Microbial Genetics

DNA Replication and the Central

Dogma
 Why do we need to know about
microbial genetics?
 Genetics is the basis of expression of certain
traits
 e.g., certain metabolic pathways in microbes or blue
eye color in humans
 If metabolism is the basis of life, and genes
encode enzymes THEN microbial genetics is the
basis of the basis of life
 Like in metabolism, many drugs used to treat
infections interfere with microbial DNA
replication or protein synthesis
 DNA forms the basis of many new diagnostic
tests for bacterial and viral infections
 Definitions
 Genetics: The science of heredity, how traits
are passed from generation to generation
 Genome: A cells genetic information (usually
DNA)
 Chromosomes: Structures that contain DNA
 Genes: segments of DNA (generally) that code
for functional products
 Genotype: the genes of an organism (potential
properties)
 Phenotype: actual expressed properties
(phenotype is the manifestation of genotype)
 DNA and chromosomes
 Bacterial
chromosome
 Length of the
E. coli
chromosome =
1.7 mm
 Length of the
E. coli cell = 2 μm
 DNA is
supercoiled
(topoisomerase)
The flow of genetic information
 DNA has directionality O T
5
CH2 O
 Polymer of nucleotides: sugar 4 1

3 2
(deoxyribose), phosphate, and
O
nitrogenous bases adenine (A),
O P O-
guanine (G), thymine (T),

5’-3’ direction
O A
cytosine (C). 5
CH2 O
 "Backbone" is deoxyribose- 4 1

3 2
phosphate
O
 Start at the Carbon 5 of the first
O P O-
sugar: PO4-5’-3’-PO4-5’-3’-PO4- C
O
5’-3’-OH 5
CH2 O
 This is referred to as the 5’-3’ 4 1

3 2
direction
OH
 DNA is
double stranded
 Strands held together by
hydrogen bonds between
A-T and C-G
 Strands are directional,
complementary and
antiparallel
 DNA replication
 One “parental” double
stranded DNA molecule to
two identical “daughter”
molecules (semi-
conservative)
 How
 DNA unwinds and strands
are separated (replication
fork)
 Free nucleotides are
matched to exposed bases
 DNA polymerase joins
nucleotides
 New and original strands
rewind
 Addition of nucleotides
 DNA polymerase
only adds
nucleotides to
the 3’ end of
DNA
 Synthesis
requires energy
(gained from
hydrolysis of
phosphate
bonds)
 Events at the replication fork
3’
5’

1 Helicase separates Synthesis of the leading strand

the double-stranded proceeds continuously: 5’ to 3’
molecule
Synthesis of the lagging strand is
3’
Discontinuous: also 5’ to 3’
DNA Polymerase III
RNA primer

2 Primase Okazaki fragments

Synthesizes an RNA
primer fragment

3 DNA polymerase III adds 5 DNA ligase seals

nucleotides onto the 3' the gaps between
end of the fragment adjacent fragments
4 DNA polymerase I replaces the
RNA primer with deoxynucleotides
3’
5’
 Summary of events at the
replication fork
 DNA is unwound and replication fork forms
 Leading strand is synthesized continuously
 Lagging strand is synthesized in small
segments (~1000bp,Okazaki fragments)
 DNA synthesis is primed by RNA primers
 RNA primers and replaced by DNA
 DNA fragments are joined together
(topoisomerase)
 Replication
of bacterial DNA
 Bidirectional
replication (two
replication forks)
 Following replication
 Each origin binds
to opposite poles of
the cells
 Each daughter cell
receives one DNA
molecule
 The Central
Dogma

 Information in DNA
is transcribed into
RNA
 Information in RNA
is translated into
proteins
 Transcription

 An RNA copy of the DNA encoding a gene or

genes is made
 RNA is like DNA except the sugar (ribose not
deoxyribose) and uses uracil (U) instead of T
 Three types of RNA
 mRNA: (messenger) contains the information from DNA
to make protein by translation
 tRNA: (transfer) acts as a “translator” between the
sequence of the mRNA and the sequence of the protein
 rRNA: (ribosomal) structural part of the ribosome
 Transcription initiation

 Begins when RNA polymerase binds to the

promoter sequence
 A copy of the coding DNA strand is made
using the other strand as a template
 Transcription

 Proceeds in the 5  3 direction

 U in the RNA pairs with A in the DNA
 Transcription termination

 DNA rewinds as RNA transcript leaves

 Transcription proceeds until a terminator is
reached
 Signals the RNA polymerase to detach from the DNA
 Eucaryotic mRNA
One of several big differences ……
The presence of introns = sequences that
are non-coding located within coding
regions (exons) of mRNA
5’ 3’

Processing in the nucleus:

5’ 3’
What purpose do introns serve?
 Translation
 mRNA is translated in
codons (3 nucleotides)
 Each codon specifies
an amino acid in the
protein
 The genetic code is
degenerate, that is
more than one codon
codes for one amino
acid
 Translation occurs on ribosomes
 Translation occurs on ribosomes
 The ribosome recognizes the
mRNA through a
ribosome binding site
on the mRNA
 Starts (AUG) and stops
(UAA, UAG, UGA)
 tRNAs are used to
translate codons into
amino acid sequence
 The first codon AUG is
recognized by the tRNA
carrying formyl
methionine. This tRNA
moves to the P site and
the second tRNA moves
into the A site
 An enzyme catalyzes the
formation of a peptide
bond between amino
acids 1 and 2 on tRNA 2
 The empty tRNA 1 is
released from via the
E site. The mRNA
moves along one
codon so that tRNA 2
moves from the A site
to the P site

 The third tRNA then

enters the A site
using its anticodon to
recognize the codon
 A peptide bond is formed
between the growing
peptide chain and amino
acids 3 on tRNA 3

 The empty tRNA 2 leaves

the ribosome through the E
site. tRNA 3 with the
peptide attached moves
the P site. Codon 4 on the
mRNA moves into the A
site and is recognized by
the anticodon on tRNA 4
  Amino acid 4 is added
to the peptide chain on
tRNA 4. The process is
repeated for each new
amino acid added until
a stop codon moves
into the A site

 When a stop codon enters the A site

 Signals an enzyme to cleave the polypeptide from
the final tRNA
 Components are then released for another round of
translation
The protein assembly line

 Bacterial cells can

translate mRNA
molecules into
proteins as they are
being transcribed
 Allows rapid
response of protein
production in
response to
changing conditions
 Antibiotics that interfere with
transcription or translation
 Rifamycins bind to bacterial transcriptase
and mRNA synthesis is inhibited
 Erythromycin and Azithromycin bind to the
bacterial ribosome, blocking mRNA
attachment.
 Tetracycline binds to the bacterial
ribosome blocking mRNA elongation