Isolation of mutator strains to identify genes for DNA repair
Mutator strains have a mutation in a gene that is involved in DNA repair, and as a result have a higher
rate of mutation than the wild type.
To identify mutator strains, a Lac- strain was use
The genetic code
The initiation and termination codons determine an open reading frame (ORF).
For any area of DNA sequence, there are 2 possible RNAs that could be made, each of
which has 3 possible reading frames, for a total of 6.
Mutations can be complemented to restore
the WT phenotype by supplying a WT copy
of the gene to the mutant.
Complementation by introduction of a library of
WT DNA on a replicating plasmid to the mutant
can be used to identify the site of
Mechanism of Conjugation
Transfer of DNA from one cell to another
Donor cell transfers DNA to a recipient cell
Recipient that has received DNA is called a transconjugant
Plasmids that encode genes for their transfer are called
Uptake of naked, free DNA by cell
Bacteria capable of being transformed are said to be competent
Some bacteria are naturally competent (Bacillus subtilis,
Streptococcus pneumoniae, Haemophilus influenzae, Neisseria
gonorrhoeae, and some
Complementation tests with phage
Recombination tests with phage
Infect a nonpermissive host with two mutant
phage that have lost the ability to infect this bacterium
Use a high multiplicity of infection (MOI; 5 phage
per 1 bacterial cell is considered h
Methyl-directed mismatch repair (MDMR)
General repair mechanism that recognizes
minor distortion of the DNA helix.
Responsible for repair of:
incorporation of base analogs
alkylation of bases
Has both lytic and lysogenic lifestyles
About 1% of infected host cells will become a lysogen, which refers to a bacterium in which a phage
genome replicates as part of the bacterial chromosome or as a plasmid.
The phage genome in a lysogen is r
Partitioning of plasmids
As with the chromosome, the system that partitions plasmids to each
daughter cell is called the par system.
Plasmids, like the chromosome, replicate at the center of the cell.
Partitioning systems for low copy plasmids fall int
Suppressors in phage
Test to distinguish suppressor mutations
Crick et al. used T4 to uncover the nature of
the genetic code.
First, they isolated frameshift mutations in a phage
Second, they isolated suppressor mutations in the
Lysis or Lysogeny?
The decision lies in CII abundance.
Lysis or Lysogeny?
The decision lies in CII abundance.
Induction of from the lysogenic to the lytic
phase is triggered by DNA damage in the host.
The phage is thought to jump ship from
Regulation of transposition
Transposons negatively regulate their acivity to ensure that they transpose infrequently.
In Tn3, TnpR is a repressor of the transposase.
In Tn10, translation of the transposase is inhibited by an antisense RNA.
The trp operon of E. coli
A negatively regulated biosynthetic operon
Regulation of a biosynthetic operon is the opposite
of a catabolic (degradative) operon such as the lac
Enzymes of a biosynthetic operon should not be
made in the presence of t
Regulation of gene expression
Reasons for a cell to regulate gene expression:
To avoid wasting energy on RNA and proteins that are
To turn off genes whose products may interfere with other
processes in the cell at that time.
To regulate the
RNA degradation and decay
RNA degradation can be divided into two different stages: Functional decay occurs when an endonuclase
cleaves the RNA into smaller pieces. The RNA is non-functional at this point, but the same mass of RNA
still exists. Mass deca
Post-translational regulation - localization
Inner membrane, outer membrane, periplasmic,
and secreted proteins must pass through a
membrane to get to their final location.
A signal sequence, which is cleaved off in the
case of proteins that pass throug
Not all DNA is transcribed into RNA. Instead, cis-acting
elements on the DNA called promoters direct RNA
polymerase to regions of DNA to be transcribed.
The strength of a promoter - its ability to attract RNA
polymerase - determin
Building blocks of DNA: deoxyribonucleotidetriphosphates (dNTPs)
The precursors for DNA synthesis are
dNTPs, nucleotides (1) that are composed
of a base, a sugar, and a phosphate group.
Ribose (2) is the sugar in DNA and RNA.
In DNA, the 2-hydroxyl
Transposons are DNA elements that can hop, or transpose from one place to another (below).
Discovered by Barbara McClintock in corn.
Transposase is the enzyme that that promotes transposition.
Transposons have inverted repeat (IRs) sequenc
Linear plasmid ends
Extrachromosomal DNA that replicates separately from chromosome
All have origin of replication (oriV)
Encode proteins and RNA molecules
Usually do not encode essential functions
Usually have genes that confer an advantage
Global regulatory mechanisms
are used to regulate numerous
operons at once to adjust to
major changes in the
Often a single regulatory gene
that encodes a DNA-binding
protein is involved.
Restriction systems and endonucleases
Cut DNA at a specific sequence
Recognition sequence is often palindromic (reads the same 5 to 3 on both strands)
Specific to bacteria
Thought to have evolved to defend against bacteriophage
2 part systems
Topoisomerases are enzymes responsible for adding or relieving supercoils in DNA.
In Type I topoisomerases, 1 DNA strand is broken, the other strand is passed through the
cut, and the break is repaired.
In Type II topoisomerases, both st
Model for replication
restart after replication
fork encounters a nick
PriA, B and DnaT proteins load
DnaB and replication complex.
PolIII extends the invading 3
strand for leading strand synthesis,
DnaG provides an RNA prmer
for lagging strand synthes
Model of replication must account for:
Initiation of replication (Timing, location)
Opposite polarity polymerization (5 to 3 and 3 to 5 - Prescott and Kuempel)
Unwinding of the helix (To expose template)
Protection of single-stranded DNA and preve
Breaking of two DNA molecules in regions of similar or identical sequence and subsequent joining of one DNA
to the other resulting in a crossover.
Can change the order of genes on a chromosome or move genes to a different replic
Fidelity of replication
1. Thermodynamics of base pairing
The difference in free energy between correct (G-C and A-T) base pairing and incorrect
(G-T for example) predicts 1/100 mistakes.
2. Base selection by polymerase
If the addit
2 types of terminators: factor-dependent terminators
and factor-independent terminators.
Factor-independent terminators rely on DNA sequence
A GC-rich inverted repeat separated by at least 4 bp
Terms used in bacterial genetics
Mutant - an organism that is known to be the direct offspring of a member of the same species but is
different in some way.
Phenotype - any observable characteristic of an organism.
(His+, His-, Rif+, Rif-)
Genotype - t