Microbial Genetics

Review on Basic Genetics

by
Prokaryotic Genome
Mechanism of gene Transfer
conjugation.wmv
Mechanisms of gene transfer
Transformation.wmv
Transduction.wmv
DNA structure
RNA
Types:
1. mRNA (5%)
2. rRNA (80%)
3. tRNA (15%)
Structure
1. Primary
2. Secondary (e.g. Hairpin config.)
3. Tertiary (3 dimensional config.)
DNA Replication
Proposed theory
Central Dogma of Life
1. Semi-conservative
- the daughter DNA contains new and
old and new strand

2. Conservative
- the old strands rewind after serving as
template

3. Dispersive
- daughter DNA is a mixture of old new strands
Unidirectional Fork Movement
a. Leading strand (3’ -------- 5’ )
- requires few primers
- continuous directional movement

b. Lagging strand (5’ -------- 3’ )

- requires several primers (1000 – 2000
nucleotides)
- short burst of discontinuous synthesis
(opposite direction to unwinding)
Reaction mechanisms
1. Unwinding
a. Helicase
- separates the DNA helix template
b. DNA binding proteins
- stabilize the DNA chain
c. DNA topoisomerase
- relieves supercoil infront of replication
fork
2. Priming and Polymerization
a. Primase
- catalyzed the synthesis of RNA primer

b. DNA polymerases
- assembles DNA chains on primers
- add DNA nucleotides sequentially
- has proofreading mechanisms

c. DNA Ligase
- seals in the DNA strand
Prokaryotic DNA polymerase
1. DNA Polymerase I
- fill the gaps left after removal of primers after
DNA polymerization

2. DNA Polymerase II
- back up of DNA Pol I

3. DNA Polymerase III

- responsible for polymerization
 a. Polypeptide alpha
-carry out the fundamental polymerization
of nucleotide into the chain

b. Asymmetric dimmer
- allows DNA Pol III to carry out replication
of leading and lagging strand
simultaneously
Transcription
- transfer of genetic information from DNA to RNA
1. Initiation
- random collision between RNA Polymerase
and DNA in the promoter region (IFs)

2. Elongation
- sequential addition of RNA nucleotides
(EF are activated, IFs are downgraded)

3. Termination
- TFs destabilized the transcription complex
- release of transcript for processing
Bacterial RNA Polymerase

- contains 3 different polypeptide as core enzymes

(alpha-2, Beta and B’)

- efficient transcription initiation, RNA polmerase

should bind sigma factor (increases the
selectivity of bacterial RNA Polymerase)
Bacterial mRNA processing

- 5’ end is marked with phosphates

- methylation of some base pairs
- short poly A tail added to 3’ end
Translation
1. Initiation
- prior to initiation, activation of amino acid
(attachment of amino acid to their respective
tRNA’s mediated by amino acyl synthetase)

- attachment of formylated methionine (fmet)

to 30S sub-unit ribosome with the action of
IF1
- orientation of fmet to AUG codon b IF3

- completion of 70S ibosomes (binding of 50S to

the 30S)

- Peptidyl binding site (P-site), Amino acyl site

(A-site)
2. Elongation
- new aa-tRNA enters the A-site (EFTu)

- release of tRNA in P-site, movement of

ribosome along the mRNA using the EF-G

- the cycle is repeated until the whole length of

mRNA is translated

- the elongation is from 5’ to 3’ relative to the

mRNA
3. Termination
- the ribosome encounter one of the terminating
codons (UAA, UAG, UGA)

- the terminating codon, once in A-site, codes

for the releasing factor (RF1 and RF2)

- RFs dissociates the polypeptide and the tRNA

from the ribosome and the 70S sub-unit
APPLICATIONS OF MICROBIAL GENETICS

1. Bacteria test systems

2. Strain improvement
3. Contributions to recombinant DNA
technology
Ames Test

determines mutagenicity of a substance

 uses mutant tester strains,, each with a known
point mutation

Bacterial biosensors (Microbe-based sensors; MBS)

detectors of environmental pollutants

 living micro-organisms genetically engineered to produce
specific output in response to target chemicals
 reporter proteins are produced by the cell after specific
contact or interaction with a target analyte or condition
Bioremediation

- use of
biological agents to degrade toxic
environmental pollutants

Strain Improvement

- genetically modified to produce a specific

compound or over-produce a desired product
(high-yielding strains)
Recombinant DNA Technology

 also known as genetic engineering,

molecular cloning

 encompasses a number of experimental

protocols leading to transfer of DNA from
one organism to another
CLONING TOOLS: RESTRICTION ENZYMES

 recognize specific nucleotide sequences and cleave

both strands of the DNA containing those sequences
 recognition sequences for many enzymes are the same
on both strands (palindromic)
 uses:
 to map DNA molecules physically
 to analyze population polymorphisms
 to rearrange DNA molecules
 to prepare molecular probes
 to create mutants
 to analyze the modification status of the DNA
Cloning Vectors: Plasmids

 Natural plasmids simplified & modified for DNA

cloning
 Requirements:
 Autonomous replication (ori site)
 Selectable marker (drugR)
 Multiple unique restriction enzyme cut sites in non-
essential regions
 Small size (~4 kb): easier to isolate & use & can add
larger DNA fragments
 High copy number: ensure high yield of clones
END