Organization of bacterial genomes

All living organisms contain DNA. Each deoxynucleotide contains a

phosphate, a 5-carbon sugar (2-deoxyribose) and one of four
nitrogenous bases: adenine, cytosine, thymine or guanine.
The phosphate and sugar make up the backbone of each strand of
DNA, while the bases are responsible for holding the two strands
together via hydrogen bonds in a structure called the double helix.
The order of the bases in a DNA strand contains the coded genetic
information.
All of the DNA found in an organism is collectively referred to as the
genome
 The human genome is comprised of 23 pairs of linear
chromosomes, and approximately 3000 megabases (Mb) of
DNA.

While the genome of the bacterium Escherichia coli consists

of a single 4.6 Mb circular chromosome.

By studying the genomes of bacteria we are able to better

understand their metabolic capabilities, their ability to cause
disease and also their capacity to survive in extreme
environments.

Many of the well-studied bacterial model organisms, such

as E. coli, have a single circular chromosome.
 Advances in molecular genetics have shown that bacteria
possess more complex arrangements of their genetic material
than just a single circular chromosome per cell.
Some bacterial genomes are comprised of multiple
chromosomes and/or plasmids and many bacteria harbor
multiple copies of their genome per cell. The following are a
few examples of bacteria with unusual genomes.
Azotobacter vinelandii

Deinococcus radiodurans

 The genetic information in a cell is called the genome

or a genome is the complete set of genes in an
organism.

A bacterial cell's genome includes its chromosomes

and plasmids. Chromosomes are structures containing
DNA that physically carry hereditary information; the
chromosomes contain the genes.

Genes are segments of DNA (except in some viruses,

in which they are made of RNA) that code for
functional products.
 D N A and C hrom osom es
Bacteria typically have a single circular chromosome
consisting of a single circular molecule of DNA with
associated proteins.
The chromosome is looped and folded and attached at one
or several points to the plasma membrane.
The DNA of E. coli, the most-studied bacterial species,
has about 4.6 million base pairs and is about 1000 times
longer than the entire cell.
The chromosome takes up only about 10% of the cell's
volume because the DNA is supercoiled
 DNA REPLICATION - IN
PROKARYOTES

It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic
material. - Watson and Crick
 Replication is an enzymatic process in
which synthesis of a daughter or
progeny duplex DNA molecule,
identical to the parental duplex DNA
occurs.
Rate of replication in E.coli (prokaryotic
cell) is 1500 nucleotides per second.
To complete replication of whole E.coli
genome it takes 40 minutes
Overview
Semi conservative
Demonstrated in 1958 by Matthew Meselson and Franklin Stahl.
When DNA replicates, each polynucleotide strand acts as a template for the
formation of a complementary strand through base pairing interactions.
 DNA Replication Occurs at Replication Forks

Autoradiogram of a replicating E. coli

chromosome
Directed by DNA polymerase
Replication Is Bidirectional & Semi
discontinuous
 DNA Synthesis Extends RNA Primers
Prokaryotic DNA Replication - Movie
Enzymes Involved in replication:
1. DNA-dependent DNA polymerases
DNA Polymerases III
2. Primase - synthesizes the RNA primers for both lagging and
leading strand synthesis.

3. DNA Ligase - joins the 5 phosphate of one DNA molecule to

the 3 OH of another.

4. DNA Helicase (dnaB gene) hexameric protein, unwinds

DNA strands, uses ATP

5. SSB single-strand DNA binding protein, prevents strands from

re-annealing and from being degraded, stimulates DNA Pol III.

6. Gyrase / Topoisomerase II, keeps DNA ahead of fork from over

winding (i.e., relieves torsional strain).
Three Main Steps in DNA Replication

Initiatio
n

Terminati
on

Elongati
on
The Mechanism of DNA Replication in E.coli

Replication of the E.colichromosome begins at a

single replication origin and proceeds
bidirectionally.

At each replication fork, both leading and lagging strand synthesis are catalyzed by a
single multiprotein replication machine, the replisome,which consists of
DNA-unwinding proteins; the priming apparatus, or primosome,which is
needed to initiate, or prime, DNA replication; and DNA polymerase III
holoenzyme with two equivalents of core polymerase, one for the leading
strand and one for the lagging strand.

As this replisome follows the replication fork, the template for

lagging strand synthesis (the strand running 5'-3' in the direction of
fork movement) must be looped around so that it can be read in the
3'-5' direction
 A Replisome
5' Primase Primosome
3'

Helicase (DnaB)
Gyrase
DNA Polymerase I extends 5'
one Okazaki fragment and
removes the RNA from
another. S p in n in g
3'
DNA Ligase then joins at 10,000
fragments together. 5'
rp m

DNA Polymerase III

acts here

ssDNA binding
protein (SSB)
5' A p ro k ary o tic fo rk is trav ellin g at 50 to 100 k b / m inu te.

3' Eu k ary o tic fo rk s trav el at 0.5 - 5 k b / m inu te.

 Initiat
ion
specific initiation point called
OriC

DnaA protein recognizes and binds up

to four 9bp repeats in OriC to form a
complex of negatively supercoiled OriC
DNAwrapped around a central core of
Dna Aprotein monomers. This process
requires the presence of the histone like
HU or 1 HC proteins to facility DNA
bending.
The Dna A protein then guides a Dna B-
Dna C complex into the melted region
to form a prepriming complex. The Dna
C is subsequently released. Dna B
further unwinds open complex to form
prepriming complex

DNA gyrase, single stranded binding

protein (SSB), Rep protein and Helicase - II
are bound to prepriming complex and now
complex is called as priming complex
Replisome
assembly
at each
replication
fork
Elonga
tion
 Termination
Located diametrically opposite
fromoriCon the E.colicircular map is a
terminus region, theTer,or locus
Ter A, D, E stop counterclockwise
movingreplicationforks
Ter C, B, F, G stop clockwise
movingreplicationforks
Tus proteins bind to
thetermination sequences,
blocking helicases
 After the complete synthesis, two
duplex DNA are found to be
catenated .
This catenation removed by the
action of topoisomerase.
Finally, from single parental duplex
DNA, two progeny duplex DNA
synthesized
DNA Is Replicated with High Fidelity
Perfect fidelity is required to accurately
transmit genetic information.

The 3 - 5 exonuclease functions of Pol I and Pol III

DNA repair systems
Cells maintain balanced levels of dNTPs
The protein conformational change constitutes a double-
check for correct WatsonCrick base pairing between the
dNTP and the template.