OCPG

Structure of prokaryotes

Double stranded DNA genome, bacterial chromosomal DNA usually circular

Plasmids: extrachromosomal DNA, double stranded

Structure of genome

One origin of replication

Bacterial DNA bind to DNA-binding proteins (non-histone) to form loop domains, which are supercoiled to further compact

Contain operons

Plasmids

Small, double stranded DNA molecules existing independently of the chromosome

Genes are not essential, but beneficial and confer selective advantage

Binary Fission process

(1) Chromosome replication begins

(2) DNA replication proceeds bidirectionally from origin of replication, replicated loops separate into two independent circular chromosomes. Replication continues, one copy of the origin is now at each end of the cell while the cell elongates

(3) Replication finishes, plasma membrane is pinched inward by a tubulin-like protein, and a new cell wall is deposited

(4) Two daughter cells result

Why does bacteria genomes differ?

• Bacteria have short generation time due to rapid process of binary fission

• Fast reproduction process leads to considerable number of mutations in a bacteria population, contributing to overall genetic diversity

Transformation process

(1) A competent bacterium can bind exogenous DNA and transport it into the cell

(2) Entering of exogenous DNA

• Exogenous DNA binds to receptor complex by competence proteins ComEA and ComG

• As DNA is pulled through the channel, one strand of DNA is degraded by a deoxyribonuclease

• Surviving strand of DNA is stabilised by single-strand DNA binding protein and RecA protein

(3) Integration

• Single strand of donor DNA is integrated into chromosome, producing a DNA heteroduplex with different alleles in the 2 strands

• Replaced recipient strand will be degraded

Conjugation

(1) Mating bridge formation

• Cell carrying F plasmid (donor cell) uses sex pilus to attach to recipient cell, which retracts and pulls both cells together to form a mating bridge

(2) Transferring of plasmid

• One strand of plasmid DNA breaks

• Broken strand peels off and one end enters the F- cell, where synthesis of complementary strand begins

• Unbroken strand is used as a template to synthesise a new strand, known as rolling circle replication

• Genes involved in pilus formation are transferred

(3) DNA Replication

• DNA replication continues in both donor and recipient cells, as transferred plasmid strand moves farther into the recipient cell

(4) Formation of 2 F+ cells

• Once DNA transfer and synthesis complete, plasmid in recipient cell circularises

• Recipient cell (F-)  is now a F+ cell too

Generalised transduction

(1) Phage infects a bacterial cell containing alleles A+ and B+

(2) Host DNA is fragmented and hydrolysed by phage enzymes after infection, phage DNA and proteins are made in the donor cell

(3) Packaging

• Bacterial DNA fragment containing A+ allele may be accidentally packaged into phase capsid

• Bacterial cell is lysed, releasing virions into the surrounding

(4) Transducing phage

• Transducing phage containing A+ allele infects another bacterial cell containing A- allele

• Recombination occurs where homologous region of recipient cell’s chromosome is broken, exchanging corresponding sections with donor DNA, and is rejoined

(5) Recombinant bacteria

• Genotype of resulting recombinant cell differs from genotype of both donor and recipient (A+ B-)

Specialised transduction

(1) Bacterial cell has prophage integrated between genes A and B

(2) Improper excision 

• Occasionally, prophage DNA exits incorrectly, taking adjacent segment of bacterial DNA with it during an induction event

(3) Packaging into phage

• Phage-bacterium hybrid DNA is packaged into a capsid head

• During cell lysis, defective phage will infect another bacterium and inject bacterial DNA into new bacterium

(4) Joining

• New alleles from previous bacterial cell can be incorporated into genome of new host by homologous recombination or integration of phage-bacterium hybrid DNA

(5) Recombinant bacteria

• Genotype of resulting recombinant cell differs from genotype of both donor and recipient (A+ B-)

Structural genes

Encodes for proteins or noncoding RNAs such as tRNA and rRNA

Regulatory genes

Encode proteins that regulate expression of genes

Constitutive gene expression

Continually expressed as proteins encoded are essential for survival (eg tRNA, rRNA, ribosomal proteins)

Inducible gene expression

Turned on in response to a substance in the environment, usually catabolic enzymes (degradative)

Aim: save energy and not make unnecessary proteins

Repressible gene expression

Turned off in response to a substance in the cell, usually anabolic enzymes (biosynthetic

Aim: avoid accumulation of products that are not needed

Positive control

regulated by activator, a protein that increases rate of transcription

Negative control

regulated by repressor, a protein that inhibits transcription

Inducer

Increase rate of transcription

Binds to repressor to prevent from binding to DNA OR bind to activator to cause it to bind to DNA

Corepressor

Inhibition of transcription

Binds to repressor to cause it to bind to DNA

Inhibitor

Inhibition of transcription

Binds to activator to prevent from binding to DNA

lac operon structure

lacZ: encodes b-galactosidase, catalyses hydrolysis of lactose to glucose and galactose and isomerisation of lactose to allolactose

LacY: encodes lactose permease

lacA: encodes galactosidase transacetylase (not needed to know)

CAP site: DNA sequence recognised by activator protein catabolite activator protein (CAP)

lacP: DNA sequence recognised by RNA polymerase

lacO: DNA sequence recognised by repressor protein lac repressor

Absence of lactose in environment

1. lacI encodes for lac repressor, a protein that regulates lac operon by binding to lacO

2. lac repressor binds to lacO and prevents RNA polymerase from transcribing genes

Presence of lactose in environment

1. basal transcriptional level allows for small amount of proteins to still be encoded by lacY, lacZ and lacA

2. lacZ encodes b-galactosidase, which converts lactose to allolactose, the inducer molecule

3. alloclactose binds to lac repressor and causes conformational change, preventing lac repressor from binding to operator

4. RNA polymerase can transcribe the operon

Presence of glucose + lactose

1. transport of glucose into the cell stimulates a signalling pathway that causes intracellular concentration of cyclic-AMP (cAMP) to decrease

2. cAMP is produced from ATP catalysed by adenylyl cyclase

3. low concentration of cAMP prevents cAMP from binding to CAP, hence CAP is inactive

4. inactive CAP does not bind to CAP site, hence transcription of lac operon is low

Presence of lactose + absence of glucose

1. allolactose and cAMP levels are high

2. allolactose binds to lac repressor and prevents it from binding to operator

3. cAMP binds to CAP, CAP binds to CAP site

4. CAP interacts with RNA polymerase which facilitates binding of RNA polymerase to lacP

5. transcription of lac operon is high

trp operon structure

trpE, trpD, trpC, trpB, trpA: encodes for polypeptides that form the 3 enzymes involved in biosynthesis of tryptophan

trpP: DNA sequence recognised by RNA polymerase

trpO: DNA sequence recognised by repressor protein trp repressor

low tryptophan levels

1. tryptophan does not bind to repressor, preventing repressor from binding to trpO

2. trp operon not repressed, RNA polymerase is free to transcribe operon

high tryptophan levels

1. tryptophan acts as a co-repressor and binds to repressor protein

2. causes conformational change in repressor protein, allowing it to bind to operator

3. trp operon is repressed, RNA polymerase cannot transcribe operon