BCH 3310: MICROBIAL GENETICS AND

MOLECULAR BIOLOGY

KANO UNIVERSITY OF SCIENCE AND

TECHNOLOGY,WUDIL
LECTURE 1 & 2
 COURSE OUTLINE
 Structure and Function of Genetic Material
 DNA replication

 RNA and Protein Synthesis

 Regulation of Gene Expression in Bacteria

 Mutation

 Genetic Transfer and Recombination

 Genetic Response

 Tools used for molecular studies

STRUCTURE AND FUNCTION OF GENETIC MATERIAL
 INTRODUCTION
 Cell is the structural and functional unit of life, it

provide basis for heredity

 Cells may be divided into two categories:

Prokaryotes(e.g Bacteria) and Eukaryotes(e.g

Fungi, yeast, Protozoa and Higher Animals)
 STRUCTURE AND FUNCTION OF THE GENETIC
MATERIAL
 Chromosomes are cellular structures
made up of genes that carry hereditary
information.
 A gene is a segment of DNA that codes

for a functional product.

 Genetics is the study of genes carry

information, how they are replicated

and passed to other generations, and
how they affect the characteristics of
an organism.
DNA and Chromosomes
 DNA in chromosomes is in the form of long
double helix.
 In prokaryotes, DNA is not found within a

nuclear membrane.
 The chromosome takes up only about 10% of

the cell’s volume because the DNA is

supercoiled by an enzyme called DNA
gyrase.
 WHAT IS MOLECULAR
BIOLOGY?
 The study of gene structure and function
at the molecular level
 It is the study of molecular basic of the
process of replication, transcription and
translation of the genetic material.
 learning how these interactions are
regulated.
 GENOTYPE AND PHENOTYPE
 The genotype is an organism’s genetic makeup, the
information that codes for all the characteristics and
potential properties of the organism.
 The genotype is its gene collection- its DNA.
 The phenotype refers to an organism’s actual
expressed properties, such as its ability to perform a
chemical reaction.
 The phenotype is the collection of enzymatic or
structural proteins.
NUCLEIC ACIDS
Two types

Deoxyribonucleic Acid

Ribonucleic Acid
DEOXYRIBONUCLEIC ACID

Is responsible for all cellular

activity.

Directs the production of

proteins.

Is double stranded and helical.

Is maintained by hydrogen bonds

(weak bonds)

Is very stable and can survive

 Temperatures as high as 70 C

 High salt concentrations

 Acid environments
 DNA IS A DOUBLE HELIX
 In the double helix, the two chains are coiled
around a common axis
 called the axis of symmetry in an anti parallel

manner, that is, the 5'-end of one strand is paired

with the 3'-end of the other strand
 DNA STRUCTURE CONT…

 The spatial relationship between the two strands in the

helix creates a major (wide) groove and a minor (narrow)
groove, which provide access for the binding of regulatory
proteins to their specific recognition sequences along the
DNA chain
 In the DNA helix, the hydrophilic deoxyribose–
phosphate backbone of each chain is on the
outside of the molecule, whereas the hydrophobic
bases are stacked inside
 BASE PAIRING
 The bases of one strand of DNA are paired
with the bases of the second strand, so that
an adenine is always paired with a thymine
and a cytosine is always paired with a
guanine.
 The base pairs are held together by

hydrogen bonds; two between A and T and

three between G and C . These hydrogen
bonds, plus the hydrophobic interactions
between the stacked
bases, stabilize the structure of the double
helix
RIBONUCLEIC ACID
 Ribonucleic acid (RNA) is a
biologically important type of
molecule that consists of a long
chain of nucleotide units.

 Each nucleotide consists of a

nitrogenous base, a ribose sugar,
and a phosphate. RNA is very
similar to DNA, but differs in a few
important structural details: in the
cell, RNA is usually single-stranded.
RIBONUCLEIC ACID

Three types of RNA

 mRNA messenger

 tRNA transfer

 rRNA ribosomal
RIBONUCLEIC ACID
mRNA messenger

Is complementary to one strand

of DNA and functions to carry
the genetic material from the
chromosome to the ribosome.

Transcription
RIBONUCLEIC ACID
tRNA transfer

Is responsible to transfer
information from mRNA to
rRNA.

Translation
RIBONUCLEIC ACID
rRNA ribosomal

Is associated with the ribosome

and accepts information from
tRNA and correlates the
information to synthesize
proteins.

Protein Synthesis
 NUCLEIC ACIDS
Are constructed from a string of
small molecules called
Nucleotides.

Nucleotides consist of a
5-carbon sugar (pentose), one or
more phosphate groups, and a
base containing nitrogenous
rings.
BASE TYPES
Purines

Contain 2 nitrogenous rings

Adenine and Guanine

BASE TYPES
Pyrimidines

Contain 1 nitrogenous ring

Cytosine and Thymine in DNA

Uracil replaces Thymine in RNA

RULES FOR BASE PAIRINGS
Adenine always pairs with
Thymine in DNA (A-T)

Uracil replaces Thymine in RNA

Guanine always pairs with

Cytosine (G-C) and are stronger
bonds.
 CENTRAL DOGMA THEORY
The central dogma theory of
molecular biology is
represented
by a simple pathway: DNA—
>RNA-->protein, which
demonstrates the flow of
genetic information in a
living cell.

The major processes involved

in this pathway are
replication, transcription,
and translation.
 CENTRAL DOGMA THEORY
In DNA replication, the DNA
polymerase enzyme replicates
all
the DNA in the nuclear genome
in a semi-conservative manner,
meaning that the double
stranded DNA is separated into
two and a template is made by
DNA polymerase.

This allows genomic material to

be duplicated so it can be evenly
partitioned between two somatic
cells (daughter cells) upon
division.
CENTRAL DOGMA THEORY SUMMARY
1. DNA guides the synthesis of
mRNA which in turn directs the
order in which amino acids are
assembled into proteins.

2. DNA directs its own

replication by giving rise to two
complete, identical DNA
molecules.

This replication is necessary

because each cell must inherit a
complete set of all genes in
order to carry out the cell’s life
processes.
REVERSE TRANSCRIPTASE

Another process in this

pathway is reverse
transcription, which involves
copying RNA information into
DNA using reverse transcriptase.
REVERSE TRANSCRIPTASE
Recently, this processes has
been defined and may expand
the central dogma.

For example, retroviruses use

the enzyme "reverse
transcriptase" to transcribe DNA
from a RNA template.

The viral DNA then integrates into the

nucleus of the host cell. Then it is
transcribed, and further translated into
proteins.

This biological process effectively adds

another pathway to the central dogma
of molecular biology.
 DNA REPLICATION IN BACTERIA
Bacteria contain 1 chromosome

Many contain plasmids

When bacterial chromosomes

replicate both strands are
duplicated. Each strand functions
as a template.

During replication, enzymes known

as polymerases transport
nucleotides from the cytoplasm
that are complimentary to the
template and fit them into place,
resulting in two strands, one
parental and one new one
DNA REPLICATION IN BACTERIA
During replication, enzymes known
as polymerases transport
nucleotides from the cytoplasm
that
are complimentary to the template
and fit them into place, resulting in
two strands, one parental and one
new one.

The replication is said to be semi-

conservative because the parental
strand is conserved (remains the
same ).
STEPS OF DNA REPLICATION
 DNA unwound with enzyme (replication fork)

 Complementary bases added to template (parent

strand) using enzyme

 Replication fork moves down strand

 Newly replicated DNA rewinds

 Process called Semiconservative Replication

 STEPS OF DNA REPLICATION
Copied in 5’ to 3’ direction
Polymerase can only add nucleotides to 3’ end

In Prokaryotes, replication begins at specific site in

chromosome called the origin of replication

Replication of DNA begins a specific site on the DNA template

termed the origin and proceeds in both directions from the
origin until nuclear division and cytokinesis take place.

Replication speed = 1000 nucleotides/sec

 INITIATION
 The initiation of DNA synthesis occurs at a site
called origin (also called consensus sequence, In
case of prokaryotes, there is a single site( in E.coli is
called oriC,) whereas in eukaryotes, there are
multiple sites of origin These sites mostly consist of
a short sequence of A-T base pairs.
 For example, in E. coli oriC,has a unique 245-bp

chromosomal site that contains 11 GATC

tetranucleotide sequences along its length.
 MELTING OF DSDNA
 In order for the two strands of the parental double helical DNA
to be replicated, they must first separate (or “melt”) over a
small region, because the polymerases use only ssDNA as a
template
 A specific protein called dna A binds to specific nucleotide
sequences at the origin of replication, causing short, tandemly
arranged (one after the other) AT-rich regions in the origin to
melt. Melting is ATP-dependent,
 DNA B -helicases : These enzymes bind to SSDNA
at the replication fork. then move into the
neighboring double stranded region, forcing the
strands apart—in effect, unwinding the double helix.
Helicases require energy provided by ATP. Their
function is comparable with a zip opener
 FORMATION OF THE REPLICATION
FORK
The separation of the two strands of parent DNA
results in the formation of a V shape called
replication fork. Where The active synthesis of
DNA occurs . The replication fork moves along
the parent DNA as the daughter DNA molecules
are synthesized.
 Replication of dsDNA is bidirectional—that is,

the replication forks move in opposite directions

from the origin, generating a replication
bubble, Multiple replication bubbles are formed
in eukaryotic DNA molecules, which is essential
for a rapid replication process.
 RNA PRIMER
 DNA polymerases cannot initiate synthesis of a complementary
strand of DNA on a totally single-stranded template. Rather,
they require RNA primer.
 RNA primer is a short, double-stranded region consisting of RNA
base-paired to the DNA template, with a free hydroxyl group on
the 3'-end of the RNA strand This hydroxyl group serves as the
first acceptor of a deoxynucleotide by action of DNA
polymerase
 A specific RNA polymerase, called primase (DnaG),
synthesizes the short stretches of RNA (approximately ten
nucleotides long) that are complementary and antiparallel to
the DNA template. In the resulting hybrid duplex, the U in RNA
pairs with A in DNA
 The enzyme primase in association with single-stranded
binding proteins forms a complex called primosome
 primases prefer to initiate RNA synthesis using an ssDNA
template containing a particular trimer (GTA in the case of
Escherichia coliprimase).
 The substrates for RNA primer synthesis are 5'-ribonucleoside
triphosphates, and pyrophosphate is released as each
ribonucleoside monophosphate is added through formation of a
3'→5' phosphodiester bond
USE OF RNA PRIMER TO INITIATE DNA
SYNTHESIS
 ELONGATION
 The elongation phase of replication includes
two distinct but related operations: leading
strand synthesis and lagging strand
synthesis
 CHAIN ELONGATION CONT..
 Prokaryotic (and eukaryotic) DNA polymerases
elongate a new DNA strand by adding
deoxyribonucleotides, one at a time, to the 3'- end of
the growing chain
 The sequence of nucleotides that are added is dictated
by the base sequence of the template strand with
which the incoming nucleotides are paired.
 DNA chain elongation is catalyzed by DNA
polymerase III. Using the 3'-hydroxyl group of the
RNA primer as the acceptor of the first
deoxyribonucleotide, DNA polymerase III begins to
add nucleotides along the single-stranded template
that specifies the sequence of bases in the newly
synthesized chain
 The nucleotide substrates are 5'-deoxyribo nucleoside
triphosphates.
 Pyrophosphate (PPi) is released when each new
deoxynucleoside monophosphate is added to the
 LEADING STRAND SYNTHESIS
 The DNA polymerases responsible for copying the
DNA templates are only able to “read” the parental
nucleotide sequences in the3'→5' direction, and they
synthesize the new DNA strands only in the 5'→3'
(antiparallel) direction
 The DNA strand (leading strand) with its 3'-end (3'-
OH) oriented towards the fork can be elongated by
sequential addition of new nucleotides
 Hint: Free hydroxyl group of the 3'-end of the RNA
primer strand serves as the first acceptor of a
deoxynucleotide by action of DNA polymerase
 LAGGING STRAND SYNTHESIS
 The other DNA strand (lagging strand) with 5'-end presents
some problem, as there is no DNA polymerase enzyme (in
any organism) that can catalyze the addition of nucleotides
on the 5'end ( i .e.3 ' → 5'direct ion)of the growing chain.
 This problem however is solved by synthesizing this strand as
a series of small discontinuous DNA fragments 1000 to 2000
nucleotides in length, called the Okazaki fragments
 new primers are needed for each Okazaki fragment ;short
RNA sequences are constantly being synthesized at the
replication fork on the lagging strand
 Okazaki fragments are eventually joined (ligated) to become
a single, continuous strand.
 PROOFREADING OF NEWLY SYNTHESIZED
DNA:
 To ensure replication fidelity, DNA polymerase III has, in
addition to its 5'→3' polymerase activity, a “proofreading”
activity (3'→5' exonuclease, As each nucleotide is added
to nthe chain, DNA polymerase III checks to make
certain the added nucleotide is, in fact, correctly matched
to its complementary base on the template.
 If it is not, the 3'→5' exonuclease activity corrects the
mistake. For example, if the template base is cytosine and
the enzyme mistakenly inserts an adenine instead of a
guanine into the new chain, the 3'→5' exonucleaseactivity
hydrolytically removes the misplaced nucleotide.
 The 5' →3‘ polymerase activity then replaces it with the
correct nucleotide containing guanine
 EXCISION OF RNA PRIMERS AND THEIR
REPLACEMENT BY DNA
 DNA polymerase III continues to synthesize DNA on the
lagging strand until it is blocked by proximity to an RNA
primer. When this occurs, the RNA is excised and the gap
filled by DNA polymerase I.
 DNA polymerase I has a 5'→3' exonuclease activity that is
able to hydrolytically remove the RNA primer First,.
 Next, DNA polymerase I hydrolytically removes the RNA
nucleotides “ahead” of itself, moving in the 5'→3' direction
(5'→3' exonuclease activity).
 As it removes the RNA, DNA polymerase I replaces it with
deoxyribonucleotides, synthesizing DNA in the 5'→3'
direction (5'→3' polymerase activity).
 As it synthesizes the DNA, it also “proofreads” the new
chain using 3'→5' exonuclease activity.
 This removal/synthesis/proofreading continues, one
nucleotide at a time, until the RNA primer is totally
degraded and the gap is filled with DNA
 DNA LIGASE
 the RNA primers used for initiation must be removed and
replaced with DNA
 The final phosphodiester linkage between the 5'-phosphate
group on the DNA chain synthesized by DNA polymerase III
and the 3'-hydroxyl group on the chain made by DNA
polymerase I is catalyzed by DNA ligase ,The joining of
these two stretches of DNA requires energy, which in most
organisms is provided by the cleavage of ATP to AMP + PPi.
DNA REPLICATION TERMINATES AT THE TER REGION
 Located diametrically opposite from oriC on the E. coli circular
map is a terminus region, the Ter, or t, locus. The
oppositely moving replication forks meet here, and
replication is terminated.
 The Ter region contains a number of short DNA sequences,
with a consensus core element 5-GTGTGTTGT. These Ter
sequences act as terminators;
 clusters of three or four Ter sequences are organized into two
sets inversely oriented with respect to one another. One set
blocks the clockwise-moving replication fork, and its inverted
counterpart blocks the counterclockwise-moving replication
fork.
Termination requires binding of a specific replication
termination protein, Tus protein, to Ter Tus protein
prevents the DNA duplex from unwinding by blocking
progression of the replication fork and inhibiting the ATP
dependent DnaB helicase activity.