Organisation of prokaryotic genome

• Structure of prokaryotic cell wall vs euk cell wall (cellulose)

  Peptidoglycan

  Gram positive bacteria stain purple in gram staining

  Gram negative bacteria stain red/pink in gram staining
• 
  

  Organisation of prok genome

  1. Large circular chromosome
  2. Double stranded DNA associated with non-histone proteins/DNA binding protein
  3. Absence of introns within genes
  4. No telomeres ; no end-replication problem ; circular DNA completely replicating daughter DNA molecules
  5. No centromeres
  6. Structural genes coding for enzymes in the same metabolic pathway organised together into operons under control of single promoter
  7. Plasmids self-replicating and confer advantages to bacterias survival in stressful environments.
  8. One origin of replication per chromosome
• Structure of bacterial chromosome

  1. Formation of looped domains anchored by non-histone proteins/DNA binding proteins
  2. Negative supercoiling further compacts looped bacterial chromosomes (by DNA gyrase and topoisomerase I.
• Overall structure of bacterial cells

  1. Peptidoglycan cell wall
  2. Lacks true nucleus/nuclear envelope
  3. Circular chromosomes not membrane bound/floating freely in cytoplasm/in nucleoid (euk nuclear membrane bound)
  4. No membrane bound organelles
  5. 70S ribosomes
  6. Presence of plasmids
  7. DNA free in cytoplasm
• Binary fission (prok reproduction)

  1. Semi-conservative replication of parental DNA at origin of replication gives rise to 2 origins
  2. Each origin moves rapidly to the ends of the cell and adheres to cell surface membrane as chromosome replicates
  3. Cell elongates, separating two identical copies of chromosome
  4. Cell wall materials deposited as cell surface membrane invaginates. Two daughter cells genetically identical to parent cells produced.
• How genetic variation arises in proks

  1. Spontaneous mutations due to mutagen, leading to change in nucleotide sequence
  2. Horizontal gene transfer(trf btwn bacterial cells) -> transformation, transduction, conjugation
• Transformation

  1. Naked foreign DNA released from lysed bacteria into environment is taken up by bacteria through specific cell surface proteins (recognize and transport DNA into cell)
  2. Foreign DNA incorporated into genome homologous recombination via crossing over.
• Example of bacterial transformation

  1. E.coli in high conc of calcium ions stimulates cell to take up small pieces of DNA. Biotechnical application to introduce genes coding for human insulin in e. coli.
• Main stages in transduction

  1. Phage infects donor bacterial cell and injects phage DNA into bacterium
  2. Donor DNA packaged in phage capsid, and phage induces lysing of donor DNA cell, releasing phages that infect recipient bacteria cell and inject donor bacteria cell's DNA genes into recipient bacterial cell.
  3. Genes transferred to recipient bacterial cell by homologous recombination.
• Generalised transduction

  1. Bacterial genes transferred randomly from one bacterial cell to another by virulent and temperate phage
  2. Error in lytic cycle results in small piece of host cell's DNA to be accidentally packaged into phage capsid instead of phage DNA.
  3. Phage infects and injects a piece of donor bacterial DNA into recipient bacteria cell.
  4. Bacteria is incoporated into recipient bacteria's DNA by homologous recombination
• Specialised transduction

  1. Bacteria genes adjacent to prophage site transferred by temperate phage.
  2. Error during lysogenic cycle causes prophage to be incorrectly excised such that excised phage DNA contains donor bacterial DNA.
  3. Phages are released from host cell via lytic cycle and phage infects and injects a piece of donor bacterial DNA into recipient bacteria cell.
  4. Donor bacterial DNA incorporated into recipient bacteria's DNA by homologous recombination.
• Conjugation, involving direct physical interaction btwn 2 bacterial cells

  1. Donor cell attaches to recipient using sex pili.
  2. Upon contact with recipient cell, sex pilus retracts, drawing the donor and recipient cells closer together
  3. Temporary cytoplasmic bridge forms between 2 cells
  4. One strand of F factor DNA cut at origin of transfer, travelling through the cytoplasmic mating bridge into recipient cell.
  5. Replication of strand remaining in donor cell, restoring F factor DNA to double-stranded condition
• Conjugation 2

  1. Two ends of F factor DNA in recipient cell join to form circular molecule, followed by replication to become double-stranded
  2. Each parental strand acts as a template for synthesis of double stranded F factor DNA by complementary base pairing
• Role of F plasmid (structure related to function)

  1. Small circular double stranded DNA
  2. Contains F factor genes required for production of sex pili
  3. Allow bacteria to mate with each other via conjugation, transferring genetic material from donor to recipient, allowing for genetic variation.
  4. Contains its own origin of replication allowing for replication independent of bacterial chromosome and origin of transfer, where single strand is cut and transferred to recipient cell
• Organisation of genes into operons:

  1. Structural genes encoding enzymes of the same metabolic pathway organised together under control of
  2. Single promoter and operator as well as terminator (REGULATORY SEQUENCES)
  3. All regulatory sequences and structural genes make up operon
• Organisation of regulatory genes:

  Codes for regulatory protein product that increases or decreases expression of structural genes
• Advantages of arrangement of bacterial genes into operons in bacteria (structural genes; same metabolic pathway under control of single promoter in operon)

  1. More efficient control of gene expression, allowing for bacteria to respond quickly to changes in environment
  2. Presence of chemical substances in environment can influence control of operon e.g. presence of cAMP leads to upregulation of transcription
• Role of operon

  1. Structural genes coding for enzymes in the same metabolic pathway organised together under control of single promoter, to produce polycistronic mRNA coding for enzymes
  2. More efficient control by responding rapidly and appropriately to changes in envt
  3. Transcriptional level control of gene expression as transcription and translation occur simultaneously due to no nuclear envelope
  4. Minimizing wastage of energy and resources, when bacteria produces enzymes only when required
  5. Allows bacteria to use variety of sugars
  6. Conferring selective advantage
• Difference between plasmids and bacterial chromosome

  1. Plasmids fewer base pairs, fewer genes than bacterial chromosome
  2. Genes coding for antibiotic resistance in plasmids vs genes coding for cell metabolism for bacterial chromosome
• Benefits of conjugation

  1. Plasmid may contain genes coding for antibiotic resistance, conferring selective advantage in envt w antibiotics
  2. Plasmid may contain genes encoding enzymes for bacteria to metabolize a new metabolite, conferring selective advantage in envt w that metabolite
  3. Plasmid may contain xenobiotic resistance genes, conferring selective advantage in envt w foreign chemicals (not naturally produced or expected within bacteria)
• Similarities between inducible and repressible operons:

  1. Both contain structural genes that encode enzymes of the same metabolic pathway and promotor and operator
  2. Both transcribe structural genes to form polycistronic mRNA
• Differences between inducible (lac) and repressible (trp) operons:

  1. Default operon expression: Inducible operon off vs repressible operon on
  2. Inducible operon synthesizes enzymes involved in a catabolic (break down) pathway vs repressible operon anabolic pathway (synthesize/build up)
  3. Regulatory Gene product: Inducible: active lac repressor vs repressible :inactive trp repressor
  4. Inducible operon, transcription turned on when inducer allolactose binds allosterically to repressor vs repressible operon transcription turned off when corepressor tryptophan binds to repressor
• Function of promoter:

  1. Site where RNA Polymerase binds to DNA for transcription of structural genes
  2. Found upstream of structural genes
• Function of Operator(like silencer of euk):

  1. Binding site for repressor to lower rate of transcription
  2. Found between promoter and structural genes
• Function of terminator:

  1. Site which signals end of transcription
  2. Found downstream of structural genes
• Function of CAP Site only in lac operon (DNA sequence) (like enhancers in euk)

  1. Binding site for activator, catabolite activator protein (CAP), to increase rate of transcription.
• Structural genes of lac operon:

  1. lacZ gene, code for B-galactosidase, which hydrolysis lactose into glucose and galactose. A side rxn converts small percentage of lactose into allolactose (increases rate of transcription)
  2. lacY gene, codes for lactose permease to transport lactose into the cell
  3. lacA gene, codes for galactoside transacetylase prevents toxic buildup of non-metabolizable lactose analogues in cytoplasm
• How a single mRNA can produce multiple enzymes

  1. Polycistronic mRNA is synthesized from structural genes
  2. Polycistronic mRNA contains 3 start and 3 stop codons, signaling the coding sequence where each polypeptide starts and ends
• Action of lacI regulatory gene in absence of lactose

  1. Codes for active lac repressor, which binds to operator site and blocks RNA polymerase from transcribing the structural genes, inhibiting transcription
  2. lac repressor allosterically regulated by allolactose inducer (effector)
  3. lacI gene constitutively expressed at fairly low levels, binding of repressor to operator is weak, basal transcriptional levels
  4. Contains its own i promoter
• Action of allolactose inducer on lac repressor and operon

  1. Small amount of lactose is transported into cytoplasm by lactose permease
  2. B-galactosidase converts lactose to allolactose
  3. Allolactose binds to repressor, resulting in conformational change of repressor, preventing it from binding to operator site
  4. RNA polymerase is able to bind to promoter to transcribe structural genes
  5. B-galactosidase catalyses hydrolysis of lactose into glucose and galactose, to be used in respiration
• Action of catabolite activator protein/cAMP receptor protein on rate of transcription

  1. Low glucose concentration, causes cAMP concentration to be high which binds to CAP , activating it
  2. Activated CAP protein binds to CAP site
  3. Because CAP is an activator, it increases rate of transcription
• How is lac operon regulated

  1. Lac repressor determines whether transcription of structural genes occur or not
  2. Activation of CAP determines rate of transcription of structural gene, only when operator is not bound by repressor
• Action of trp regulatory gene

  1. Codes for inactive trp repressor
  2. Upon production of sufficient tryptophan, tryptophan binds to trp repressor, activating it, causing it to bind to operator
  3. Transcription of structural genes in trp operon is inhibited
• Significance of tryptophan in regulation of trp operon

  1. Tryptophan acts as a corepressor which binds to repressor to change it to active form.
  2. Active repressor binds to operator, which inhibits transcription of structural genes
  3. Minimises waste of energy and resources through end-product inhibition of metabolic process that the structural genes of the trp operon are involved in
• Translational control:

  1. Binding of translational repressors to mRNA near ribosome-binding site or start codon to block ribosome from initiating translation
  2. Synthesis of antisense RNA, complementary to strand of mRNA. mRNA forms a duplex with complementary antisense RNA sequence, preventing ribsomes from gaining access to nucleotides in mRNA, preventing translation
• How to prove inducible operon

  1. Enzymes coded for by inducible operon are involved in breakdown of
  2. When substrate is absent, transcription of structural genes are usually 'off' and when substrate is present, transcription of structural genes become turned 'on'
• What happens if mutation occurs in trp C gene

  1. No functional trp C produced
  2. Trp C is an enzyme involved in the synthesis of tryptophan
  3. No production of tryptophan and the bacteria dies
• Differences between binary fission and mitosis

  1. DNA replication occurs prior to mitosis, in S phase of interphase vs DNA replication occurs during binary fission
  2. Cell elongates in binary fission but not mitosis
  3. Cell wall formation in binary fission but not mitosis
  4. Chromosomes attach to spindle fibres via kinetochore microtubules whereas in binary fission, chromosomes do not attach to spindle fibres
  5. In binary fission, single chromosome does not line up along the metaphase plate vs in mitosis, Chromosomes line up along the metaphase plate;
• Action of penicillin on bacteria

  1. Penicillin inhibits transpeptidase by acting as a competitive inhibitor
  2. Penicillin inhibits formation of covalent cross-links between peptidoglycan chains
  3. Bacterial cell wall synthesis is inhibited
  4. Bacterial cell wall is weakened because of high osmotic pressure inside the bacterium when bacteria takes in water by osmosis
  5. Increased turgor pressure causes cell to swell and lyse
• How substitution mutation in gene coding for enzyme involved in DNA replication avoids antibiotic action

  1. Change in mRNA codon, change in amino acid
  2. Change in R group, change in bonds formed
  3. Change in specific three-dimensional conformation
  4. Change in binding site for antibiotic thus antibiotic cannot bind