Genetics
What is genetics?
• The science of heredity; includes the
study of genes, how they carry
information, how they are replicated,
how they are expressed
Adaptation and Natural
Selection
• How do organisms adapt to change?
– Two basic options: regulate gene
expression or change the genetic code
– Change in genetic code = mutation
Why use bacteria to study
mutations?
• Only have one chromosome…one copy
of each gene
• Easy to grow
Direct selection
• Testing for traits that are easily
identified
– Colony color
– Motility
– Resistance to antibiotics
Indirect selection
• A way to look at traits that are not easily
identified, at changes in metabolic
pathways
• Replica plating
– A way to identify AUXOTROPHS from
PROTOTROPHS
Vertical Gene transfer
Horizontal gene transfer
Chapter 7
What do you know about
DNA?
• Chromosomes made of DNA
make up an organism’s
genome
• DNA codes for genes =
functional unit of the genome
• Genes code for proteins
• Chemical composition =
nucleotides
Replication: duplication of the
genome prior to cell division
Gene expression: decoding of
DNA in order to synthesize gene
products (proteins):
Transcription: DNA →RNA
Translation: RNA → protein
Diagrammatic representation
of DNA
DNA Structure
• Double helix formed by
complementary strands
• Strands composed of
deoxyribonucleotide
subunits = nucleotides
• Antiparallel strands held
together by hydrogen
bonds between base
pairs
– 5’ P04 binds to 3’ OH
– Thymine pairs with adenine
– Guanine pairs with cytosine
DNA Replication
Enzymes necessary for DNA
replication
•
•
•
•
Primase: synthesizes the RNA primer
Helicase: “unzips” 2 strands of DNA
DNA Polymerase: synthesize 5’→3’
DNA gyrase: releases tension during
uncoiling of circular DNA
– Produced by prokaryotes and some simple eukaryotic
organisms only, so potential target for antibiotics
**target of quinolones and aminocoumarins**
• DNA ligase: seals the gaps between
Okazaki fragments (forms covalent bonds)
Gene Expression
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•
•
•
Transcription
Post-transcriptional modification
Translation
Post-translational modification
Transcription: DNA to RNA
• RNA polymerase
– Does not require a primer to initiate
synthesis
– Recognition of the promoter via sigma
factor (bacterial transcription factor)
• Process begins at the promoter region
and ends at the terminator sequence
• Process proceeds in the direction 5’→3’
• Base pairing: thymine replaced with
uracil; U-A, G-C
RNA synthesis
What are the possible
products from transcription?
• Messenger RNA (mRNA)
• Transfer RNA (tRNA)
• Ribosomal RNA (rRNA)
Translation: RNA to protein
• What is needed for the process?
– mRNA: has the code
– Ribosomes: present the codons to tRNA,
align the amino acids
• Protein + rRNA
– Amino acids
– tRNA: anticodon ; initiates the
protein sythesis at the P-site
brings the correct amino acid to
add at the A-site
Translation: RNA to protein
• What is needed for the process?
– mRNA
– Ribosomes
– Amino acids
– tRNA
Initiation of Translation
• Ribosome binds ribosome binding site
– on mRNA molecule
– In bacteria: binding occurs during mRNA
synthesis – so translation and transcription
occur simultaneously
• Ribosome completes assembly while
bound to the mRNA
• Initiating tRNA binds to start codon: AUG
– N-formylmethionine = f-Met)
– Also codon for normal methionine
Elongation of the
Polypeptide Chain
• 2 binding sites on ribosome for tRNA:
– P-site:
– A-site:
• Initiation tRNA binds to P-site and
provides f-Met
• tRNA recognizing the next codon binds
to A-site and provides coded AA
• Ribosomal enzyme creates a peptide
bond between
Termination of Translation
• Ribosome gets to stop codon
• No tRNA recognizes the stop codon
→enzymatic cleavage of bond that
binds the polypeptide to the mRNA
• Ribosome falls off and dissociates into 2
subunits
• Subunits are ready to reassemble and
initiate translation at another site
Post-Translational
Modification
• Synthesized polypeptides are straight
chains of amino acids
• Modifications to make them into
functional proteins, ready them for
transport out of the cell = PTMs
• Folding: chaperone-assisted
• Tag removal: export signal sequence is
removed in the process of crossing the
cytoplasmic membrane
The reading frame determines
the protein
The Genetic code
Translation
Both processes occur at the
same time in bacteria (why not
in eukaryotic cells?)
Eukaryotic cells differ in
transcription and translation
• Ribosomes are 80s – 40s and 60s subunits
• 5’ end of mRNA is capped
– Methylated guanine added to pre-mRNA
– Stabilizes transcript, enhances translation
• Polyadenylation of 3’ end of mRNA
– Poly A tail added to pre-mRNA
– Stabilizes transcript , enhances translation?
• Splicing: removal of non-coding sequences
= introns; exons spliced together
• Translation is monocystronic
Is protein synthesis regulated?
• Three types of protein regulation
– Enyme inhibition (ex: feedback inhibition)
– Repression (ex: tryptophan operon)
– Induction (ex: lactose operon)
Does regulation occur at the
level of transcription?
• Some gene expression is constitutive:
proteins encoded by these genes are
continuously synthesized
• Other genes are induced: proteins only
made when needed
• Other genes are repressed: proteins
produced routinely, but turned off when
not needed
Models for transcriptional
regulation with repressors
Transcriptional regulation by
activators
Lactose Operon as a model
• Used to understand control of gene
expression in bacteria
• Operon consists of three genes needed
to degrade lactose
• Repressor gene (codes for repressor
protein) outside of operon coding region
inhibits transcription unless something
else binds to the repressor protein
Lactose Operon
What conditions are needed for the
lactose operon to be turned “on”?
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•
•
•
No glucose
Lactose present
Increasing levels of cAMP
cAMP binds to CAP, then complex
binds next to lactose operon promoter
at the activator region
• RNA polymerase binds to promoter
If E. coli is growing in a flask with glucose
and lactose…
Gene regulation systems in bacteria
• Signal transduction:
transmission of
information from
outside to inside cell
– Quorum sensing: ability
to sense the density of
cells within the same
population
– Communication occurs
via molecular signals
– In quorum sensing,
response to the signal is
concentration dependent
– Critical level → induction
of gene expression
Chapter 8
Adaptation and Natural
Selection
• How do bacteria adapt to change?
• Like any organisms, they have 2 basic
options:
– Regulate gene expression
– Change the genetic code
• Change in genetic code = mutation
• Bacteria can also utilize HORIZONTAL
GENE TRANSFER
Vertical Gene transfer
Horizontal gene transfer
What are mutations?
• Changes in the base sequence of the
DNA
• Do they always change the genetic
code?
What can cause mutations?
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•
•
•
Chemicals (nitrous acid)
Physical mutagens (uv light)
Biological mutagens (transposons)
Spontaneous mutations (errors in
replication)
– Random occurrences
– Low frequency; usually at a constant within
a given population
– Essential for a population to adapt to
change
Causes of mutations in
bacteria
• Most are spontaneous
– Errors made by DNA Polymerase
• UV light exposure
• Oxidative injury induced by reactive
oxygen species (ROS) – superoxide,
hydrogen peroxide
Types of Mutations
• Base substitution: replacement of one
nucleotide base with another
– Missense mutation: altered codon specifies a different
amino acid
– Nonsense mutation: altered codon is a stop codon,
resulting in formation of a truncated, usually nonfunctional protein
– Silent mutation: the strict definition = a change in the
codon does not change the encoded amino acid; a more
broad definition = a change that does not change the
function of the encoded protein
• by this definition a silent mutation could be any of these types of
base substitions, as long as the function of the protein
(phenotype) was not affected)
Base-pair mutation:
missense
Results of base-pair mutations
Types of Mutations
• Frameshift: deletion or addition of a
nucleotide base
– Changes the reading frame
– Most result in a truncated, non-functional
protein = knockout mutation
Frameshift mutation
Induced mutations: transposition
Transposons =
segments of DNA
- Barbara
that can move
McClintock: “jumping
from one location genes” biological
mutagen
in a cell’s
- Most contain
genome to
transcriptional
another
terminators
Induced mutations:
Chemical mutagens
• Nucleobase
modifiers
• Intercalating agents
• Base analogs
Nitrous acid as a chemical
mutagen
Nucleoside analogs are mutagens
Intercalating agents
Induced mutations: Radiation
• Ultraviolet light: introduction of thymine
dimers
– Covalent bonds form between adjacent thymine
molecules
– Alters shape (distorts) double helix
– Replication and transcription can’t proceed past
the site of distortion
– SOS repair is initiated →increased risk of errors
• X rays: double and single strand breaks
in DNA + nucleobase alterations
UV light as a mutagen
Repair mechanisms
• Wrong nucleotide inserted
– Proofreading by DNA polymerase
– Mismatch repair: fixes errors missed in
proofreading
1. recognition of mismatch (i.e., A-G)
* the non-methylated DNA strand is the new
strand and therefore the one that is incorrect
if a mismatch is present
2. protein binds to site
3. enzymatic cleavage of DNA strand
4. enzymatic degradation of region of strand the
includes the incorrect nucleotide
Repair: Mismatch
Repair of UV damage
• Two repair mechanisms
– Photoreactivation (light repair):
• Enzymatic cleavage of covalent bonds between
thymine molecules
• Uses energy from visible light to break the
bonds
• Restores original DNA molecule
– Excision repair (dark repair):
• Removal of strand of DNA containing thymine
dimers
• DNA polymerize synthesizes replacement
• DNA ligase binds the segments together
Photoreactivation
Excision Repair
SOS Repair
• Last ditch effort: fix or die
• DNA polymerase synthesized in
response to severe DNA damage does
not proofread – quick and dirty
transcription, error prone →
SOS mutagenesis
DNA-mediated Transformation
• Transduction
– Specialized
– Generalized
• Conjugation
– Plasmid transfer
– Chromosome transfer
Plasmid transfer
• Making contact: F pilus of donor binds
to receptor on cell wall of recipient
bacterium
• Initiation of transfer
• Transfer of DNA
• Transfer complete
Chromosome transfer
• Hfr cells: have F plasmid integrated into
chromosome
• Hfr cells produce an F pilus
• F plasmid DNA directs transfer
• A small amount of regional
chromosomal DNA is also shared in the
transfer