcription.
However, there are also some differences, although these differences are often a matter of degree. First,
eukaryotic genes are not organized into operons and are rarely transcribed together into a single mRNA
molecule; instead, each structural gene typically has its own promoter and is transcribed separately.
Second, chromatin structure affect gene expression in eukaryotic cells; DNA must unwind from
the histone proteins before transcription can take place.
Third, although both repressors and activators function in eukaryotic and bacterial gene regulation,
activators seem to be more common in eukaryotic cells. Finally, the regulation of gene expression in
eukaryotic cells is characterized by a greater diversity of mechanisms that act at different points in the
transfer of information from DNA to protein.
There are several methods used by eukaryotes.
 Altering the rate of transcription of the gene. This is the most important and widely-used
strategy and the one we shall examine here.
 However, eukaryotes supplement transcriptional regulation with several other methods:
 Altering the rate at which RNA transcripts are processed while still within the
nucleus.
 Altering the stability of messenger RNA (mRNA) molecules; that is, the rate at
which they are degraded.
 Altering the efficiency with which ribosomes translate the mRNA into a polypeptide.
Protein-Coding Genes Have
 exons whose sequence encodes the polypeptide;
 introns that will be removed from the mRNA before it is translated
 a transcription start site
 a promoter
 a basal or core promoter located within about 40 base pairs (bp) of the start site
 an "upstream" promoter, which may extend over as many as 200 bp farther
upstream
 enhancers
 silencers
 Adjacent genes are often separated by an insulator which helps them avoid cross-talk
between each other's promoters and enhancers (and/or silencers).
Transcription Start Site
This is where a molecule of RNA polymerase II (pol II, also known as RNAP II) binds. Pol II is
a complex of 12 different proteins. The start site is where transcription of the gene into RNA begins.
The Core Promoter
All eukaryotic genes contain a core promoter. One common example is a sequence of bases
(e.g., TATAAAAAA) called the TATA box. It is bound by a large complex of some 50 different proteins,
including
 Transcription Factor IID (TFIID) which is a complex of
 TATA-Binding Protein (TBP), which recognizes and binds to the TATA box
 13 other protein factors which bind to TBP - and each other - but not to the DNA.
 Transcription Factor IIB (TFIIB) which binds both the DNA and pol II.
A core promoter, with little variation in its structure and binding factors, is found in all protein-
coding genes. This is in sharp contrast to the upstream promoter whose structure and associated
binding factors differ from gene to gene.
Many different genes and many different types of cells share the same transcription factors not
only those that bind at the core promoter but even some of those that bind upstream. What turns on a
particular gene in a particular cell is probably the unique combination of promoter sites and the transcription
factors that are chosen.

An Analogy
To open any particular box in the room requires two keys:
 your key, whose pattern of notches fits only the lock of the box assigned to you (= the
upstream promoter), but which cannot unlock the box without
 a key carried by a bank employee that can activate the unlocking mechanism of any box
(= the core promoter) but cannot by itself open any box.
Enhancers
Some transcription factors ("Enhancer-binding protein") bind to regions of DNA that are thousands
of base pairs away from the gene they control. Binding increases the rate of transcription of the gene.
One possibility is that enhancer-binding proteins in addition to their DNA-binding site, have sites that bind
to transcription factors ("TF") assembled at the promoter of the gene.
Silencers : Silencers are control regions of DNA that, like enhancers, may be located thousands
of base pairs away from the gene they control. However, when transcription factors bind to them,
expression of the gene they control is repressed.
Insulators
Insulators are stretches of DNA (as few as 42 base pairs may do the trick). They located between
the enhancer(s) and promoter or silencer(s) and promoter of adjacent genes or clusters of adjacent
genes.
Fig. Working of Insulators

Example
The enhancer for the promoter of the gene for the delta chain of the gamma/delta T-cell receptor
for antigen (TCR) is located close to the promoter for the alpha chain of the alpha/beta TCR (on
chromosome 14 in humans). A T-cell must choose between one or the other. There is an insulator
between the alpha gene promoter and the delta gene promoter that ensures that activation of one does
not spread over to the other.
Another Example : In mammals (mice, humans, pigs), only the allele for insulin-like growth
factor-2 (IGF-2) inherited from one's father is active; that inherited from the mother is not a phenomenon
called imprinting.
5. Mutation
In genetics, a mutation is a permanent change of the nucleotide sequence of the genome of an
organism, Mutation can result in several different types of change in sequences. Mutations in genes can
either have no effect, alter the product of a gene, or prevent the gene from functioning properly or
completely.
Four classes of mutations are (1) spontaneous mutations (molecular decay), (2) mutations due
to error prone replication bypass of naturally occurring DNA damage (also called error prone translesion
synthesis), (3) errors introduced during DNA repair, and (4) induced mutations caused by mutagens.
Scientists may also deliberately introduce mutant sequences through DNA manipulation for the sake of
scientific experimentation.
1. Spontaneous Mutation : Spontaneous mutations on the molecular level can be caused
by :
 Tautomerism : A base is changed by the repositioning of a hydrogen atom, altering
the hydrogen bonding pattern of that base, resulting in incorrect base pairing during
replication.
 Depurination : Loss of a purine base (A or G) to form an apurinic site (AP site).
 Deamination : Hydrolysis changes a normal base to an atypical base containing
a keto group in place of the original amine group. Examples include C  U and
A  HX (hypoxanthine), which can be corrected by DNA repair mechanisms; and
5MeC (5-methylcytosine)  T, which is less likely to be detected as a mutation
because thymine is a normal DNA base.
 Slipped Strand Mispairing : Denaturation of the new strand from the template
during replication, followed by renaturation in a different spot ("slipping"). This can
lead to insertions or deletions.
2. Error Prone Replication By-Pass : There is increasing evidence that the majority of
spontaneously arising mutations are due to error prone replication (translation synthesis)
past a DNA damage in the template strand. The naturally occurring DNA damages arise
about 60,000 to 100,000 times per day per mammalian cell. In mice, the majority of
mutations are caused by translesion synthesis.
3. Errors Introduced During DNA Repair : Although naturally occurring double-strand breaks
occur at a relatively low frequency in DNA, their repair often causes mutation. Non
homologous end joining (NHEJ) is a major pathway for repairing double-strand breaks.
NHEJ involves removal of a few nucleotides to allow somewhat inaccurate alignment of
the two ends for rejoining followed by addition of nucleotides to fill in gaps.
4. Induced Mutation : Induced mutations on the molecular level can be caused by :
 Chemicals
 Hydroxylamine NH2OH
 Base analogs (e.g., BrdU)

 Alkylating agents (e.g., N-ethyl-N-nitrosourea): These agents can mutate

both replicating and non-replicating DNA. In contrast, a base analog can
mutate the DNA only when the analog is incorporated in replicating the
DNA. Each of these classes of chemical mutagens has certain effects that
then lead to transitions, transversions, or deletions.
 Agents that form DNA adducts (e.g. ochratoxin A metabolites)
 DNA intercalating agents (e.g., ethidium bromide)
 DNA crosslinkers
 Oxidative damage
 Nitrous acid converts amine groups on A and C to diazo groups,
altering their hydrogen bonding patterns, which leads to incorrect
base pairing during replication.
 Radiation : Ultraviolet radiation (nonionizing radiation). Two nucleotide bases in
DNA - cytosine and thymine are most vulnerable to radiation that can change their
properties. UV light can induce adjacent pyrimidine bases in a DNA strand to
become covalently joined as a pyrimidine dimer. UV radiation, in particular longer-
wave UVA, can also cause oxidative damage to DNA.
6. Classification of Mutation
i. By Effect on Structure
a. Small-scale mutations, such as those affecting a small gene in one or a few nucleotides,
including :
 Point Mutations : Exchange a single nucleotide for another. These changes are
classified as transitions or transversions. Most common is the transition that
exchanges a purine for a purine (A  G) or a pyrimidine for a pyrimidine, (C 
T). A transition can be caused by nitrous acid, base mis-pairing, or mutagenic
base analogs such as 5-bromo-2-deoxyuridine (BrdU).
Less common is a transversion, which exchanges a purine for a pyrimidine or a
pyrimidine for a purine (C/T  A/G). An example of a transversion is the conversion
of adenine (A) into a cytosine (C). Point mutations that occur within the protein
coding region of a gene may be classified into three kinds, depending upon what
the erroneous codon codes for:
 Silent mutations, which code for the same (or a sufficiently similar) amino
acid.
 Missense mutations, which code for a different amino acid.
 Nonsense mutations, which code for a stop and can truncate the protein.
 Insertions : add one or more extra nucleotides into the DNA. They are usually
caused by transposable elements, or errors during replication of repeating elements
(e.g., AT repeats. Insertions in the coding region of a gene may alter splicing of the
mRNA (splice site mutation), or cause a shift in the reading frame (frameshift),
both of which can significantly alter the gene product. Insertions can be reversed
by excision of the transposable element.
 Deletions remove one or more nucleotides from the DNA.
b. Large-scale mutations in chromosomal structure, including:
 Amplifications (or gene duplications) leading to multiple copies of all chromosomal
regions, increasing the dosage of the genes located within them.
 Deletions of large chromosomal regions, leading to loss of the genes within those
regions.

 Mutations whose effect is to juxtapose previously separate pieces of DNA, potentially

bringing together separate genes to form functionally distinct fusion genes (e.g.,
bcr-abl). These include:
 Chromosomal translocations: interchange of genetic parts from nonhomologous
chromosomes.
 Interstitial deletions: an intra-chromosomal deletion that removes a segment of
DNA from a single chromosome, thereby apposing previously distant genes.
 Chromosomal inversions: reversing the orientation of a chromosomal segment.
 Loss of heterozygosity: loss of one allele, either by a deletion or a recombination
event, in an organism that previously had two different alleles.
Fig. : Chromosome Structure after Mutation

ii. By effect on Function
 Loss-of-Function Mutations result in the gene product having less or no function. When
the allele has a complete loss of function (null allele) it is often called an amorphic
mutation. Phenotypes associated with such mutations are most often recessive. Exceptions
are when the organism is haploid, or when the reduced dosage of a normal gene product
is not enough for a normal phenotype (this is called haploinsufficiency).

 Gain-of-Function Mutations change the gene product such that it gains a new and
abnormal function. These mutations usually have dominant phenotypes. Often called
aneomorphic mutation.
 Dominant Negative Mutations (also called antimorphic mutations) have an altered
gene product that acts antagonistically to the wild-type allele. These mutations usually
result in an altered molecular function (often inactive) and are characterized by a dominant
or semi-dominant phenotype. In humans, dominant negative mutations have been implicated
in cancer (e.g., mutations in genes p53, ATM, CEBPA and PPARgamma).
 Lethal mutations are mutations that lead to the death of the organisms that carry the
mutations.
 A back mutation or reversion is a point mutation that restores the original sequence and
hence the original phenotype.
iii. By Effect on Fitness
It is usual to speak of mutations as either harmful or beneficial.
 A harmful, or deleterious, mutation decreases the fitness of the organism.
 A beneficial, or advantageous mutation increases the fitness of the organism. Mutations
that promotes traits that are desirable, are also called beneficial. In theoretical population
genetics, it is more usual to speak of mutations as deleterious or advantageous than
harmful or beneficial.
 A neutral mutation has no harmful or beneficial effect on the organism. Such mutations
occur at a steady rate, forming the basis for the molecular clock. In the neutral theory of
molecular evolution, neutral mutations provide genetic drift is the basis for most variation
at the molecular level.
 A nearly neutral mutation is a mutation that may be slightly deleterious or advantageous,
al though most nearly neutral mutations are slightly deleterious.
iv. By Impact on Protein Sequence
 A Frameshift Mutation : Caused by insertion or deletion of a number of nucleotides that
is not evenly divisible by three from a DNA sequence. Due to the triplet nature of gene
expression by codons, the insertion or deletion can disrupt the reading frame, or the
grouping of the codons, resulting in a completely different translation from the original. The
earlier in the sequence the deletion or insertion occurs, the more altered the protein
produced is. In contrast, any insertion or deletion that is evenly divisible by three is termed
an in-frame mutation.

Fig. : Mutation Impact on Protein Sequence

 A nonsense mutation is a point mutation in a sequence of DNA that results in a premature
stop codon, or a nonsense codon in the transcribed mRNA, and possibly a truncated, and
often nonfunctional protein product.
 Missense mutations or nonsynonymous mutations are types of point mutations where
a single nucleotide is changed to cause substitution of a different amino acid. This in turn
can render the resulting protein nonfunctional. Such mutations are responsible for diseases
such as Epidermolysis bullosa, sickle-cell disease, and SOD1 mediated ALS.
 A neutral mutation is a mutation that occurs in an amino acid codon that results in the
use of a different, but chemically similar, amino acid. The similarity between the two is
enough that little or no change is often rendered in the protein. For example, a change
from AAA to AGA will encode arginine, a chemically similar molecule to the intendedlysine.
 Silent Mutations : That type of mutation do not result in a change to the amino acid
sequence of a protein, unless the changed amino acid is sufficiently similar to the original.
They may occur in a region that does not code for a protein, or they may occur within a
codon in a manner that does not alter the final amino acid sequence.
v. By Inheritance
Diploid organisms (e.g., humans) contain two copies of each gene - a paternal and a maternal
allele. Based on the occurrence of mutation on each chromosome, we may classify mutations into three
types.
 A heterozygous mutation is a mutation of only one allele.
 A homozygous mutation is an identical mutation of both the paternal and maternal
alleles.
 Compound heterozygous mutations or a genetic compound comprises two different
mutations in the paternal and maternal alleles. A wildtype or homozygous non-mutated
organism is one in which neither allele is mutated.

Regulation of Transcription in Eukaryotes

The control of gene expression is far more complex in eukaryotes than in bacteria and is primarily
controlled at the level of initiation of transcription. As in bacteria, transcription in eukaryotic cells is
controlled by proteins that bind to specific regulatory sequences and modulate the activity of RNA
polymerase. In addition, the packaging of DNA into chromatin and its modification may also control the
eukaryotic gene expression.
Table : Overview of differences between prokaryotic and eukaryotic genome and gene expression
Features Prokaryotes Eukaryotes
Nature of genome Generally single circular Most genome present in
bacterial chromosome the of linear chromath
and plasmids within nucleus
Size of genome Relatively small Relatively large
Transcription and transiation Coupled; in cytosol Nuclear transcription and
cytoplasmic translation
Transcription unit Mostly polycistronic Mostly monocistronic
Default state of transcription On Off
Influence of Chromatin Structure on Transcription
Changes in chromatin structure can result in repression or stimulation of gene expression. As
genes become more transcriptionally active, chromatin shows increased sensitivity to DNase I digestion
also, suggesting that the chromatin structure is more open. DNase I hypersensitivity sites are typically
found near regulatory promoter elements, approximately 1000 nucleotides upstream of a transcription start
site, and occasionally, at the 3’ end of the gene as well. This sensitivity to DNase I suggests a more relaxed
or open conformational state for the chromatin, allowing access to transcriptional factors as well as DNase
I. Essentially, the DNA becomes more exposed due to the lack of nuclesome structure. The transcription
factors responsible for transcription seem unable to assemble onto a promoter that is packaged in a
conventional nucleosome. Thus, the local alterations in chromatin structure facilitate the assembly of the
transcription factors and the RNA polymerase holoenzyme at the promoter. Two most important ways for
altering chromatin structure locally are: covalent histone modifications and nucleosome remodeling.
Covalent Histone Modifications
The N-terminal tails of histones are subject to a wide range of post-translational covalent
modifications. These modifications play a significant role in the ability of nearby genes to be transcribed.
There are four main types of modifications to histone tails known to play a role in regulating gene
expression: acetylation of lysines, methylation of lysines and arginine, ubiquitinylation of lysines, and
phosphorylation of serines and threonines. Other less common modifications are ADP-ribosylation of
glutamic acid and sumoylation of lysine residues.
Histone acetylation and deacetylation control chromatin activity. Specific lysines in the tails of
histones are often found to be acetylated in chromosomal regions containing genes that are active or
potentially active. Enzymes that acetylate histones are called histone acetyl transferases (HATs). Acetylation
appears to function at several levels to influence gene expression. One of the first HAT discovered was
P55 in Tetrahymena. HATs function as transcriptional co-activators. Due to the loss of positive charges
when the charged lysine side chains are modified, the affinity between histones and DNA have reduced.
The net effect is that RNA polymerase and transcription factors find it easier to access the promoter
region. Histone deacetylases (HDACs) have the opposite effect of removing acetyt groups and promoting
transcriptional repression. HDACs like HAT, are contained in multiprotein complexes.
Methylation also reversibly modify histones. Histone methylation is carried out by methyl
transferases targeting certain arginine and lysine residues in histones H3 and H4. Histone methylation
acts as a signal for both transcriptional repression and activation. For example, methylation of histone

H3 at lysine 9 (abbreviated as dimethylated H3K9) and methylation of histone H3 at lysine 27 (H3K27)

are both largely associated with gene silencing and heterochromatin formation. In contrast, methylation
of H3K4, H3K36 or H3-K79 is associated with active chromatin.
Fig. : Strands of DNA are wrapped around histone octamers, forming nucleosomes, which to be organized
Into chromatin, the building block of a chromosome. Schematic of the reversible changes in
chromatin organization that Influence gene expression: genes are expressed (switched on) when
the chromatin is open (active), and they are inactivated (switched off) when the chromatin is
condensed (silent).
Nucleosome Remodeling
The second type of chromatin modification that can influence genome expression is nucleosome
remodeling. Nucleosome remodeling does not involve covalent modification to histone proteins. Remodeling
is induced by an energy dependent process that weakens the contact between the nucleosome and the
DNA with which it is associated. Remodeling in the strict sense, involves a change in the structure of
the nucleosome but no change in its position. In some cases, the remodeling can involve cis and trans
displacement that involves movement of nucleosome. ATP-dependent chromatin remodeling complexes
responsible for nucleosome remodeling. Two best studied classes of these complexes are the swi/snf
family and the ISWI-based family. Remodeling complex swi/snf (pronounced switch-sniff) is made up of
>10 proteins. The name derives from the catalytic ATPase subunit, which were first described in mutants
that were mating-type switching defective (Swi2) and sucrose nonfermenters (Snf2). In addition to carrying
ATPase domains, it exhibits bromodomains which interact with acetylated lysine residues in histones.
Fig. : Histone acetylation and nucleosme remodeling generally render the chromatin DNA more accessible
to other proteins required for transcription initiation.

DNA Methylation and Gene Regulation

In eukaryotes, DNA methylation occurs in cytosine residues and specific for CpG sequence. This
methylation is catalyzed by enzyme DNA methyl transferases. DNA methylation is an important heritable
epigenetic modification of the genome and is involved in the gene regulation. The regions of the genome
with a high number of methylated cytosine are usually transcriptionally inactive. The absence of DNA
methylation is a prerequisite for transcriptionally active regions.
Fig. : Methylation of cytosine.

In bacteria, both adenine and cytosine can be methylated. The main function of DNA methylation
in bacteria is to provide protection from own restriction endonucleases. It also plays an important role
in the mismatch repair. Cytosine methylation is relatively rare in lower eukaryotes, but in vertebrates up
to 10% of the total number of cytosine bases are methylated and in plants up to 30%. There are two
types of methylation processes known in eukaryotic cells: maintenance methylation and de novo
methylation. In maintenance methylation, addition of methyl groups occurs in a newly synthesized strand
of DNA at positions opposite methylated sites on the parent strand. The maintenance activity ensures
that the two daughter DNA molecules retain the methylation pattern of the parent molecule. In de novo
methylation, addition of methyl groups occur at totally new positions.
Fig. : (a) Maintenance methylation and (b) de novo methylation. In case of maintenance methylation
addition of methyl groups occurs in a newly synthesized strand of DNA at positions opposite
methylated sites on the parent strand. In de novo methylation, addition of methyl groups occurs
at totally new positions.
CpG Islands
CpG is present in the vertebrate genome at only about one-fifth of its randomly expected frequency.
During the evolution, the sequence CpG has been progressively eliminated from the genome due to
deamination of 5- methylcytosines to thymines. For example, in humans this dinucleotide is present only
5 to 10% of its predicted frequency. In 70 to 80% these CpG dinucleotides are methylated. These
methylated regions are typical of the bulk chromatin that constitutes most nontranscribed DNA. In contrast

to the rest of the genome, smaller regions of DNA termed CpG islands have maintained the expected
frequency of CpG content. Most frequently these islands are located within upstream regions of many
genes. Genes which contain CpG Island In their promoter are usually ‘housekeeping genes’, which have
a broad tissue pattern of expression. Many relatively tissue specific genes are also regulated by CpG
island methylation. It is important to note that nonmethylated CpG island within the promoter region is
not always associated with actively transcribed gene. However, the lack of methylation of the CpG island
within the promoter region of the gene is required for transcription of the gene.
Regulation of Transcription by Methylation
DNA methylation is persistent throughout genomes, and is missing only in regions such as CpG
islands, promoters and possibly enhancers. DNA methylation switches off eukaryotic gene expression,
particularly when it occurs in the promoter regions upstream of a gene’s transcribed sequences. The
way in which DNA methylation prevents gene expression is poorly understood. However, in many cases,
DNA methylation is recognized by a family of proteins that contain a conserved methyl-CpG binding
domain (MBD). Since the methyl groups of m5C residues extend into dsDNA’s major groove, MBDs can
bind to them without perturbing DNA’s double helical structure. MBD-containing proteins inhibit the
transcription of their bound promoter-methylated genes by recruiting protein complexes that induce the
alternation of the local chromosome structure in a way that prevents the transcription of the associated
gene. An important regulatory role of DNA methylation has been established in the phenomenon known
as genomic imprinting (also called gametic or parental imprinting), which controls the expression of
some genes involved in the development of mammalian embryos. In most cases, both the paternal and
maternal alleles of a gene are expressed in diploid cells. However, there are a few imprinted genes
whose expression depends on whether they are inherited from the mother or from the father. In some
cases, only the paternal allele of an imprinted gene is expressed, and the maternal allele is transcriptionally
inactive. For other imprinted genes, the maternal allele is expressed and the paternal allele is inactive.
This is a form of allelic exclusion. In such cells, the relevant gene is said to exhibit functional hemizygosity.
DNA methylation is involved in genomic imprinting. Imprinting is controlled by imprint control elements,
DNA sequences that are found within a few kilobases of clusters of imprinted genes. These centers
mediate the methylation of the imprinted regions. An example of an imprinted gene in humans is Igf2
(insulin like growth factor-2), which codes for a growth factor. Only the paternal copy of Igt2 is transcribed.
Igf2 is not expressed from the maternally inherited chromosome.
Post-Transcriptional Gene Regulation
Regulation of gene expression at the level of transcription is most efficient and most common
process. Regulation of genes also occur post-transcriptionally. Post-transcriptional regulation may occurs
at the level of translation. However regulation at this level is less efficient. It is consequently less frequent.
Just as regulatory proteins may bind to DNA and either promote or repress transcription, proteins may
also bind to specific sequences on a mRNA and regulate its translation. Examples of both positive and
negative translational regulation are known.
The response of mRNA to Iron is used below as an example of translational repression. The level
of ferritin Is regulated in response to the iron supply. When iron concentration is low, translation of ferritin
mRNA is reduced. When iron concentration is high, ferritin synthesis Increases. In animals, an RNA-
binding protein is responsible for translational repression of ferritin mRNA. The 5’UTR of ferritin mRNA
contains a special recognition sequence known as an Iron-responsive element (IRE), which forms a
stem loop structure. When iron concentration is low, an iron regulatory protein (IRP) binds to the IRE and
prevents translation. The free iron concentration in the cytoplasm is directly monitored by the iron
regulatory proteins. Although there are several IRPs, the major one is cytosolic enzyme, aconitase.
Surplus iron results in the detachment of IRP from the ferritin mRNA, which can then be translated. The
protein transferrin receptor responsible for the transport of Iron binding protein transferrin is also regulated
by IRP binding to an IRE present at the 3’UTR of the mRNA, but in this case mRNA stability Is affected.

Low [Iron] High [Iron]

Ferritin mRNA translation Repressed Activated
Transferrin receptor mRNA stability Increased Decreased
Fig. : Production of the ferritin and transferrin receptor is regulated at the level of mRNA by iron
regulatory proteins (JRPs), which bind to iron response elements (IREs) on the 5’ and 3’-
untranslated regions of their respective mRNAs. In iron deficiency, the IRPs bind to the IREs,
protecting the transferrin receptor mRNA from nuclease digestion and preventing the synthesis
of ferritin. When iron concentration is high, the IRP no longer binds to the IREs, allowing transferrin
receptor mRNA to be destroyed and allowing the expression of ferritin.
RNA Interference
RNA interference (abbreviated RNAI) is an evolutionarily conserved mechanism of gene regulation
that is induced by small silencing RNA in a sequence-specific manner. In 1998, Fire and Mello first
established this in C. elegans. Historically, RNA interference was known by other names, including post
transcriptional gene silencing (PTGS), transgene silencing and quelling. RNAi has been observed in all
eukaryotes, from yeast to mammals. RNA interference has an important role in post-transcriptional gene
regulation, transposon regulation and defending cells against viruses. Two types of small silencing RNA
molecules - small interfering RNA (siRNA) and microRNA (miRNA) — are central to RNA interference.
siRNAs Mediated RNAi
In the siRNAs mediated RNAi pathway, the dsRNAs are processed into siRNAs duplexes comprised
of two ~21 nucleotides long strands with two nucleotides overhangs at the 3’ ends by an enzyme called
Dicer. Dicer is a ~200 kDa multidomain, an RNase III family enzyme that functions In processing dsRNA
to siRNA. The Dicer includes an ATPase/RNA helicase domain, catalytic RNase III domains, and dsRNA
binding domain. Dicer and a dsRNA binding protein (together form the RISC loading complex) then load
the RNA duplex into RISC. The siRNA is thought to provide target specificity to RISC through base pairing
of the guide strand with the target mRNA. Only one of the two strands, which is known as the guide
strand, directs the gene silencing. The other anti-guide strand or passenger strand is degraded during
RISC activation. The active components of an RNA-induced silencing complex (RISC) are endonucleases
called argonaute proteins, which cleave the target mRNA strand complementary to their bound siRNA.

Fig. : dsRNA precursors are processed by Dicer to generate siRNA duplexes containing guide and
passenger strands. RISC-loading complex loads the duplex into RISC. The passenger strand is
later destroyed and the guide strand directs RISC to the target RNA.
miRNA Mediated RNAi
miRNAs (microRNAs) are small, non-coding RNA molecules encoded in the genomes of plants,
animals and their viruses. These highly conserved, 20–25 mer RNAs appear to regulate gene expression
post-transcriptionally by binding to the 3’-untranslated regions (3’-UTR) of specific mRNAs. Victor Ambros
and colleagues identified the first miRNA, lin-4, in C. elegans over a decade ago. The Iin-4 gene was
unusual in that it did not encode a protein but rather a small RNA that imperfectly base-paired to
complementary sequences on target mRNAs in order to block gene expression. The Iin-4 controls the
expression of Iin-14 gene. The lin-4 transcripts are complementary to sequence present in the 3’ UTR
of lin-14 mRNA. In 2000, a second miRNA, Iet-7, was discovered by Gary Ruvkun’s group in C. elegans
that works in a similar manner to lin-4. These small RNAs have been shown to play critical roles in
developmental timing, hematopoietic cell differentiation, cell death, cell proliferation and oncogenesis.
miRNAs are synthesized in the nucleus as long (up to 1000 nts) RNA polymerase II transcripts, called
pri-miRNAs, that are characterized by imperfect hairpin structures. RNA polymerase III also transcribes
some pri-miRNAs An RNase III enzyme, drosha acts as a dsRNA-specific endonuclease, in conjunction
with a dsRNA-binding protein, called pasha (in Drosophila) and DGCR8 (in mammal), processes the pri-
miRNA into hairpin RNAs 70-100 nts in length, called pre-miRNAs. Complex of DGCR8 or pasha with
the enzyme drosha is called microprocessor complex. Pre-miRNAs also derive from introns and known
as mirtrons. However, mirtrons not involve processing by microprocessor complex. Pre-miRNAs are
transported to the cytoplasm. It is mediated by exportin-5.

Fig. : Translational silencing by miRNA.

In the cytoplasm, dicer processes the pre-miRNA into miRNA duplex (miRNA-miRNA*) and load
it into the RISC. Members of the Argonaute (Ago) protein family are central to RISC function. Argonautes
are needed for miRNA- induced silencing and contain two conserved RNA binding domains: a PAZ
domain that can bind the single stranded 3’ end of the mature miRNA and a PIWI domain that structurally
resembles ribonuclease-H.
miRNA biogenesis in plants differs from animal biogenesis mainly in the steps of nuclear processing
and export. First, in plants, single enzyme DCL1 present inside the nucleus fills the roles of both Drosha
and Dicer, converting pri-miRNAs to mature miRNA. Second, before plant miRNA-miRNA* duplexes are
transported out of the nucleus, its 3’ overhangs are methylated by a S-adenosyl methionine-dependent
methyltransferase called HEN1.
Gene silencing may occur either via mRNA degradation or preventing mRNA from being translated.
miRNAs function via base-pairing with complementary sequences within target mRNA molecules. If there
is complete complementation between the miRNA and target mRNA sequence, Ago can cleave the
mRNA and lead to direct mRNA degradation. If there isn’t complete complementation the silencing is
achieved by preventing translation. Plant miRNAs usually have perfect or near-perfect pairing with their
target mRNA and induce gene repression through degradation of their target transcripts. In contrast,
animal miRNAs are able to recognize their target mRNAs by as little as 6-8 nucleotides (seed region)
at the 5’ end of an animal miRNA. A given miRNA may have multiple different mRNA targets.
siRNA versus miRNA
The small RNAs that provide target specificity to the silencing machinery-siRNAs and miRNAs-
can be distinguished by their origin. siRNAs are processed from dsRNA precursors made up of two
distinct strands of perfectly base– paired RNA, while miRNAs originate from a single, long transcript that
forms imperfectly base-paired hairpin structures. siRNAs were originally identified as intermediates in the
RNAi pathway after induction by exogenous dsRNA; however, endogenous sources of siRNAs have now
been recognized. miRNAs were discovered through their critical roles in development and cellular regulation.
miRNAs have always been recognized as being of endogenous origin.
miRNA appeared to be processed from stem-loop precursors with incomplete double-stranded
character, whereas siRNAs derive from long, fully complementary double-stranded RNAs (dsRNAs).
The siRNAs derived from long dsRNA precursors also differ from miRNAs in that miRNAs,
typically have an incomplete base pairing to a target and inhibit the translation of many different mRNAs
with similar sequences. In contrast, siRNAs typically base-pair perfectly and induce mRNA cleavage only
in a single, specific target.
piRNA
piRNA (Piwi-interacting RNAs) are the most recently discovered class of longer small RNAs
(~25–30 nucleotides) which bind to the Piwi clade of argonaute proteins. piRNAs are thought to derive
from ssRNA precursor and are made without a dicing step (dicer independent). They are mostly antisense,
but a small fraction is in the sense orientation. They are 2’- O-methylated at their 3’ termini unlike
miRNAs, but similar to siRNAs. piRNAs probably direct cleavage of transposon mRNA or chromatin
modification at transposon loci.
Table : Types of small silencing RNAs
Types Organism Length Features
miRNA Animals, plants, protists 20-25 Dicer/Drosha-dependent
siRNA
Exo-siRNA Animals, plants, fungi, protist ~21 Dicer-dependent
Endo-siRNA Animals, plants, fungi, protist ~21 Dicer-dependent
piRNA Metazoans 24-30 Dicer-independent

SAMPLE QUESTIONS
1. DNA exists in a double-stranded form whereas RNA is mainly a single stranded molecule. What
is the likely reason for DNA being double stranded ?
(A) RNA strands cannot form base pairs
(B) Double stranded DNA is a more stable structure
(C) DNA cannot exist in the single stranded form
(D) It is easier to replicate double stranded DNA than single stranded RNA Double stranded
DNA is a more stable structure
2. What is the role of topoisomerases in eukaryotic DNA replication ?

(A) Topoisomerise enzymes cut, uncoil and reseal the double stranded DNA
(B) Topoisomerise enzymes bind to the origin of replication sites within double stranded DNA
(C) Topoisomerise enzymes open up the double stranded DNA at the replication fork
(D) Topoisomerise enzymes join the Okazaki fragments together with phosphodiester bonds
3. Which of the following is true regarding translation ?

(A) mRNA is exposing its anticodon to the ribosome
(B) DNA is being replicated in the nucleus
(C) mRNA is exposing its codon to the tRNA
(D) tRNA remains attached to the amino acid after the peptide chain is formed
4. Why does the genetic code have to be a triplet code ?

(A) because there are more base pairs than amino acids
(B) so there are enough codes for the 20 amino acids
(C) so that each amino acid is coded by one base
(D) there is no reason
5. Which of the following is MISMATCHED ?

(A) rRNA - recode (B) tRNA - anticodon
(C) mRNA - codon (D) none of these
6. Which of the following is mismatched ?

(A) RNA polymerase II : mRNA (B) RNA polymerase III : snoRNAs
(C) RNA polymerase I: most rRNAs (D) RNA polymerase I: tRNAs
7. Unwinding of DNA is done by

(A) Helicase (B) ligase
(C) Hexonuclease (D) Topoisomerase
8. Eukaryotes differ from prokaryote in mechanism of DNA replication due to :

(A) Different enzyme for synthesis of lagging and leading strand
(B) Use of DNA primer rather than RNA primer
(C) Unidirectional rather than bidirectional replication
(D) Discontinuous rather than semidiscontinuous replication
9. At a replication fork, the leading strand is synthesized :

(A) continuously (B) discontinuously
(C) first (D) when the lagging strand is complete
10. Which of the following describes a primer used in DNA replication ?

(A) A short segment (about 10 nucleotides) of DNA
(B) A short segment (about 10 nucleotides) of RNA
(C) A short segment (about 10 amino acids) of a polypeptide
(D) None of these
ANSWER KEYS
1 2 3 4 5 6 7 8 9 10
B A C B A D A D A B

SOLUTIONS
1. (B) Double stranded DNA is much more stable than single stranded RNA and this helps to
protect our genetic code. Having a second copy of our genetic code means that there is
a reference for repair in the event of a mutation or damage.
2. (A) Helicase enzymes function to unwind the double helix, locally opening up the DNA to allow
replication to occur. However, this results in increased coiling ahead of the replication fork.
To reduce this coiling, topoisomerise enzymes work ahead of the replication fork by
cutting, uncoiling, and resealing the DNA strand.
3. (C) mRNA is exposing its codon to the tRNA. As the mRNA passes through the ribosome
tRNAs leave the ribosome, a piece of the mRNA codon is exposed so that the next tRNA
can attach.
4. (B) The genetic code is made of three base sequences called codons so that there are
enough codons for the 20 different amino acids.
5. (A) Ribosomal RNA (rRNA) associates with a set of proteins to form ribosomes. tRNA used
for anticodon and mRNA work on codon.
6. (D) RNA polymerase I (also known as Pol I) is, in higher eukaryotes, the polymerase that only
transcribes ribosomal RNA (but not 5S rRNA, which is synthesized by RNA polymerase
III).
7. (A) Helicases are often used to separate strands of a DNA double helix or a self-annealed
RNA molecule using the energy from ATP hydrolysis, a process characterized by the
breaking of hydrogen bonds between annealed nucleotide bases.
8. (D) Eukaryotes differ from prokaryote in mechanism of DNA replication due to discontinuous
rather than semidiscontinuous replication present.
9. (A) On the leading strand DNA replication proceeds continuously along the DNA molecule as
the parent double-stranded DNA is unwound, but on the lagging strand the new DNA is
made in installments, which are later joined together by a DNA ligase enzyme.
10. (B) synthesizes a short RNA primer approximately 11 ±1 nucleotides long, to which new
nucleotides can be added by DNA polymerase.

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CSIR-UGC (NET)

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1. The lac Operon - an inducible system

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Positive Control of Transcription : CAP

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Fig. : Tryptophan Operon System

either domain 2 (nucleotides 74-94) or domain 4 (nucleotides 126-134). If domain 3 pairs

Fig. : Attenuation of The trp Operon mRNA

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Fig. Working of Insulators

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 Alkylating agents (e.g., N-ethyl-N-nitrosourea): These agents can mutate

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 Mutations whose effect is to juxtapose previously separate pieces of DNA, potentially

Fig. : Chromosome Structure after Mutation

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Fig. : Mutation Impact on Protein Sequence

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Regulation of Transcription in Eukaryotes

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H3 at lysine 9 (abbreviated as dimethylated H3K9) and methylation of histone H3 at lysine 27 (H3K27)

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DNA Methylation and Gene Regulation

Fig. : Methylation of cytosine.

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Low [Iron] High [Iron]

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Fig. : Translational silencing by miRNA.

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2. What is the role of topoisomerases in eukaryotic DNA replication ?

3. Which of the following is true regarding translation ?

4. Why does the genetic code have to be a triplet code ?

5. Which of the following is MISMATCHED ?

6. Which of the following is mismatched ?

7. Unwinding of DNA is done by

8. Eukaryotes differ from prokaryote in mechanism of DNA replication due to :

9. At a replication fork, the leading strand is synthesized :

10. Which of the following describes a primer used in DNA replication ?

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