DNA REPLICATION AND

TRANSCRIPTION
 DNA replication involves separation of the parent strands to
form 2 daughter strands identical to the parent strand
(semiconservative replication).
 Each parent strand serves as a template for the synthesis of
a new strand.
 Each template strand is complementary to the daughter
strand which is dependent on the Watson and crick base
pairing.
 In this way genetic information is duplicated and
transmitted to the next generation.
Stages of DNA replication

1. Initiation
 Begins with the correct assembly of the replication proteins at the
site where DNA replication is to start.
 This phase starts with the unwinding of the chromosome at a single
origin site, termed the oriC locus.
 It consists of a unique sequence of 245 base pairs that is very rich
in A-T pairs, presumably to facilitate strand separation.
 A class of proteins termed helicases accomplishes separation. A
complex of up to 30 subunits of the 52-kD Helicase DnaA protein
binds to oriC and causes a segment of DNA to melt open.
Proteins involved in initiation of replication
A. Helicases
 Strand separation by helicases is brought about
through dissolution of hydrogen bonds holding the
two DNA strands together.
 Initial separation at the initiation site by DnaA
protein is continued further by DnaB protein, which
is the major strand separating protein. It separates the
DNA strands in both directions, resulting in the
formation of two replication forks which move in
opposite directions.
B. SSB protein
 Once separated, the single-stranded regions associate
with SSB protein, which prevents reannealing.
 As the helicase moves in advance of the replication
fork, the SSB proteins move on and off the DNA and
are recycled for use on another site.
DNA gyrase (topoisomerase)
 The strand separation at the replication fork applies a
turning force to the double helix, which may soon
result in positive supercoiling (overwinding) of the
remaining unseparated DNA and cessation of further
separation.
 This is prevented by DNA gyrase, a DNA-
topoisomerase that relaxes positive supercoils
passively and introduces negative supercoils by an
ATP-dependent mechanism.
 Gyrase, SSB and helicases are collectively known as
unwinding proteins; together they create single-
stranded DNA for replication.
2. Elongation

 The principal enzymes for the synthesis of new strands are

DNA polymerases (III) which catalyze polymerization of
deoxyribonucleotides under direction of DNA template.
 These enzymes catalyze the stepwise addition of
deoxyribonucleotide residues to the free 3’-hydroxyl end
of a pre-existing DNA or RNA primer strand; thus the
replication is propagated in the 5’-3’ direction.
 DNA polymerases need participation of the following:
DNA template: Each separated parental polynucleotide strand serves as template and copied
by DNA polymerases according to Watson–Crick base pairing rules.
Precursors: Any of the four deoxyribonucleotide-5’-triphosphate (dATP, dGTP, dCTP and
dTTP) can serve as a precursor molecule for DNA synthesis.
Primer: DNA polymerase is incapable of assembling the first nucleotide of a new chain. It
needs a pre-existing oligonucleotide sequence called primer, containing a 3’-hydroxy group at
end. This group attacks the innermost phosphate of the incoming precursor molecule and
forms a phosphodiester bond at the end of the growing chain.
A primase forms a short stretch (approximately 3–5 ribonucleotide units) of RNA in the
replication fork. This small RNA, base paired with the template strand, is extended by the
DNA polymerase III. The primer is hydrolyzed soon after by DNA polymerase I. The same
enzyme then fills the gap so created with a new deoxyribonucleotide (DNA) sequence. DNA
ligase then joins the loose ends.
 Leading and lagging strands: DNA polymerase can synthesize a new
chain only in the 5’-3’ direction, reading the template in 3’-5’.
 Only on one of the strands, continuous synthesis takes place in 5’-3’
direction, which is also direction of movement of the replication fork. This
strand is termed as the leading strand.
 In the other strand, known as the lagging strand, synthesis takes place in
short DNA fragments, named Okazaki fragments. As the replication fork
moves, the primase comes into action repeatedly, each time synthesizing
short oligonucleotide sequences which serve as primers for DNA
polymerase III.
 DNA fragments, each approximately 1000 nucleotides long, are then added
by DNA polymerase III at the 3’ ends of these primers. The RNA primer is
removed soon by 5’-exonuclease activity of DNA polymerase I and the
gap is filled by its polymerase activity: it synthesizes a fragment of DNA
that replaces the RNA primer.
 Finally, the enzyme DNA ligase catalyzes formation of a continuous strand
of DNA by joining the 3’-OH at the end of one DNA fragment to the 5’-
monophosphate of the adjacent fragment.
3. Termination

 This involves the disassembly of the protein machinery and the separation of daughter
molecules. The two replication forks moving in opposite directions from the origin meet at
the opposite side of the circular DNA. This is the terminal event in DNA replication.
Roles of DNA polymerases

1. All DNA polymerases select the nucleotide that is to be added to the 3'-OH end of the growing chain and catalyze
formation of the phosphodiester bond. The substrates for DNA polymerases are the four deoxynucleoside-5'-triphosphates
(dATP dCTP, dGTP, and dTTP) and a single-stranded template DNA.
2. No DNA polymerase can catalyze the reaction between two free nucleotides, even if one has a 3'-OH group and the other a
5'-triphosphate. Polymerization can occur only if the nucleotide with the 3'-OH group is hydrogen-bonded to the template
strand. Such a nucleotide is called a primer.
3. The primer can either be a single nucleotide or the terminus of a hydrogen-bonded oligonucleotide. When an incoming
nucleotide is joined to a primer it supplies another free 3'-OH group, so that the growing strand itself is a primer. Since
polymerization occurs only at the3'-OH end, strand growth is said to proceed in the 5'- 3' direction.
4. All known polymerases (both DNA and RNA) are capable of chain growth only in the 5'-3' direction. This unidirectional
feature of polymerases complicates the simultaneous replication of both strands of DNA.
5. Although present in very low concentration in the cell, polymerase III, also called replicase, is the polymerase that
elongates both strands of DNA in the replication fork. Polymerase I is primarily a DNA repair enzyme and is responsible
for excision of the short RNA primer that is required to initiate DNA synthesis on both the leading and lagging strands of
DNA during replication. It also can remove mismatched base pairs during replication and fill in gaps in single-stranded
DNA that is joined in a double helix.
Role of DNA polymerase 1

1. Pol I Can Edit Its Mistakes via 3’-5’ and 5’-3’ exonuclease activity (proof reading, editing.
Pol I therefore has the ability to proofread or edit a DNA chain as it is synthesized so as to
correct its mistakes.
2. Pol I Functions Physiologically to Repair DNA. Damaged DNA is detected by a variety of
DNA repair systems. Many of them endonucleolytically cleave the damaged DNA on the
5’ side of the lesion, thereby activating Pol I’s 5’-3’ exonuclease. While excising this
damaged DNA, Pol I simultaneously fills in the resulting single-strand gap through its
polymerase activity.
3. Pol I’s 5’ - 3’ Exonuclease Functions Physiologically to Excise RNA Primers. Pol I’s 5’ –
3’ exonuclease also removes the RNA primers at the 5’ ends of newly synthesized DNA
while its DNA polymerase activity fills in the resulting gaps.
DNA polymerase III

 It is a component of the repliosome and helps in replication of leading and lagging strand.
Alpha subunit is responsible for the polymerase activity and epsilon subunit has
proofreading exonuclease activity.
 DNA Polymerase III is the major enzyme of DNA replication. Its processivity is high: it
can catalyze polymerization of thousands of nucleotides at a rate of almost 1000
nucleotides per second.
 Pol III is similar to pol I in that it has a requirement for a template and a primer.
TRANSCRIPTION

 Transcription is a cellular process during which RNA is synthesized using DNA as a

template. All three types of cellular RNAs are copied from DNA template in the process of
transcription. Only mRNA contains instructions for protein synthesis. The other two types
(rRNA and tRNA) are not translated, but perform other vital functions during protein
synthesis.
 It differs from replication in two important aspects:
 1. Transcription does not require a primer. RNA polymerase does not need a primer and RNA
polymerase lacks the ability to excise mismatched nucleotides.
 2. It involves only short segments of DNA. Within these segments, one of the separated strands of
DNA serves as a template (i.e. the template strand); the other strand of DNA is the coding strand.
Synthesis of RNA is similar to DNA synthesis in several aspects.
1. Direction of synthesis is 5’ to 3’.
2. The mechanism of elongation is similar; ribonucleotides are added to the 3’-OH group at the
terminus of the growing chain.
RNA polymerase (RNAP) is the principal enzyme of transcription which synthesizes all three types
of cellular RNAs—mRNA, tRNA and rRNA. It acts according to instructions given by a DNA
template and does not need a primer. It is a complex enzyme (500 kD), consisting of various subunits.
RNA polymerase requires the following components:
3. A template. Double stranded DNA or single stranded DNA.
4. Activated precursors. All four ribonucleoside triphosphates-ATP, GTP, UPT and CTP.
5. A divalent metal ion either Mg 2+ or Mn2+ is effective.
 Stages of transcription

1. Initiation
 Transcription is initiated when RNA Polymerase (RNAP) interacts
with the recognition sequences on the coding strand of DNA, called
the promoters.
 Then the enzyme slides along DNA. As it encounters the promoter
sequence, it stops moving further. The transcription initiation complex
is now formed, called the closed complex; it consists of the enzyme
plus the duplex DNA.
 The duplex DNA must be unwound at this stage, so that one of its
strands may serve as template. This unwinding is brought about by the
enzyme, starting at the conserved AT-rich sequence. This sequence,
called the Pribnow box (after David Pribnow who described it in
1975) in prokaryotes, lies in a region about 10 base pairs upstream of
the site where mRNA synthesis starts (i.e. transcription initiation site).
A segment of nearly 17 base pairs of DNA is unwound in this manner.
 The promoter sequences are clustered approximately 10 base pairs and
35 base pairs upstream of the transcriptional initiation site.
 The –10 sequence has the consensus TATAAT.
The –35 sequence has the consensus TTGAGA
and is important in DNA-unwinding during
transcriptional initiation.
 Eukaryotic genes encoding proteins have promoter
sites with a TATAAA consensus sequence called a
TATA box of Hogness box centered at -25. Many
eukaryotic promoters also have a CAAT box with
a GGCNCAATCT consensus sequence at about
-75.
 The promoter sequences are located on the coding
strand and not on the template strand of the DNA.
2. Elongation
 RNA polymerase plays a pivotal role in the elongation phase, catalyzing the
polymerization. New nucleotide units are incorporated in the emerging RNA, one at a
time, according to the base pairing rule. Thus, A in DNA is transcribed to U in mRNA, G is
transcribed to C.
 The polymerization occurs in the 5’-3’ direction like in case of replication. RNAP is
processive, i.e. a single enzyme molecule can remain attached to the template and carry out
transcription till the end. The region containing RNA polymerase, DNA and emerging
RNA is called a transcription bubble.
 Length of the DNA-RNA hybrid, and the extent of unwound area in duplex remains
constant as the RNA polymerase moves along the DNA template. This indicates that DNA
is rewound upstream at the same rate as it is unwound downstream. Furthermore, as RNA
polymerase causes local unwinding of the duplex DNA, its movement is associated with
generation of waves of positive supercoiling ahead of it and of negative supercoiling
behind it. Such transcription driven supercoilings are relieved by action of topoisomerases.
 Sense (+) and antisense (–) strands: only one strand of the duplex DNA is copied,
directing synthesis of new RNA in a given region of genome; it is the template or non-
coding strand, also called the antisense (–) strand. The other strand is sense strand, also
called coding (or non-template) strand. The RNA produced has same sequence as the the
sense (+) strand (except that T replaces U).
Termination
 RNAP must know the defined site at which to stop RNA synthesis, so that the appropriate size of
transcript is produced. It occurs by two well characterized mechanisms.
 The first mechanism, rho-dependent termination, requires the action of a protein factor, called rho
factor, which recognizes certain termination signals. This halts movement of RNAP along the DNA
template.
 The other mechanism does not require participation of the rho factor i.e. (rho-independent
termination). A consistent feature of all transcriptional termination sites is the presence of a
palindrome i.e. the base sequence of one DNA strand, read in one direction is same as that of the
other
 When palindrome is transcribed, the base sequence of the RNA transcript is self-complementary.
Therefore, the mRNA transcript of this region forms a self-complementary “hairpin” structure due to
internal base pairing. The hairpin loop is often rich in GC.
 The GC rich region is often followed by a sequence of 6–8 uridine (U) residues. Formation
of this secondary loop-structure dislodges the RNAP from the DNA template, resulting in
termination of the RNA synthesis in the U stretch.
Post transcriptional modifications
 Most prokaryotic mRNA transcripts participate in translation without
further alteration. However, most primary transcripts in eukaryotes
require extensive post-transcriptional modifications to become
functional.
 For mRNAs, these modifications include the addition of a 7-
methylguanosine-containing “cap” that is enzymatically appended to
the transcript’s 5’ end and 250-nucleotide polyadenylic acid [poly(A)]
“tail” that is enzymatically appended to its 3’ end.
 However, the most striking modification that most eukaryotic
transcripts undergo is a process called gene splicing in which one or
more often lengthy RNA segments known as introns (for
“intervening sequences”) are precisely excised from the RNA and
the remaining exons (for “expressed sequences”) are rejoined in
their original order to form the mature mRNA.
 Different mRNAs can be generated from the same gene through the
selection of alternate transcriptional initiation sites and/or alternative
splice sites, leading to the production of somewhat different proteins,
usually in a tissue-specific manner.