biotechnology, genetics

1. DNA REPLICATION
A KAVITHA
DEPARTMENT OF PHYSIOLOGY

2. • DNA Replication, the process by which a cell
doubles its DNA before division.
• Basic mechanism evolved from Watson and Crick
model of DNA .
In this chapter :
Modes of replication
Requirements of replication
Participating enzymes and the proteins
Knowing the essentiality for the segregation of
homologous chromosomes, for the production of
genetic variants and for DNA repair

3. MODELS OF REPLICATION
• From the three dimensional structure of DNA
proposed by Watson and Crick . Several important
genetic implications were immediately apparent.
• Initially, three alternative models for DNA replication
were proposed.
• Conservative replication
• Dispersive replication
• Semi-conservative replication

4. • In conservative replication,
the entire double-stranded DNA molecule
serves as a template for a whole new molecule
of DNA, and the original DNA molecule is fully
conserved during replication.
• In dispersive replication
both nucleotide strands break down (disperse)
into fragments, which serve as templates for the
synthesis of new DNA fragments, and then
somehow reassemble into two complete DNA
molecules.
In this model, each resulting DNA molecule is
interspersed with fragments of old and new DNA;
none of the original molecule is conserved.

5. Semi-conservative replication
• The complementary nature of the two nucleotide strands in a DNA
molecule suggested that,
During replication, each strand can serve as a template for the
synthesis of a new strand.
The specificity of base pairing (adenine with thymine; guanine with
cytosine) implied that only one sequence of bases can be specified
by each template
So two DNA molecules built on the pair of templates will be identical
with the original.
• This process is called semiconservative replication, because
each of the original nucleotide strands remains intact
(conserved), despite no longer being combined in the same
molecule; the original DNA molecule is half (semi) conserved
during replication.

7. These three models allow different predictions to be
made about the distribution of original DNA and newly
synthesized DNA after replication.
• With conservative replication,
after one round of replication, 50% of the
molecules would consist entirely of the original DNA
and 50% would consist entirely of new DNA. After a
second round of replication,25% of the molecules
would consist entirely of the original DNA and 75%
would consist entirely of new DNA.
With each additional round of replication, the
proportion of molecules with new DNA would
increase, although the number of molecules with the
original DNA would remain constant.

8. • Dispersive replication would always produce hybrid
molecules, containing some original and some new
DNA, but the proportion of new DNA within the
molecules would increase with each replication event.
• With semiconservative replication, one round of
replication would produce two hybrid molecules,
each consisting of half original DNA and half new
DNA. After a second round of replication half the
molecules would be hybrid, and the other half would
consist of new DNA only. Additional rounds of
replication would produce more and more molecules
consisting entirely of new DNA, and a few hybrid
molecules would persist.

9. Meselson and Stahl’s Experiment
To determine which of the three models of replication
applied to E. coli cells, Mathew Meselson and Franklin
Stahl needed a way to distinguish old and new DNA.
• They did so by using two isotopes of nitrogen, 14N
(the common form) and 15N (a rare, heavy form).
• Meselson and Stahl grew a culture of E. coli in a
medium that contained 15N as the sole nitrogen
source; after many generations, all the E. coli cells had
15N incorporated into the purine and pyrimidine
bases of DNA.
• Meselson and Stahl took a sample of these bacteria,
switched the rest of the bacteria to a medium that
contained only 14N, and then took additional samples
of bacteria over the next few cellular generations

10. • In each sample, the bacterial DNA that was synthesized
before the change in medium contained 15N and was
relatively heavy, whereas any DNA synthesized after the
switch contained 14N and was relatively light.
• Meselson and Stahl distinguished between the heavy
15N-laden DNA and the light 14N-containing DNA with
the use of equilibrium density gradient centrifugation.
• In this technique, a centrifuge tube is filled with a heavy
salt solution and a substance whose density is to be
measured—in this case, DNA fragments.
• The tube is then spun in a centrifuge at high speeds. After
several days of spinning, a gradient of density develops
within the tube, with high density at the bottom and low
density at the top.

11. • The density of the DNA fragments matches that of the
salt: light molecules rise and heavy molecules sink.

13. • Meselson and Stahl found that DNA from bacteria
grown only on medium containing 15N produced a
single band at the position expected of DNA
containing only 15N

14. • DNA from bacteria
transferred to the
medium with 14N
and allowed one
round of replication
also produced a
single band, but at a
position
intermediate
between that
expected of DNA
containing only 15N
and that expected of
DNA containing only
14N

15. • This result is inconsistent with the conservative
replication model, which predicts one heavy band
(the original DNA molecules) and one light band (the
new DNA molecules).
• A single band of intermediate density is predicted by
both the semiconservative and the dispersive models.
• To distinguish between these two models, Meselson
and Stahl grew the bacteria in medium containing
14N for a second generation.
• After a second round of replication in medium with
14N, two bands of equal intensity appeared, one in
the intermediate position and the other at the
position expected of DNA that contained only 14N

17. • All samples taken after additional rounds of
replication produced two bands, and the band
representing light DNA became progressively
stronger.

18. • Meselson and Stahl’s results were exactly as expected
for semiconservative replication and are incompatible
with those predicated for both conservative and
dispersive replication.

19. concept
•Replication is semiconservative: each
DNA strand serves as a template for the
synthesis of a new DNA molecule.
•Meselson and Stahl convincingly
demonstrated that replication in E. coli
is semiconservative.

20. Modes of Replication
• Following Meselson and Stahl’s work,
investigators confirmed that other organisms
also use semiconservative replication.
• No evidence was found for conservative or
dispersive replication.
• There are, however, several different ways that
semiconservative replication can take place,
differing principally in the nature of the
template DNA—whether it is linear or circular.

21. •Individual units of replication are called
replicons, each of which contains a
replication origin.
•Replication starts at the origin and
continues until the entire replicon has
been replicated.
•Bacterial chromosomes have a single
replication origin, whereas eukaryotic
chromosomes contain many.

22. Modes of replication
Theta
Rolling
circle
Linear
takes place in
some viruses and in
the F factor (a small
circle of extra
chromosomal
DNA
Theta replication is a
type of replication
common in E. coli and
other organisms
possessing circular
DNA
Linear DNA
replication takes
place in eukaryotic
chromosomes

23. Theta replication
• A Structure that resembles the Greek letter theta ().
• In theta replication, double-stranded DNA begins to
unwind at the replication origin, producing single-
stranded nucleotide strands that then serve as templates
on which new DNA can be synthesized.
• The unwinding of the double helix generates a loop,
termed a replication bubble.
• Unwinding may be at one or both ends of the bubble,
making it progressively larger.
• DNA replication on both of the template strands is
simultaneous with unwinding.
• The point of unwinding, where the two single
nucleotide strands separate from the double-
stranded DNA helix, is called a replication fork.

24. • If there are two replication forks, one at each end of
the replication bubble, the forks proceed outward in
both directions in a process called bidirectional
replication,
• simultaneously unwinding and replicating the DNA
until they eventually meet. If a single replication fork
is present, it proceeds around the entire circle to
produce two complete circular DNA molecules, each
consisting of one old and one new nucleotide strand.

26. • John Cairns provided the first visible evidence of theta
replication in 1963 by growing bacteria in the presence of
radioactive nucleotides.
• After replication, each DNA molecule consisted of one “hot”
(radioactive) strand and one “cold” (nonradioactive) strand.
• Cairns isolated DNA from the bacteria after replication and
placed it on an electron microscope grid, which was then
covered with a photographic emulsion.
• Radioactivity present in the sample exposes the emulsion and
produces a picture of the molecule (called an autoradiograph),
similar to the
• way that light exposes a photographic film.
• Because the newly synthesized DNA contained radioactive
nucleotides.
• Cairns was able to produce an electron micrograph of the
replication process

28. ROLLING CIRCLE REPLICATION
• Another form of replication, called rolling-circle replication , takes place in some viruses and in
the F factor ( a small circle of extra chromosomal DNA that controls mating, of E. coli.
• This form of replication is initiated by a break in one of the nucleotide strands that creates a 3′-
OH group and a 5′-phosphate group. New nucleotides are added to the 3′ end of the broken
strand, with the inner (unbroken) strand used as a template.
• As new nucleotides are added to the 3′ end, the 5′ end of the broken strand is displaced from
the template, rolling out like thread being pulled off a spool. The 3′ end grows around the
circle, giving rise to the name rolling-circle model.
• The replication fork may continue around the circle a number of times, producing several
linked copies of the same sequence.
• With each revolution around the circle, the growing 3′ end displaces the nucleotide strand
synthesized in the preceding revolution.
• Eventually, the linear DNA molecule is cleaved from the circle, resulting in a double stranded
circular DNA molecule and a single-stranded linear DNA molecule.
• The linear molecule circularizes either before or after serving as a template for the synthesis of
a complementary strand.

30. Linear eukaryotic replication
• The large linear chromosomes in eukaryotic cells however, contain far too much
DNA to be replicated speedily from a single origin.
• Eukaryotic replication proceeds at a rate ranging from 500 to 5000 nucleotides per
minute at each replication fork (considerably slower than bacterial replication).
• Even at 5000 nucleotides per minute at each fork, DNA synthesis starting from a
single origin would require 7 days to replicate a typical human chromosome
consisting of 100 million base pairs of DNA.
• The replication of eukaryotic chromosomes actually takes place in a matter of
minutes or hours, not days.
• This rate is possible because replication takes place simultaneously from thousands
of origins.
• At each replication origin, the DNA unwinds and produces a replication bubble.
• Replication takes place on both strands at each end of the bubble, with the two
replication forks spreading outward.
• Eventually, replication forks of adjacent replicons run into each other, and the
replicons fuse to form long stretches of newly synthesized DNA.

31. • Replication and fusion of
all the replicons leads to
two identical DNA
molecules.

32. Characteristics of theta, rolling-circle, and linear eukaryotic
replication

33. Requirements of Replication:
The process of replication includes many components,
they can be combined into three major groups:
• a template consisting of single-stranded DNA,
• raw materials (substrates) to be assembled into a
new nucleotide strand, and
• enzymes and other proteins that “read” the template
and assemble the substrates into a DNA molecule.

34. • Which acts as a template ? - a double-stranded DNA
molecule must unwind to expose the bases that act as
a template for the assembly of new polynucleotide
strands, which are made complementary and
antiparallel to the template strands.
• The raw materials from which new DNA molecules
are synthesized are deoxyribonucleotides
triphosphates (dNTPs), each consisting of a
deoxyribose sugar and a base (a nucleoside) attached
to three phosphates

35. • In DNA synthesis, nucleotides are added to the 3′-OH
group of the growing nucleotide strand.
• The 3′-OH group of the last nucleotide on the strand
attacks the 5′-phosphate group of the incoming dNTP.
• Two phosphates are cleaved from the incoming dNTP,
and a phosphodiester bond is created between the
two nucleotides.
Thus, DNA synthesis requires a single-stranded DNA
template, deoxyribonucleoside triphosphates, a
growing nucleotide strand, and a group of enzymes
and proteins.

37. DNA polymerases the enzymes that synthesize DNA, can add
nucleotides only to the 3′ end of the growing strand (not the
5′ end), and so new DNA strands always elongate in the
same 5′-to-3′ direction (5′-3′).
This new strand which undergoes continuous replication, is called the leading
strand.

38. DNA synthesis must start anew
at the replication fork and
proceed in the direction
opposite that of the movement
of the fork until it runs into the
previously replicated segment
of DNA.
This process is repeated again
and again, and so synthesis of
this strand is in short,
discontinuous bursts. The
newly made strand that
undergoes discontinuous
replication is called the lagging
strand.
The short lengths of DNA
produced by discontinuous
replication of the lagging strand
are called Okazaki fragments,
after Reiji Okazaki, who
discovered them.

39. • All DNA synthesis is 5′to3′, meaning that new
nucleotides are always added to the 3′ end of the
growing nucleotide strand. At each replication fork,
synthesis of the leading strand proceeds continuously
and that of the lagging strand proceeds
discontinuously.

48. • The Mechanism of Replication
• Replication takes place in four stages:
• initiation,
• unwinding,
• elongation,
• termination.
• Initiation The circular chromosome of E. coli has a single replication origin
(oriC). The minimal sequence required for oriC to function consists of 245 bp
that contain several critical sites. Initiator proteins bind to oriC and cause a
short section of DNA to unwind. This unwinding allows helicase and other
single-strand-binding proteins to attach to the polynucleotide strand.
• Unwinding Because DNA synthesis requires a singlestranded template and
because double-stranded DNA must be unwound before DNA synthesis can
take place, the cell relies on several proteins and enzymes to accomplish
the unwinding.

49. • DNA helicases break the hydrogen bonds that
• exist between the bases of the two nucleotide strands
of a DNA molecule. Helicases cannot initiate the
unwinding of double-stranded DNA; the initiator
proteins first separate
• DNA strands at the origin, providing a short stretch of
single-stranded DNA to which a helicase binds.
Helicases bind to the lagging-strand template at each
replication fork and move in the 5′B3′ direction along
this strand, thus also moving the replication fork.

50. • After DNA has been unwound by helicase, the single stranded nucleotide
chains have a tendency to form hydrogen bonds and re- anneal (stick back
together).
• Secondary structures, such as hairpins also may form between
complementary nucleotides on the same strand. To stabilize the single-
stranded DNA long enough for replication to take place, single-strand-
binding proteins (SSBs) attach tightly to the exposed single-stranded DNA.
• Another protein essential for the unwinding process is the enzyme DNA
gyrase, a topoisomerase.
• topoisomerases control the supercoiling of
• DNA. In replication, DNA gyrase reduces torsional strain (torque) that builds
up ahead of the replication fork as a result of unwinding.
• It reduces torque by making a double-stranded break in one segment of the
DNA helix, passing another segment of the helix through the break, and
then resealing the broken ends of the DNA. This action removes a twist in
the DNA and reduces the supercoiling.

51. • Replication is initiated at a replication origin, where
an initiator protein binds and causes a short stretch of
DNA to unwind.
• DNA helicase breaks hydrogen bonds at a replication
fork, and single-strand-binding proteins stabilize the
separated strands.
• DNA gyrase reduces torsional strain that develops as
the two strands of double-helical DNA unwind.

52. •Primers
• All DNA polymerases require a nucleotide with a 3′-OH group to which a new
nucleotide can be added.
•
• Because of this requirement, DNA polymerases cannot initiate DNA synthesis on a
bare template; rather, they require a primer—an existing 3′-OH group—to get
started.
• An enzyme called primase synthesizes short stretches of nucleotides (primers) to
get DNA replication started.
• Primase synthesizes a short stretch of RNA nucleotides (about 10–12 nucleotides
long), which provides a 3′-OH group to which DNA polymerase can attach DNA
nucleotides.
• Because primase is an RNA polymerase, it does not require an existing 3′-OH group
to which nucleotides can be added.
• All DNA molecules initially have short RNA primers imbedded within them; these
primers are later removed and replaced by DNA nucleotides.

53. • On the leading strand, where DNA synthesis is
continuous, a primer is required only at the 5′ end of
the newly synthesized strand.
• On the lagging strand, where replication is
discontinuous, a new primer must be generated at
the beginning of each Okazaki fragment.
• Primase forms a complex with helicase at the
replication fork and moves along the template of the
lagging strand.
• The single primer on the leading strand is probably
synthesized by the primase–helicase complex on the
template of the lagging strand of the other replication
fork, at the opposite end of the replication bubble.

55. Elongation
• After DNA is unwound and a primer has been added,
DNA polymerases elongate the polynucleotide strand
by catalyzing DNA polymerization.
• The best-studied polymerases are those of E. coli,
which has at least five different DNA polymerases.
• Two of them, DNA polymerase I and DNA polymerase
III, carry out DNA synthesis in replication;
• the other three have specialized functions in DNA
repair

57. that the major
enzymatic
components of
elongation—DNA
polymerases,
helicase,
primase, and ligase

58. how these components interact at
the replication fork?
• Because the synthesis of both strands takes place
simultaneously,
• two units of DNA polymerase III must be present at the
replication fork, one for each strand.
• In one model of the replication process, the two units
of DNA polymerase III are connected.
• the lagging-strand template loops around so that it is in
position for 5′B3′ replication as the polymerase moves
down the DNA.
• In this way, the DNA polymerase III complex is able to
carry out 5′B3′ replication simulaneously on both
templates, even though they run in opposite directions.

60. • After about 1000 bp of new DNA has been
synthesized, DNA polymerase III releases the
lagging strand template, and a new loop forms
Primase synthesizes a new primer on the lagging
strand and DNA polymerase then synthesizes a new
Okazaki fragment

61. In summary, each active replication fork requires five
basic components:
• 1. helicase to unwind the DNA,
• 2. single-strand-binding proteins to keep the
nucleotide strands separate long enough to allow
replication,
• 3. the topoisomerase gyrase to remove strain ahead
of the replication fork,
• 4. primase to synthesize primers with a 3′-OH group
at the beginning of each DNA fragment, and
• 5. DNA polymerase to synthesize the leading and
lagging nucleotide strands.

62. Termination
• In some DNA molecules, replication is terminated
whenever two replication forks meet.
• In others, specific termination sequences block
further replication.
• In some DNA molecules, replication is terminated
whenever two replication forks meet.
• In others, specific termination sequences block
further replication.

81. TRANSCRIPTION

82. • RNA differs from DNA in that RNA possesses a hydroxyl group on the 2-
carbon atom of its sugar, contains uracil instead of thymine, and is normally
single stranded.
• Several classes of RNA exist within bacterial and eukaryotic cells.

85. WHAT IS TRANSCRIPTION ?
• SYNTHESIZING RNA FROM A DNA MOLECULE
• All cellular RNAs are synthesized from a DNA template through the process
of transcription.
• Transcription is in many ways similar to the process of replication, but one
fundamental difference relates to the length of the template used.
• During replication, all the nucleotides in the DNA template are copied, but,
during transcription, only small parts of the DNA molecule—usually a single
gene or, at most, a few genes—are transcribed into RNA.
• Transcription is, in fact, a highly selective process—individual genes are
transcribed only as their products are needed.
• Like replication, transcription requires three major components:

86. 1. a DNA template;
2. the raw materials (substrates) needed to build a new RNA molecule; and
3. the transcription apparatus, consisting of the proteins
necessary to catalyse the synthesis of RNA.

87. 1. The transcribed strand The template for RNA synthesis, as for DNA synthesis, is a single
strand of the DNA double helix.
2. Unlike replication, however, transcription typically takes place on only one of the two
nucleotide strands of DNA.
3. The nucleotide strand used for transcription is termed the template strand.
4. The other strand, called the nontemplate strand, is not ordinarily transcribed.
5. Thus, in any one section of DNA, only one of the nucleotide strands normally is transcribed
into RNA .
Evidence that only one DNA strand serves as a template came from several experiments carried
out by Julius Marmur and his colleagues in 1963 on the DNA of bacteriophage SP8, which
infects the bacterium Bacillus subtilus.
Within a single gene, only one of the two DNA strands, the template strand, is generally
transcribed into RNA.

88. 1. The transcription unit A transcription unit is a stretch of DNA that codes for an RNA
molecule and the sequences necessary for its transcription.
2. Within a transcription unit are three critical regions: a promoter, an RNA-coding
sequence, and a terminator.
3. The promoter is a DNA sequence that the transcription apparatus recognizes and binds.
4. It indicates which of the two DNA strands is to be read as the template and the direction
of transcription.
5. The promoter also determines the transcription start site, the first nucleotide that will be
transcribed into RNA.
6. In most transcription units, the promoter is located next to the transcription start site but
is not, itself, transcribed.
7. The second critical region of the transcription unit is the RNA-coding region, a sequence
of DNA nucleotides that is copied into an RNA molecule.
8. The third component of the transcription unit is the terminator, a sequence of
nucleotides that signals where transcription is to end.

89. • Terminators are usually part of the coding sequence; that is, transcription stops
only after the terminator has been copied into RNA.
• Molecular biologists often use the terms upstream and downstream to refer to the
direction of transcription and the location of nucleotide sequences surrounding the
RNA coding sequence.
• The transcription apparatus is said to move downstream during transcription: it
binds to the promoter (which is usually upstream of the start site) and moves
toward the terminator (which is downstream of the start site).
A transcription unit is a piece of DNA that encodes an RNA molecule and the
sequences necessary for its proper transcription. Each transcription unit
includes a promoter, an RNA-coding region, and a terminator.
SUBSTRATE
• RNA is synthesized from ribonucleoside triphosphates. Transcription is 5 B 3:
each new nucleotide is joined to the 3-OH group of the last nucleotide added
to the growing RNA molecule.

90. The Transcription Apparatus
• Single enzyme—RNA polymerase—carries out all the required steps of
transcription on closer inspection, the processes are actually similar.
• The action of RNA polymerase is enhanced by a number of accessory
proteins that join and leave the polymerase at different stages of the
process.
• Each accessory protein is responsible for providing or regulating a special
function.
• Thus, transcription, like replication, requires an array of proteins.
Bacterial RNA polymerase
• Bacterial cells typically possess only one type of RNA polymerase, which
catalyses the synthesis of all classes of bacterial RNA: mRNA, tRNA, and
rRNA. Bacterial RNA polymerase is a large, multimeric enzyme (meaning
that it consists of several polypeptide chains).
• In bacterial RNA polymerase, the core enzyme consists of five subunits:
two copies of alpha, a single copy of beta , a single copy of beta prime ,
and a single copy of omega .
• The omega subunit is not essential for transcription, but it helps stabilize
the enzyme.

91. • The core enzyme catalyzes the elongation of the RNA
molecule by the addition of RNA nucleotides. The sigma
factor () joins the core to form the holoenzyme, which is
capable of binding to a promoter and initiating
transcription.

92. • The core enzyme catalyzes the elongation of the RNA
molecule by the addition of RNA nucleotides.
• Other functional subunits join and leave the core enzyme at
particular stages of the transcription process.
• The sigma () factor controls the binding of RNA polymerase
to the promoter.
• Without sigma, RNA polymerase will initiate transcription at
a random point along the DNA.
• After sigma has associated with the core enzyme (forming a
holoenzyme)
• RNA polymerase binds stably only to the promoter region
and initiates transcription at the proper start site.
• Sigma is required only for promoter binding and initiation;
when a few RNA nucleotides have been joined together,
sigma usually detaches from the core enzyme.

93. • Many bacteria possess multiple types of sigma. E. coli,
for example, possesses sigma 28 (28), sigma 32 (32),
sigma 54 (54), and sigma 70 (70), named on the basis
of their molecular weights.
Each type of sigma initiates the binding of RNA
polymerase to a particular set of promoters.
For example, 32 binds to promoters of genes that
protect against environmental stress,
54 binds to promoters of genes used during nitrogen
starvation,
70 binds to many different promoters.
• Other subunits provide the core RNA polymerase with
additional functions. Rho () and NusA,
for example, facilitate the termination of transcription.

94. • Eukaryotic RNA polymerases Eukaryotic cells possess
three distinct types of RNA polymerase, each of which is
responsible for transcribing a different class of RNA:
• RNA polymerase I transcribes rRNA
• RNA polymerase II transcribes pre-mRNAs, snoRNAs, and
some snRNAs
• RNA polymerase III transcribes small RNA molecules—
specifically tRNAs, small rRNA, and some snRNAs .
• All three eukaryotic polymerases are large, multimeric
enzymes, typically consisting of more than a dozen
subunits.
• Some subunits are common to all three RNA
polymerases, whereas others are limited to one of the
polymerases.
• As in bacterial cells, a number of accessory proteins bind
to the core enzyme and affect its function.

95. Bacterial cells possess a single type of RNA polymerase consisting of a core enzyme
and other subunits that participate in various stages of transcription. Eukaryotic cells
possess three distinct types of RNA polymerase: RNA polymerase I transcribes rRNA;
RNA polymerase II transcribes pre-mRNA, snoRNAs, and some snRNAs; and RNA
polymerase III transcribes tRNAs, small rRNAs, and some snRNAs.

96. The Process of Bacterial Transcription
• Transcription can be conveniently divided into three
stages:
1. initiation, in which the transcription apparatus assembles
on the promoter and begins the synthesis of RNA;
2. elongation, in which RNA polymerase moves along the
DNA, unwinding it and adding new nucleotides, one at a
time, to the 3 end of the growing RNA strand.
3. termination, the recognition of the end of the
transcription unit and the separation of the RNA molecule
from the DNA template.

97. INITIATION
The steps necessary to begin RNA synthesis,
(1) promoter recognition,
(2) formation of the transcription bubble,
(3) creation of the first bonds between rNTPs,
(4) escape of the transcription apparatus from the
promoter.

98. the transcription apparatus recognize and bind to the
promoter.
• the binding of RNA polymerase to the promoter
determines which parts of the DNA template are to
be transcribed and how often.
• Different genes are transcribed with different
frequencies, and promoter binding is primarily
responsible for determining the frequency of
transcription for a particular gene.
• Promoters also have different affinities for RNA
polymerase.
• Even within a single promoter, the affinity can vary
over time, depending on its interaction with RNA
polymerase and a number of other factors.

99. • Promoters are DNA sequences that are recognized by
the transcription apparatus and are required for
transcription to take place.
• In bacterial cells, promoters are usually adjacent to an
RNA-coding sequence.
• The examination of many promoters in E. coli and
other bacteria reveals a general feature: although
most of the nucleotides within the promoters vary in
sequence, short stretches of nucleotides are common
to many.
• Furthermore, the spacing and location of these
nucleotides relative to the transcription start site are
similar in most promoters.
• These short stretches of common nucleotides are
called consensus sequences.

100. • The term “consensus sequence” refers to sequences
that possess considerable similarity or consensus.
• By definition, the consensus sequence comprises the
most commonly encountered nucleotides found at a
specific location.
• The most commonly encountered consensus
sequence, found in almost all bacterial promoters, is
located just
• upstream of the start site, centred on position 10.
Called the 10 consensus sequence or, sometimes, the
Pribnow box, its sequence is
5 T ATA A T 3
3 AT AT T A 5

101. • that TATAAT is just the consensus sequence—
representing the most commonly encountered
nucleotides at each of these positions.
• In most prokaryotic promoters, the actual sequence is
not TATAAT

102. Another consensus sequence common to
most bacterial promoters is TTGACA, which
lies approximately 35 nucleotides upstream of
the start site and is termed the 35 consensus
sequence.
The nucleotides on either side of the 10 and
35 consensus sequences and those between
them vary greatly from promoter to promoter,
suggesting that they are relatively
unimportant in promoter recognition.

104. Elongation
• At the end of initiation, RNA polymerase undergoes a
change in conformation (shape) and thereafter is no
longer able to bind to the consensus sequences in the
promoter.
• This allows the polymerase to escape from the
promoter and begin moving downstream.
• The sigma subunit is usually released after initiation,
although some populations of RNA polymerase may
retain sigma throughout elongation.

105. • As it moves downstream along the template, RNA
polymerase progressively unwinds the DNA at the
leading (downstream) edge of the transcription
bubble, joining nucleotides to the RNA molecule
according to the sequence on the template, and
rewinds the DNA at the trailing (upstream) edge of
the bubble.
• In bacterial cells at 37°C, about 40 nucleotides are
added per second. This rate of RNA synthesis is much
lower than that of DNA synthesis, which is more than
1500 nucleotides per second in bacterial cells.

106. • Transcription takes place within a short stretch of
about 18 nucleotides of unwound DNA—the
transcription bubble.
• Within this region, RNA is continuously synthesized,
with single-stranded DNA used as a template. About 8
nucleotides of newly synthesized RNA are paired with
the DNA-template nucleotides at any one time.
• As the transcription apparatus moves down the DNA
template, it generates positive supercoiling ahead of
the transcription bubble and negative supercoiling
behind it.
• Topoisomerase enzymes probably relieve the stress
associated with the unwinding and rewinding of DNA
in transcription, as they do in DNA replication.

107. • Concepts
• Transcription is initiated at the start site, which, in
bacterial cells, is set by the binding of RNA
polymerase to the consensus sequences of the
promoter.
• No primer is required.
• Transcription takes place within the transcription
bubble.
• DNA is unwound ahead of the bubble and rewound
behind it.

108. TERMINATION
• RNA polymerase moves along the template, adding nucleotides to
the 3 end of the growing RNA molecule until it transcribes a
terminator.
• Most terminators are found upstream of the point of termination.
• Transcription therefore does not suddenly end when polymerase
reaches a terminator, Rather, transcription ends after the terminator
has been transcribed.
• At the terminator, several overlapping events are needed to bring an
end to transcription:
• RNA polymerase must stop synthesizing RNA, the RNA molecule
must be released from RNA polymerase, the newly made RNA
molecule must dissociate fully from the DNA, and RNA polymerase
must detach from DNA templates.

109. Bacterial cells possess two major types of terminators.
• Rho-dependent terminators are able to cause the
termination of transcription only in the presence of
an ancillary protein called the rho factor.
• Rho-independent terminators are able to cause the
end of transcription in the absence of rho.

110. Rho-independent terminators have two common features.
• First, they contain inverted repeats (sequences of nucleotides
on one strand that are inverted and complementary). When
inverted repeats have been transcribed into RNA, a hairpin
secondary structure forms.
• Second, in rho-independent terminators, a string of
approximately six adenine nucleotides follows the second
inverted repeat in the template DNA.
• Their transcription produces a string of uracil nucleotides after
the hairpin in the transcribed RNA.
• The presence of a hairpin in an RNA transcript causes RNA
polymerase to slow down or pause, which creates an
opportunity for termination.
• The adenine–uracil base pairings downstream of the hairpin
are relatively unstable compared with other base pairings, and
the formation of the hairpin may itself destabilize the DNA–
RNA pairing, causing the RNA molecule to separate from its
DNA template.
• When the RNA transcript has separated from the template,
RNA synthesis can no longer continue.

111. Rho – independent termination
Rho – dependent termination

112. Rho-dependent terminators have two features:
• (1) DNA sequences that produce a pause in
transcription.
• (2) a DNA sequence that encodes a stretch of RNA
upstream of the terminator that is devoid of any
secondary structures.
• This unstructured RNA serves as binding site for the
rho protein, which binds the RNA and moves toward
its 3 end, following the RNA polymerase.
• When RNA polymerase encounters the terminator, it
pauses, allowing rho to catch up.
• The rho protein has helicase activity, which it uses to
unwind the RNA–DNA hybrid in the transcription
bubble, bringing transcription to an end.

113. Concepts
• Transcription ends after RNA polymerase
transcribes a terminator.
• Bacterial cells possess two types of terminator: a
rho-independent terminator, which RNA
polymerase can recognize by itself; and a rho-
dependent terminator, which RNA polymerase can
recognize only with the help of the rho protein.

114. The Process of Eukaryotic
Transcription
• Eukaryotic cells possess three different RNA
polymerases, each of which transcribes a different
class of RNA and recognizes a different type of
promoter. Thus, a generic promoter cannot be
described.
• promoter’s description depends on whether the
promoter is recognized by RNA polymerase I, II, or III.
• Many proteins take part in the binding of eukaryotic
RNA polymerases to DNA templates, and the different
types of promoters require different proteins.

115. Transcription and Nucleosome Structure
• Transcription requires that sequences on DNA are accessible to
RNA polymerase and other proteins.
• However, in eukaryotic cells, DNA is complexes with histone
proteins in highly compressed chromatin .
• Before transcription, the chromatin structure is modified so
that the DNA is in a more open configuration and is more
accessible to the transcription machinery.
• Several types of proteins have roles in chromatin modification.
• Acetyltransferases add acetyl groups to amino acids at the
ends of the histone proteins, which destabilizes the
nucleosome structure and makes the DNA more accessible.
• Other types of histone modification also can affect chromatin
packing.
• In addition, proteins called chromatin- remodeling proteins
may bind to the chromatin and displace nucleosomes from
promoters and the regions important for transcription.

116. Transcription Initiation:
• The initiation of transcription is a complex process in
eukaryotic cells because of the variety of initiation
sequences and because numerous proteins bind to
these sequences.
• Two broad classes of DNA sequences are important
for the initiation of transcription: promoters and
enhancers.
• A promoter is always found adjacent to (or sometimes
within) the gene that it regulates and has a fixed
location with regard to the transcription start point.
• An enhancer, in contrast, need not be adjacent to the
gene; enhancers can affect the transcription of genes
that are thousands of nucleotides away, and their
positions relative to start sites can vary.

117. • A significant difference between bacterial and
eukaryotic transcription is the existence of three
different eukaryotic RNA polymerases, which
recognize different types of promoters.
• We saw that, in bacterial cells, the holoenzyme (RNA
polymerase plus sigma) recognizes and binds directly
to sequences in the promoter.
• In eukaryotic cells, promoter recognition is carried out
by accessory proteins that bind to the promoter and
then recruit a specific RNA polymerase (I, II, or III) to
the promoter.

118. • One class of accessory proteins comprises general
transcription factors, which, along with RNA
polymerase, form the basal transcription apparatus
that assembles near the start site and is sufficient to
initiate minimal levels of transcription.
• Another class of accessory proteins consists of
transcriptional activator proteins, which bind to
specific DNA sequences and bring about higher levels
of transcription by stimulating the assembly of the
basal transcription apparatus at the start site.

119. concepts
• Two classes of DNA sequences in eukaryotic cells
affect transcription: enhancers and promoters.
• A promoter is near the gene and has a fixed
position relative to the start site of transcription.
• An enhancer can be distant from the gene and
variable in location.

120. RNA Polymerase II Promoters
• RNA polymerase II, which transcribes the genes
that encode proteins.
• A promoter for a gene transcribed by RNA
polymerase II typically consists of two primary
parts: the core promoter and the regulatory
promoter.

121. Core promoter :
• The core promoter is located immediately upstream
of the gene and typically includes one or more
consensus sequences.
• The most common of these consensus sequences is
the TATA box, which has the consensus sequence
TATAAA and is located from 25 to 30 bp upstream of
the start site.
• Mutations in the sequence of the TATA box affect the
rate of transcription, and a change in its position
alters the location of the transcription start site.

122. • Another common consensus sequence in the core
promoter is the TFIIB recognition element (BRE), which
has the consensus sequence G/C G/C G/C C G C C and is
located from 32 to 38 bp upstream of the start site.
• TFIIB is the abbreviation for a transcription factor that
binds to this element;
• Instead of a TATA box, some core promoters have an
initiator element (Inr) that directly overlaps the start site
and has the consensus Y Y A N A/T Y Y.
• Another consensus sequence called the downstream core
promoter element (DPE) is found approximately 30 bp
downstream of the start site in many promoters that also
have Inr; the consensus sequence of DPE is R G A/T C G T
G.
• All of these consensus sequences in the core promoter are
recognized by transcription factors that bind to them and
serve as a platform for the assembly of the basal
transcription apparatus.

124. Assembly of the basal transcription apparatus
• The basic transcriptional machinery, called the basal
transcription apparatus, that binds to DNA at the start
site is required for the initiation of minimal levels of
transcription.
• It consists of RNA polymerase, a series of general
transcription factors, and a complex of proteins
known as the mediator.
• The general transcription factors include TFIIA, TFIIB,
TFIID, TFIIE, TFIIF, and TFIIH, in which TFII stands for
transcription factor for RNA polymerase II and the
final letter designates the individual factor.

126. 1. TFIID binds to the TATA box and positions the active
site of RNA polymerase II so that it begins
transcription at the correct place.
• TFIID consists of at least nine polypeptides. One of
them is the TATA-binding protein (TBP), which
recognizes and binds to the TATA box on the DNA
template.
• The TATA-binding protein binds to the minor groove
and straddles the DNA as a molecular saddle bending
the DNA and partly unwinding it.
• Other proteins, called TBP-associated factors (TAFs),
combine with TBP to form the complete TFIID
transcription factor.

127. • The large holoenzyme consisting of RNA polymerase,
additional transcription factors, and the mediator are
thought to preassemble and bind as a unit to TFIID.
• The other transcription factors provide additional
functions:
• TFIIA helps to stabilize the interaction between TBP
and DNA, TFIIB plays a role in the selection of the
start site, and
• TFIIH has helicase activity and unwinds the DNA
during transcription. The mediator plays a role in
communication between the basal transcription
apparatus and the transcriptional activator proteins.

128. • Regulatory promoter The regulatory promoter is
located immediately upstream of the core promoter.
• A variety of different consensus sequences may be
found in the regulatory promoters, and they can be
mixed and matched in different combinations.
• Transcriptional activator proteins bind to these
sequences and, either directly or indirectly (through
the mediation of coactivator proteins, make contact
with the mediator in the basal transcription apparatus
and affect the rate at which transcription is initiated.
• Some regulatory promoters also contain repressing
sequences, which are bound by proteins that lower
the rate of transcription through inhibitory
interactions with the mediator.

130. • Enhancers DNA sequences that increase the rate of
transcription at distant genes are called enhancers.
• An enhancer may be upstream or downstream of the
affected gene or, in some cases, within an intron of
the gene itself.
• Enhancers also contain sequences that are recognized
by transcriptional activator proteins.
• The DNA between the enhancer and the promoter
loops out, allowing the enhancer and the promoter to
lie close to each other.
• Transcriptional activator proteins bound to the
enhancer interact with proteins bound to the
promoter and stimulate the transcription of the
adjacent gene.

131. • The looping of DNA between the enhancer and the
promoter explains how the position of an enhancer
can vary with regard to the start site—enhancers that
are farther from the start site simply cause a longer
length of DNA to loop out.
• Sequences having many of the properties possessed
by enhancers sometimes take part in repressing
transcription instead of enhancing it; such sequences
are called silencers.
• Although enhancers and silencers are characteristic of
eukaryotic DNA, some enhancer-like sequences have
been found in bacterial cells.

132. Concepts
• General transcription factors assemble into the
basal transcription apparatus, which binds to DNA
near the start site and is necessary for transcription
to take place at minimal levels.
• Additional proteins called transcriptional activators
bind to other consensus sequences in promoters
and enhancers, and affect the rate of transcription.

133. Elongation
• After several nucleotides have been linked together,
RNA polymerase leaves the promoter and dissociates
from some of the transcription factors, moving
downstream and continuing to synthesize RNA.
• During elongation, the RNA polymerase maintains a
transcription bubble in which about eight nucleotides
of RNA remain base paired with the DNA template
strand.

134. • The molecular structure of eukaryotic RNA polymerase II
is understood in exquisite detail, revealing many aspects
of its function.
• In the course of elongation, the DNA double helix enters a
cleft in the polymerase and is gripped by jawlike
extensions of the enzyme
• The two strands of the DNA are unwound and the RNA
nucleotides that are complementary to the template
strand are added to the growing 3 end of the RNA
molecule.
• As it funnels through the polymerase, the DNA–RNA
hybrid hits a wall of amino acids and bends at almost a
right angle; this bend positions the end of the DNA–RNA
hybrid at the active site of the polymerase, and new
nucleotides are added to the 3 end of the growing RNA
molecule.
• The newly synthesized RNA is separated from the DNA
and runs through another groove before exiting from the
polymerase.

135. Termination
• The termination of transcription is less well
understood in eukaryotic genes than in bacterial
genes.
• The three eukaryotic RNA polymerases use different
mechanisms for termination.
• RNA polymerase I requires a termination factor, like
the rho factor utilized in the termination of some
bacterial genes.
• Unlike rho, which binds to the newly transcribed RNA
molecule, the termination factor for RNA polymerase
I binds to a DNA sequence downstream of the
termination site.

136. • After transcription, the 3 end of pre-mRNA is cleaved
at a specific site, designated by a consensus
sequence, producing the mature mRNA.
• Research findings suggest that termination is coupled
to cleavage, which is carried out by a cleavage
complex that probably associates with the RNA
polymerase.
• Following behind the RNA polymerase, this complex
may suppress termination until the consensus
sequence in the RNA that marks the cleavage site is
encountered.
• The pre-mRNA is cleaved by the complex, and
transcription is then terminated downstream.