• At the same time when the cell reproduces,
the DNA also should duphcate itself to distribute equally to the daughter
cells.
• As a carrier of genetic information, DNA has
to perform two important functions:
•
When DNA directs the synthesis of chemical
molecules other than itself, then such functions of DNA are called Heterocatalytic functions.
Eg
•
Synthesis of RNA (transcription), .
•
synthesis of protein (Translation), etc.
• b) Autocatalytic function:
• When DNA directs the synthesis of DNA itself,
then such function of DNA is called autocatalytic function. E.g. .
•
Replication. -The process b which DNA
duplicates itself is called replication.
•
Through replication, it forms two copies that
are identical to it.
• In eukaryotic organisms, replication of DNA
takes place only once in the cell cycle. It occurs in the S- phase of interphase in t ell cycle.
• DNA
Replication through Semiconservative
method.
• The model for Semiconservative replication
was proposed by Watson and Crick, on the basis of antiparallel and
complementary nature of DNA strands.
• The process of semiconservative replication
is as below
Process of semiconservative method of DNA replication-
1)Activation of Nucleotides:
i)The four types of n Q €0tides of DNA i.e. dAMP, dOMP, dCMP and dTMP are present in the nucleoplasm.
ii)They are activated by ATP in presence of an enzyme phosphorylase.
iii)This results in the formation of deoxyribonucleotide triphosphates i.e. dATP, dOTP, dCTP and dTTP.
The process is known as Phosphorylation.
2)Point of Origin or Initiation point:
i)It begins at specific point '0' -origin and terminates at point 'T'. Origin is flanked by 'T' sites.
ii)The unit of DNA in which replication occurs, is called replicon.
iii) In prokaryotes, there is only one replicon however in eukaryotes, there are several replicons in tandem.
iv)At the point '0', enzyme endonuclease binds to one of the strands of DNA, temporarily.
v)The binding occurs in the sugar-phosphate back bone or the phosphodiester bondi)
3)Unwinding of DNA molecule:
i)Now enzyme DNA helicase breaking weak hydrogen bonds at the origin point '0.
ii)The strands of DNA separate and unwind.
iii)This unwinding is bidirectional and continues as 'Y' shaped replication fork. Unwinding of DNA molecule:
iv)Each separated strand acts as template Unwinding of DNA molecule:
v)The two separated strands are prevented from recoiling (rejoining) by SSBP (Single strand binding proteins). Unwinding of DNA molecule:
vi)SSB proteins remain attached to both the separated strands so as to start synthesis of new polynucleotide strands
4) Replicating fork:
The point formed due to unwinding and sepa {ibn of two strands appear like a Y -shaped fork, called replicating/ replication fork. The unwinding of strands imposes strain which is relieved by super-helix relaxing enzyme.
5)Synthesis of new strands:
i)Each separated strand acts as aold or template for the synthesis of new complementary strand.
ii)It begins with the help of small RNA molecule, called RNA primer.
iii)RNA primer gets associ ·ed with the 3' end of template strand and attracts complementary nucleotides from surrounding nuoleop asm.
iv)These nucleotides molecules bind to the complementary nucleotides on the template strand by forming hydrogen bonds. (i.e. A=T or T=A; G = C or C = G).
v)The newly bound nucleotides get interconnected by phosphodiester bonds, forming a polynucleotide strand.
vi)The synthesis of new complementary strand is carried out by enzyme DNA polymerase.
vii)The new complementary strand is always formed in 5' -3' direction.
6)Leading and Lagging strand:
i)The template strand with free 3' end is called leading template and with free 5' end is called lagging template.
ii) The process of replication always starts at C-3 end of template strand and proceeds towards C-5 end.
iii)As both the strands of the parental DNA are antiparallel, new strands are always formed in 5' ~ 3' direction.
iv)One of the newly synthesized strands develops continuously towards replicating fork is called leading strand (produced on 3 'templet strand)
v)Another new strand develop discontinuously away from the replicating fork is called lagging strand (produced on 5' temp let strand).
Maturation of Okazaki fragments:
i)DNA synthesis on lagging template takes place in the form of small fragments, called Okazaki fragments (named after scientist Okazaki).Okazaki fragments are joined by enzyme DNA ligase.
ii)RNA primers are removed by DNA polymerase and replaced by DNA sequence with the help of DNA polymerase- I in prokaryotes and DNA polymerase-a in eukaryotes.
iii)Finally, DNA gyrase (topoisomerase) enzyme forms double helix to form daughter DNA molecules.
7)Formation of daughter DNA molecules:
i)At the end of the replication, two daughter DNA molecules are formed. .
ii)In each daughter DNA, one strand is parental and the other one is totally newly synthesized.
iii)Thus, 50% is contributed by mother DNA.
iv)Hence, it is described as semiconservative replication.
Experimental
confirmation:
Semiconservative
Replication:
i)In newly formed DNA molecule, one strand is old
(i.e. conserved) and another strand is newly synthesized.
ii)Thus, it is called Semiconservative mode of
replication.
iii)It is experimentally proved by Matthew Meselson
and Franklin Stahl (1958) by using equilibrium - density - gradient - centrifugation technique.
1. Meselson and Stahl in 1958 performed an
experiment to prove semi nservative nature (mode) of replication.
2. They cultured bacteria E.coli in the
medium containing 14N (lig t itrogen) and obtained equilibrium density gradient
band by using 6M CsCl2. The position of this band is: G0rded.
3. E. coli cells were then transferred to 15N medium (heavy i e opic nitrogen) and
allowed to replicate for several generations.
At equilibrium point density gradient band was
obtained by using 6M CsCl2. The position of this band is recorded.
4. The heavy DNA (15N) molecule can be distinguished
from normal DNA by centrifugation in a 6M Cesium chloride (CsCh) density
gradient. The density gradient value of 6M CsCh and 15N DNA is almost same.
Therefore, at the equilibrium point 15N DNA will form a band. In this both the
strands of DNA are labelled with 15N.
5. Such E. coli cells were transferred to
another medium containing 14N i.e. normal (light) nitrogen. After first
generation, the density gradient band for 14N 15N was obtained and its position
was recorded.
After second generation, two density gradient bands
were obtained - one at 14N 15N position and other at 14N position.
6. The position of bands after two generations
clearly proved that DNA replication is Semiconservative.
Interpretation
of results of Meselson’s experiment on the separation of DNA by equilibrium density gradient
centrifugation
Protein synthesis:
1)Proteins are very important biomolecules.
2)They serve as structural components, enzymes and hormones
3)The cell needs to synthesize new protein molecules.
4)The process of protein synthesis includes transcription and translation.
5)The process of copying of genetic information from one (template) strand of DNA into a single stranded RNA transcript, is termed as transcription.
6)During this process, synthesis of complementary strand of RNA takes place (Except that the Adenine nitrogen base pairs with the Uracil base instead of Thymine).
Double stranded DNA molecule gives rise to mRNA which acts as a messenger to programme the synthesis of a polypeptide chain (protein). This type of unidirectional flow of information from DNA to RNA to protein! proteins is referred as central dogma of molecular biology. It was postulated by F.H.C. Crick in 1958.
•
1)During transcription, information of only one
&trand of DNA is copied into RNA.
• 2)This strand of DNA acts as template. Enzyme RNA polymerase catalyses the formation
of RNA transcript.
• 3)DNA is located in the nucleoid of re aryotes and in nucleus of Eukaryotes.
• 4)DNA transcription takes place in nueleus in
eukaryotes whereas translation occurs in cytoplasm.
• 5)DNA transfers information t/m-RNA which then moves
to ribosomes.
• 6)Transcription occurs in th nuc eus during Gland G2
phases of cell cycle.
• 7)DNA has promotor an erminator sites.
• 8) Transcription starts at promotor site and stops
at terminator site.
• 9)Actually, the proeegs of transcription, in both
Prokaryotes and Eukaryotes, involves three stages-
•
Initiation
•
Elongation
•
Termination.
•
Each
transcribed segment of DNA is called transcription unit.
•
It consists of
– i) Promotor, ii)structural gene, iii)
terminator.
Two strands of DNA in the
structural gene show following features: –
•
i) Promotor
•
i)The promotor is located towards 5' end of structural gene i.e.
upstream. .
•
ii)It is a DNA
sequence that provides binding site for enzyme RNA polymerase. .
•
iii)RNA
polymerase binds to specific Promotor. .
• iv)In
prokaryotes, the enzyme recognizes the promotor by its sigma factor sub unit.
•
ii) Structural
genes -
•
a)Two strands
of DNA have opposite polarity. .
•
b)DNA
dependent RNA polymerase catalyses polymerization in 5'~3' direction. .
•
c)So, the DNA
strand having 3'~5' polarity acts as template strand. .
•
d)The other strand
of DNA having 5'~3' polarity is complementary to template strand.
•
e)The sequence
of bases in this strand, is same as in RNA (where Thymine is replaced by
Uracil). .
•
f)It is the
actual coding strand. .
•
g)The
information on this strand of DNA is copied on mRNA. This is called sense
strand.
•
iii) Terminator-
The terminator is located at 3' end of coding strand
i.e. downstream. It defines the end of the transcription process.
- •
After binding
to promoter, RNA polymerase moves along the DNA and causes local unwinding of
DNA duplex into two chains in the region of the gene.
- • Exposed ATCG bases project into nucleoplasm. Only one strand functions as template (antisense strand) and the other strand is complementary
- • which is actually a coding strand (sense strand).
- • The ribonucleoside tri phosphates join to bases of DNA template chain. .
- • As transcription proceeds, the hybrid DNA-RNA molecule dissociates and makes mRNA molecule free.
- •
When RNA polymerase reaches the terminator signal on
the DNA, it leaves DNA and fully formed mRNA (primary transcript) is released.
- •
As the mRNA grows, the transcribed region of DNA
molecule becomes spirally coiled and attains (regains) double helical form.
- •
In bacteria, m-RNA does not require any processing
because it has no introns.
- •
Prokaryotes possess only one type of RNA polymerase.
- •
In eukaryotes, there are three types RNA
polymerases. .
- •
RNA polymerase-I transcribes r-RNA. RNA
polymerase-II transcribes m-RNA (primary transcript) and heterogeneous nuclear
RNA (or hnRNA).
- •
RNA polymerase-III is responsible for transcription
oft-RNA and small nuclear RNA (snRNA).
Transcription unit and the gene:
1) The DNA sequence coding for m-RNA! t-RNA
or r- RNA is defined as a gene.
2)Cistron is a segment of DNA coding for a
polypeptide.
3)A single structural gene in transcription unit is
said to be monocistronic where as a long segment of
4)DNA having set of various structural genes in one
transcription unit is referred as polycistronic. .
5) Structural genes in eukaryotes have interrupted
non-coding sequences (introns).
6)The coding sequences or express- sequences are
defined are exons.
7) Only exons appear in processed mRNA in
Eukaryotes.
Processing of hnRNA:
1)In eukaryotes, forms of RNA transcribed from DNA
are called primary transcripts.
2)Such transcripts undergo changes called processing
or maturation before becoming functional.
3)Primary transcript is nonfunctional and contains
both exons and introns.
4)During processing only introns are removed by the
process called splicing. .
5)Exons are joined in a definit€~equence (order) by
DNA ligase enzyme. .
6)Heterogeneous nuclear RNA, undergoes the process
of c :rnmg and tailing.
7)In capping, methylated na osine tri phosphate is
added to 5' end of hnRNA. In tailing, polyadenylation
take place at 3' end.
8)It is the fully
processed hnRNA, now called
m-RNA. .
9)For translation m-RNA is transported out of the
nucleus through nuc ear pore.
