Replication, Transcription,

Translation and its

regulation

By,
Abhinava J V
University of Agricultural Sciences,
Dharwad
 DNA REPLICATION

 DNA replication is
the process by
which DNA makes a
copy of itself during
cell division.
 It is
Semiconservative
replication.
 Semiconservative
replication would
produce two copies
that each contained
one of the original
strands and one
 Initiation
 The first step in DNA replication is to ‘unzip’ the
double helix structure of the DNA molecule at Origins
of Replication region & it is mediated by is mediated
by DnaA & helicase in Prokaryotes & DNA
polymerase α in Eukaryotes.
 The separation of the two single strands of DNA
creates a ‘Y’ shape called a replication ‘fork’. The
two separated strands will act as templates for
making the new strands of DNA.
 SSB protein prevent the single strands of DNA from
forming any secondary structures and to prevent them
from reannealing and DNA gyrase is needed to
relieve the stress helicase and in eukaryotes it is
maintained by Topoisomerase.
 Before new DNA strands can form, there must be
RNA primers present to start the addition of new
nucleotides
 Primase is the enzyme that synthesizes the RNA
Primer
 DNA polymerase can then add the new
nucleotides
 Elongation
 DNA polymerase uses each strand
as a template in the 3’ to 5’ direction to
build a complementary strand in the 5’
to 3’ direction by adding the
complementary dNTP’s.
 One of the strands is oriented in the 3’
to 5’ direction, this is the leading
strand The other strand is oriented in
the 5’ to 3’ direction, this is the lagging
strand.
 As a result of their different
orientations, the two strands are
leading strand
 DNA polymerase slides along the leading
strand in the 3’ to 5’ direction synthesizing
the matching strand in the 5’ to 3’
direction.
 The RNA primer is degraded by RNase H
and replaced with DNA nucleotides by
DNA polymerase, and then DNA ligase
connects the fragment at the start of the
new strand to the end of the new strand
lagging strand
 Chunks of DNA, called Okazaki fragments, are then
added to the lagging strand also in the 5’ to 3’ direction
& it occurs in discontinuous manner
 The RNA primers are degraded by RNase H and replaced
with DNA nucleotides by DNA polymerase
 DNA ligase connects the Okazaki fragments to one another
 Termination
 Termination of DNA replication in Prokaryotes is
completed through the use of termination
sequences and the Tus protein. These
sequences allow the two replication forks to
pass through in only one direction, but not the
other.
 In eukaryotic cells the end replication problem
of telomere regions is handled by telomerase.
 Telomeres extend the 3' end of the parental
chromosome beyond the 5' end of the daughter
strand.
 This 3' addition provides a template for
extension of the 5' end of the daughter strand
by lagging strand DNA synthesis.
 Regulation
 In Prokaryotes, regulation of DNA replication
is achieved through several mechanisms.
 Mechanisms involve the ratio of ATP to
ADP, of DnaA to the number of DnaA
boxes and the hemimethylation and
sequestering of OriC.
 In Eukaryotes, DNA replication is controlled
within the context of the cell cycle.
 As the cell grows and divides, it progresses
through stages in the cell cycle; DNA
replication takes place during the S phase
(synthesis phase). The progress of the
eukaryotic cell through the cycle is
controlled by cell cycle checkpoints.
 TRANSCRIPTION
 Transcription is the synthesis of a single-stranded
RNA molecule using the DNA template.
 Regulated by gene regulatory elements within each
gene.
 RNA is transcribed 5’ to 3’ from the template (3’ to 5’).

 Similar to DNA synthesis, except:

 NTPs instead of dNTPs (no deoxy-)
 No primer
 No proofreading
 Adds Uracil (U) instead of thymine (T)
 RNA polymerase
Initiation
 RNA polymerase combines with sigma factor (a
polypeptide) to create RNA polymerase holoenzyme
which recognizes promoters and initiates transcription.
 RNA polymerase holoenzyme binds promoters and
untwists DNA
 It binds loosely to the -35 promoter and then binds
tightly to the -10 promoter and untwists the DNA
Elongation
 Elongation of RNA chain takes place by the
addition of ribonucleotides to the 3'-end of
the RNA so that the RNA chain grows in 5'-
3' direction.
 As elongation proceeds, the DNA is
continuously unwound ahead of the core
enzyme and rewound behind it .
 Since the base pairing between DNA and
RNA is not stable enough to maintain the
stability of the mRNA synthesis
components, RNA polymerase acts as a
stable linker between the DNA template
and the nascent RNA strands to ensure
that elongation is not interrupted
prematurely.
Termination
 In prokaryotes, termination of
transcription is brought about by certain
termination signals on DNA called
terminators (these are DNA sequences).
1. Intrinsic termination- In the stem of
the RNA, there is a stretch of G-C
reach segment. The G-C reach
segment results in a hair-pin loop
formation in the RNA stem.
• As a result the weak association
between A-U in the long stretch of
termination sequence break and the
RNA is released.
2. Extrinsic termination- rho protein
dependent.
 Rho factor binds to the 5'-end of
nascent m-RNA and scans down along
the length of m-RNA until it reaches the
termination point.
 At termination point when the
transcription slows down rho breaks
ATP and utilizes that energy to denature
the RNA-DNA hybrid so that the RNA is
released from the bubble.
 In Eukaryotes the termination process
differs for each of the three RNA
polymerases.
 Most of eukaryotes possess robust
methods of regulating transcription
initiation on a gene-by-gene basis.
 The transcription of a gene can be
regulated by cis-acting elements
within the regulatory regions of the
DNA, and trans-acting factors that
include transcription factors and the
basal transcription complex.
Post-Transcriptional Modification
 DNA transcription occurs in a cell's
nucleus.
 The RNA that is synthesized in this
process is then transferred to the cell's
cytoplasm where it is translated into a
protein.
 In prokaryotes, the RNA it is ready for
translation into a protein.
 Eukaryotic RNA from DNA transcription,
is not immediately ready for translation
and it needs Post-Transcriptional
Modification.
 RNA splicing - A two-step reaction in
which introns are removed from a
primary RNA transcript and exons are
joined together to form mature mRNA.
 5' Capping - occurs in cell nucleus, the
addition of a GTP molecule to the 5' end
of a primary RNA transcript forming a 5'-
5' linkage between the two.
 Poly A tail - occurs in cell nucleus, A
string of up to 500 adenines added to
the 3' end of primary RNA transcripts.
Addition catalyzed by the enzyme poly
(A) polymerase that recognizes the
sequence AAUAAA.
 Regulation
 Prokaryotic transcription is regulated by
three main sequence elements.
 Promoters are elements of DNA that
may bind RNA polymerase and other
proteins for the successful initiation of
transcription directly upstream of the
gene.
 Operators recognize repressor proteins
that bind to a stretch of DNA and inhibit
the transcription of the gene.
 Positive control elements that bind to
 In Eukaryotes 3 mechanisms. Were
involved
 Control over polymerase access to the
gene. This includes the functions of
histone, remodeling enzymes,
transcription factors, enhancers and
repressors, and many other complexes.
 Productive elongation of the RNA
transcript. Once polymerase is bound to
a promoter, it requires another set of
factors to transcribing the RNA.
 Termination of the polymerase A
number of factors which have been found
to control how and when termination
occurs, which will dictate the fate of the
 TRANSLATION
 The mechanism for translating
messenger RNA into protein in
eukaryotic cells is basically the same as
in prokaryotes. That is, messenger RNA
is read by ribosomes.
 The ribosome and its subunits are
larger in eukaryotes. 40S and 60S
subunits combine to form a functional
80S ribosome. In prokaryotes, the
analogous particles are 30S & 50S
subunits combine to form a functional
70S.
 Initiation
 Initiation of translation involves the
assembly of the ribosomal subunits and
it is mediated by the Initiation factors.
 Prokaryotes- IF1, IF2, and IF3
 Eukaryotes – eIF1, eIF2, eIF3, eIF4,
eIF5, eIF6
 In Eukaryotes Initiation usually involves
the interaction of certain key proteins
with a special tag bound to the 5' cap.
 The ribosome has three sites: the A site,
the P site, and the E site.
 The A site is the point of entry for the
aminoacyl tRNA (except for the first
aminoacyl tRNA, fMet-tRNAfMet, which
enters at the P site).
 The P site is where the peptidyl tRNA is
formed in the ribosome.
 The E site which is the exit site of the
now uncharged tRNA after it gives its
amino acid to the growing peptide chain.
 The smaller subunit binds to the mRNA in
upstream purine-rich region of the AUG
(initiation codon).
 Elongation
 Elongation of the polypeptide chain
involves addition of amino acids to the
carboxyl end of the growing chain.
 Elongation starts when the fMet-tRNA
enters the P site, causing a
conformational change which opens the
A site for the new aminoacyl-tRNA to
bind.
 Now the P site contains the beginning
of the peptide chain of the protein to be
encoded and the A site has the next
 The growing polypeptide connected to
the tRNA in the P site is detached and a
peptide bond is formed between the last
amino acids of the polypeptide and the
amino acid still attached to the tRNA in
the A site. This process, known as
peptide bond formation, is catalyzed by
a ribozyme.
 The newly formed peptide in the A site
tRNA is known as dipeptide and the
whole assembly is called dipeptidyl-
tRNA.
 The tRNA in the P site minus the amino
 In the final stage of elongation, called
translocation, the deacylated tRNA (in
the P site) and the dipeptidyl-tRNA (in
the A site) along with its corresponding
codons move to the E and P sites,
respectively, and a new codon moves
into the A site. This process is catalyzed
by elongation factor G (EF-G).
 The deacylated tRNA at the E site is
released from the ribosome during the
next A-site occupation by an aminoacyl-
tRNA it is facilitated by EF-Tu.
 Termination
 Termination occurs when termination
codons (UAA, UGA, or UAG )moves into
the A site.
 These codons are recognized by proteins
called release factors, namely RF1
(recognizing the UAA and UAG stop
codons) or RF2 (recognizing the UAA and
UGA stop codons).
 These factors trigger the hydrolysis of the
ester bond in peptidyl-tRNA and the
release of the newly synthesized protein
from the ribosome.
 RF-3 catalyzes the release of RF-1 and
RF-2 at the end of the termination process
 Regulatation
 Translation is regulated on many levels.
The joining of the two ribosomal subunits
can be blocked by RsfS.
 RsfS binds to L14, a protein of the large
ribosomal subunit, and thereby blocks
joining of the small subunit to form a
functional 70S ribosome, slowing down or
blocking translation entirely.
 RsfS proteins are found in almost all
eubacteria (but not archaea) and homologs
are present in mitochondria and
chloroplasts (where they are called
C7orf30 and iojap, respectively).
 RNA interference (RNAi) : On the
other hand to control the synthesis of
Proteins from the mRNA, DNA
synthesis the complementary RNA to
that mRNA & it is called as RNA
interference.
 It is done by small RNA called as
microRNA (miRNA) and small
interfering RNA (siRNA)
 It forms the RNA-RNA Hybrid and
thus involves in the cleavage of mRNA
was occur by the enzyme called Dicer.