The Bacterial DNA Replication
 A typical bacterial cell has anywhere from about 1 million to 4 million base pairs of DNA.
 DNA is replicated in order to create a duplicate molecule, so that the two cells generated
during binary fission can each receive one complete copy.
 The replication process is generally bidirectional, meaning it proceeds in both directions
from the starting point. This allows a chromosome to be replicated in half the time it would
take if the process were unidirectional.
 The progression of bidirectional replication around a circular DNA molecule creates two
advancing forks where DNA synthesis is occurring. These regions, called replication forks,
ultimately meet at a terminating site when the process is complete.
 The two DNA molecules created through replication each contain one of the original strands
paired with a newly synthesized strand. Because half of the original molecule is conserved
in each molecule, replication is said to be semiconservative.
 Bacteria, with their smaller genomes, DNA replication involves an incredibly sophisticated
and highly coordinated series of molecular events.
 These events are divided into several major stages: initiation, unwinding, primer synthesis,
elongation and termination.
Initiation and unwinding of DNA Replication
 To initiate replication of a DNA molecule, specific initiator proteins (DnaA) must recognize
and bind to a distinct DNA sequence called an origin of replication/oriC and proceeds in
two directions towards another specific region, the terminus.
 Prokaryotic chromosomes and plasmids typically contain only one of these initiating sites.
 A molecule that lacks this sequence will not be replicated.
 This binding by the initiator protein (DnaA) triggers events that unwind the DNA double
helix into two single-stranded DNA molecules. Several groups of proteins are involved.
 For example, the DNA helicases are responsible for breaking the hydrogen bonds that join
the complementary nucleotide bases to each other.
 Adenines pair with thymines using only two hydrogen bonds, so A-T rich segments of DNA
become single stranded more readily than do G-C rich regions.
 Because the newly unwound single strands have a tendency to rejoin, another group of
proteins, the single-strand-binding proteins (SSBs), keep the single strands separated and
prevent them from rejoining until elongation begins.
 A third family of proteins, the topoisomerases/DNA gyrase, reduce some of the torsional
strain caused by the unwinding of the double helix.
 Breaking the A-T and C-G bonds within a short stretch of DNA, allows the two strands to
separate, forming a bubble.
 Exposed bases within single-stranded regions of the bubble act as templates that are free to
pair with new bases in nucleotides to form new molecule of DNA.
 A template is a pattern for making something; DNA acts as a template because each strand
specifies the new daughter strand by base‐pairing.
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 The location at which a DNA strand begins to unwind into two separate single strands is
known as the origin of replication.
 At the end of the initiation process, two replication forks exist, going in opposite directions
from the “bubble” at the origin of replication.
 The initiation and unwinding process makes a template, but replication can't happen
because no primer yet exists.
Primer synthesis
 Primer synthesis marks the beginning of the actual synthesis of the new DNA molecule.
 Primers are short stretches of RNA of nucleotides (about 10 to 12 bases in length)
synthesized by an RNA polymerase enzyme called primase.
 Primers are required because DNA polymerases, the enzymes responsible for the actual
addition of nucleotides to the new DNA strand, can only add nucleotides to the 3'-OH group
of an existing chain and cannot initiate synthesis.
 A specialized RNA polymerase enzyme called primase can initiate synthesis.
 Later, after elongation is complete, the primer is removed and replaced with DNA
nucleotides.
Elongation and termination
 The process of DNA replication requires the coordinated action of many different enzymes
and other proteins.
 DNA polymerases require a template and primer, which is complementary to the template.
 DNA polymerase III can synthesize a continuous strand of DNA only in one direction. DNA
polymerase III links new nucleotides to the 3’ OH end of an existing nucleic acid strand,
lengthening it in the 5’-3’ direction.
 This pre-existing strand, necessary for the function of DNA polymerase III, is called a primer.
 Because the DNA strands exposed at the replication fork are antiparallel, only one of the
two DNA strands presents a 3’ primer that can be extended by DNA polymerase III.
 This strand, called the leading strand, is synthesized continuously in the direction that the
replication fork is moving.
 Synthesis of the second (lagging) strand is more complicated because it is going in the
wrong direction to serve as a template.
 No DNA polymerase exists to synthesize DNA in the 3′ to 5′ direction, so copying of the
lagging strand is discontinuous, that is, short strands of DNA are made and subsequently
matured by joining them together.
 An RNA primer, which is made by primase, initiates each of these small pieces of DNA.
 DNA polymerase III elongates the primer until it butts up against the 5′ end of the next
primer molecule.
 DNA polymerases cannot add nucleotides to the 5’ end, so as additional template is
exposed, synthesis must be reinitiated.
 Each time synthesis is reinitiated, another RNA primer must be made first. The result is a
series of small fragments, each of which has a short stretch of RNA at its 5’ end. These
fragments are called Okazaki fragments after their discoverer, Reiji Okazaki.
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 As DNA polymerase adds nucleotides to the 3’ end of one Okazaki fragment, it eventually
reaches the 5’ end of another.
 Because of the orientation of the lagging strand, this synthesis proceeds backward, away
from the replication fork.
 DNA polymerase III, like all DNA polymerase has an additional function that is critically
important proofing. Proofreading is the removal of a mismatched immediately after it has
been added; its removal must be before the next base is incorporated.
 DNA polymerase I (Pol I) then uses its polymerizing and 5′ to 3′ exonuclease activities to
remove the RNA primer from Okazaki fragments and fill in this sequence with DNA.
 Because DNA polymerase I is not very processive, it falls off the lagging strand after a
relatively short‐length synthesis. The key distinction among the enzyme forms is their
processitivity; how long a chain they synthesize before falling off the template.
 DNA polymerases can't seal up the nicks that result from the replacement of RNA primers
with DNA.
 Another enzyme, DNA ligase, seals off the nicks by using high energy phosphodiester bonds
to join a free 3′ hydroxyl with an adjacent 5′ phosphate.
 DNA replication stops when the replisome reaches a termination site (ter) on the DNA. The
Tus protein binds to the sites and halts progression of the forks.
 In many bacteria, replication stops randomly when the forks meet. The two replication forks
proceed around the chromosome, until they meet at the terminus.
 When a circular bacterial chromosome is replicated, the two replication forks eventually
meet at a site opposite the origin of replication. Two complete DNA molecules have been
produced at this point, and these can be passed on to the two daughter cells.
Component of DNA replication
Component Comments
Replisome
The complex of enzymes and other proteins that synthesize DNA
DNA gyrase Enzyme that temporarily breaks the strands of DNA, relieving the tension caused
by unwinding the two strands of the DNA helix.
DNA ligase
Enzyme that joins two DNA fragments by forming a covalent bond between the
sugar and phosphate residues of adjacent nucleotides.
DNA
Enzymes that synthesize DNA; they use one polymerases strand of DNA as a
polymerases template to make the complementary strand. Nucleotides can be added only to
the 3’ end of an existing fragment-therefore, synthesis always occurs in the 5’ to
3’ direction.
Helicases
Enzymes that unwind the DNA helix ahead of the replication fork.
Okazaki
Nucleic acid fragment produced during discontinuous synthesis of the lagging
fragment
strand of DNA.
Origin
of Distinct region of a DNA molecule at which replication is initiated.
replication
Primase
Enzyme that synthesizes small fragments of RNA to serve as primers for DNA
synthesis
Primer
Fragment of nucleic acid to which DNA Polymerase can add nucleotides (the
enzyme can add nucleotides only to an existing fragment).
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Gene expression on microorganisms
 An important function of DNA is to use the genetic information in directing synthesis of
proteins, which control the cell activities by catalyzing the biochemical reactions and by
constituting the structural elements of the cell. This is generally known as gene expression,
and is accomplished through a sequence of events consisting of two separate but
interrelated processes.These are transcription and translation.
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Transcription process
 Transcription is the first step in gene expression. In transcription, the information contained
in DNA is copied into three types of RNA: messenger RNA (mRNA), transfer RNA (tRNA), and
ribosomal RNA (rRNA). When the three types of RNA have been made by transcription, the
cell is biochemically equipped to make proteins.
 In prokaryotes, both transcription and translation take place in the cytoplasm, whereas in
eukaryotes, transcription takes place in the nucleus of the cell.
 Transcription is the process in which the information stored in the DNA is used to code for
the synthesis of RNA.
 During transcription, only one strand of DNA serves as a template and a single stranded
RNA is synthesized.
 In transcription, the enzyme RNA polymerase synthesizes single-stranded RNA molecules
from a DNA template. Nucleotide sequences in the DNA direct the polymerase where to
start and where to end.
 The DNA sequence to which RNA polymerase can bind and initiate transcription is called a
promoter; one that stops the process is a terminator.
 Like DNA polymerase, RNA polymerase can add nucleotide only to the 3’ end of a chain and
therefore synthesizes RNA in the 5’ to 3’ direction.
 Unlike DNA polymerase, however, RNA polymerase can start synthesis without a primer.
 The RNA sequence made during transcription is complementary and antiparallel to the DNA
template.
 The DNA strand that serves as the template for transcription is called the minus (-) strand/
(3′-5′) and its complement is called the plus (+) strand/ (5′-3′).
 Because the RNA is complementary to the (-) DNA strand, its nucleotide sequence is the
same as the (+) DNA strand, except it contains uracil rather than thymine.
 In prokaryotes, mRNA molecules can carry the information for one or multiple genes.
 A transcript that carries one gene is called monocistronic (a cistron is synonymous with a
gene).
 A transcript that carries multiple genes is called polycistronic. The proteins encoded on a
polycistronic message generally have related functions, allowing a cell to express related
genes as one unit.
Initiation of transcription
 Transcription is initiated when RNA polymerase binds to a promoter.
 The binding melts a short stretch of DNA creating a region of exposed nucleotides that
serves as a template for RNA synthesis.
 The portion of RNA polymerase that recognizes the promoter is a loosely attached subunit
called sigma (σ) factor.
 A cell can produce various types of σ factors, each recognizing different promoters.
 By controlling which σ factors are made, cells can transcribe specialized sets of genes as
needed.
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 The RNA polymerase of eukaryotic cells and archaea use proteins called transcription
factors to recognize promoters.
 Promoters identify the regions of a DNA molecule that will be transcribed into RNA. In doing
so, they also orient the direction of the RNA polymerase on the DNA molecule; thereby
dictating which strand will be used as a template.
Elongation of the RNA transcript
 In the elongation phase, RNA polymerase moves along DNA, using the (-) strand as a
template to synthesize a single-stranded RNA molecule.
 As with DNA replication, nucleotides are added only to the 3’ end; at the rate of about 40
nucleotides per second at 370C.
 The reaction is fueled by hydrolyzing a high-energy phosphate bond of the incoming
nucleotide.
 When RNA polymerase advances, it denatures a new stretch of DNA and allows the
previous portion to close.
 This exposes a new region of the template so elongation can continue.
 Once elongation has proceeded far enough for RNA polymerase to clear the promoter,
another molecule of the enzyme can bind, initiating a new round of transcription. Thus, a
single gene can be transcribed repeatedly very quickly.
Termination of transcription
 Just as an initiation of transcription occurs at a distinct site on the DNA, so does
termination. When RNA polymerase encounters a sequence called a terminator, it falls off
the DNA template and releases the newly synthesized RNA.
Component of DNA transcription
Component Comments
(-) strand
Strand of DNA that serves as the template forRNA synthesis; the resulting RNA
molecule is complementary to this strand. (3′-5′)
(+) strand
Strand of DNA complementary to the one that serves as the template for RNA
synthesis; the nucleotide sequence of the RNA molecule is the same as this strand,
except it has uracil rather than thymine. (5′-3′).
Promoter
Nucleotide sequence to which RNA polymerase binds to initiate transcription.
RNA
polymerase
Enzyme that synthesizes RNA using single-stranded DNA as a template; synthesis
always occurs in the 5’ to 3’ direction.
Sigma
factor
(σ) Component of RNA polymerase that recognizes the promoter regions. A cell can
have different types of factors that recognize different promoters, allowing the cell
to transcribe specialized sets of genes as needed.
Terminator
Nucleotide sequence at which RNA synthesis stops; the RNA polymerase falls off the
DNA template and releases the newly synthesized RNA.
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Summary of transcription process:
1. Promoter recognition; RNA polymerase binds to the DNA at a site called the promoter. This
is the most crucial step of RNA synthesis and results in the formation of a closed promoter
complex.
2. Local unwinding; binding of the RNA polymerase to the promoter site causes unwinding of
the DNA double helix, and the RNA polymerase is said to have formed an open promoter
complex.
3. Chain initiation; the RNA polymerase recognizes a transcription start site, which is very
close to the initial binding site. The first nucleoside triphosphate is added to this site and
synthesis begins.
4. Chain elongation; RNA polymerase then moves along the DNA in the 5’-3’ direction, adding
nucleotides to the 3‘-OH of the growing RNA chain. During the elongation phase, the newly
synthesized RNA remains bound to the template DNA for a short distance forming a
heteroduplex (DNA-RNA).
5. Chain termination; RNA synthesis continues until RNA polymerase reaches a site on the
DNA called the terminator. When this happens, RNA polymerase and the newly formed,
single-stranded RNA molecule is released from the DNA.
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Transcription differs significantly from replication.
 In transcription the point of polymerization travels only in a single direction and only one
strand of the double helix is transcribed.
 The product of transcription is a single-stranded rather than a double-stranded molecule.
 The RNA produced by transcription is much shorter than the bacterial chromosome
produced by replication. Transcription copies only one or a few genes at a time.
 A single enzyme, RNA polymerase (a different enzyme from the primase that participates in
DNA replication), is solely responsible for transcription in prokaryotes, as opposed to the
numerous enzymes needed for replication.
 During transcription, A pairs with U (Uracil).
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