OCPEG

• Genome in eukaryotes

  • large and more
  • multiple sets
  • linear
  • telomeres and centromeres present
• Genome in prokaryotes

  • small and fewer
  • one chromosome only
  • circular
  • no telomeres and centromeres present
• Packing of DNA in prokaryotes

  • circular dna folded into chromosomal looped domains by histone-like protein DNA association
  • supercoiling
• Packing of DNA in eukaryotes

  • Negatively charged DNA coiled around positively charged histone proteins, octamers of 8 histone proteins form nucleosomes linked by linker DNA
  • association of histones H2A, H2B, H3 and H4
  • Subsequent coiling forms 30nm chromatin fibre (solenoids)
  • 30nm chromatin fibre coils further with scaffold proteins and form looped domains
  • supercoiling to form chromosomes
• Promoter in eukaryote

  • upstream of transcription start side
  • contains TATA box
  • the greater the binding efficiency of general transcription factors to critical elements within promoter, the stronger the promoter
• Alternative RNA splicing

  • exons spliced out tgt with introns in diff combinations
  • Different spliceosomes (diff sequence) recognise diff splice sites and cut out and splice together diff combinations of introns and exons
  • enables larger no. of proteins to be produced relative to the no. of genes
  • Diff mRNA can be generated from one pre-mRNA
• Enhancers

  • Activator proteins bind to it
  • Activator binds spacer dna to allow direct interaction of activator with rna polymerase and general transcription factors
  • Activator recruits chromatin remodelling complex and histone acetyl to decondense chromatin
  • Increases rate
• Silencers

  • Repressor proteins bind to it
  • Repressor recruits histone deacetylase, prevents correct assembly of TIC
  • silencers interferes with correct binding of transcription factors and activator to dna by binding at/near promoter and enhancer regions
  • Silencer binds to activator proteins to prevent them from carrying out function
  • Decreases rate
• Telomeres (only eukaryotes)

  • series of short tandem repeat sequences
  • have single stranded dna at 3’ ends that loops back and displaces same sequences on upstream region (binds by comp base pairing)
• Telomerase

  • made of ribonucleoprotein and serves as reverse transcriptase (make dna from rna)
  • RNA in telomerase binds to tandem repeat in 3’ overhang, adjacent rna in telomerase used as template to add dna nucleotides to 3’ end
  • telomerase extends 3’ overhand
  • rna primer added, dna nucleotides added (like in replication)
• Importance of gene regulation

  • Allow cells to respond to changes in the environment (turn on/off when needed)
  • Cellular differentiation (diff cell types need to synthesis diff sets of proteins, diff genes are expressed at specific times during development or within a specific tissue)
• Eukaryotic gene regulation (genome level)

  • Chromatin modification
  • Chromatin remodelling complex
  • DNA methylation
  • Acetylation and deacetylation of histones
• Chromatin modification

  Heterochromatin: highly compact, wound more tightly

  Euchromatin: less compact, wound less tightly
• Chromatin remodelling complexes

  • Alters structure of nucleosomes temporarily
  • Can result in less or more tightly coiled dna around histones
• DNA methylation

  • Chemical attachment of methyl groups to cytosine nucleotides (catalyses by DNA transferase)
  • Prevents transcription (usually long term) by blocking binding of general transcription factors and RNA polymerase to promoter, preventing formation of transcription initiation complex
• Acetylation: adds acetyl groups to lysine residues to remove positive charge on histones (catalyses by histones acetyl transferase HAT)→ loosen electrostatic forces of attraction of dna to histones, decondense
• Eukaryotic gene regulation (transcriptional level)

  • Depends on efficiency of the promoter and rate of transcription initiation complex formation
  • The faster the formation, the faster the rate of transcription
• Importance of promoter (euk)

  • Critical elements within promoter can increase efficiency of promoter
  • CAAT box and GC box sequence can increase efficiency of promoter
• Eukaryotic gene regulation (post-transcriptional level)

  • 5’ cap
  • Splicing
  • Polyadenylation
• 5’ cap

  • Protects pre-mRNA from degradation from cellular nucleases
  • Recognised by translation initiation factors, promotes translation
• Polyadenylation

  • Enhances half life of mRNA by delaying degradation of nucleases
  • Poly-A-tail needed to recruit initiation factors
• Eukaryotic gene regulation (translational level)

  • mRNA stability
  • Translation initiation factors
  • Binding of small ribosomal subunit
• mRNA stability

  • Influenced by length of poly-A-tail
  • If protein is not needed in large amounts, mRNA has shorter half life
  • Poly-A-tail removed in 3’ to 5’ direction
• Translation initiation factors

  • Function: enable proper positioning of small ribosomal subunit with tRNA and then recruitment of large subunit
  • Availability of such factors determine rate
• Eukaryotic gene regulation (post translational level)

  • Proteolytic cleavage
  • Chemical modification
  • Protein degradation
• Proteolytic cleavage and activation: Cleavage of inhibitory proteins by proteases activate proteins that are initially inactive
• Prokaryotic gene regulation (transcriptional level) depends on

  • Promoter
  • Polymerase and sigma factor
• Prokaryotic promoter

  • Contains critical elements (-10 and -35)
  • Consensus sequences: comparing critical elements of 6 diff promoters on non-template DNA
  • The more the critical elements resemble consensus seq, the stronger the promoter
• Polymerase and sigma factor

  • Holoenzyme = core polymerase and sigma factor
  • Sigma factor binds to critical elements at promoter
  • Availability of sigma factors controlled allow for diff sets of genes to be transcribed by same rna polymerase core enzyme
• Prokaryotic regulation (translational level)

  • mRNA stability
  • Binding of small ribosomal subunit
  • Translation initiation factors
• mRNA stability (prok)

  • Prokaryotes generally have short half lives
  • Synthesis of antisense RNA complementary to mRNA binds to mRNA, forms duplex and reduces its half life by targeting rna for degradation or blocking translation initiation
• Binding of small ribosomal subunit

  • Shine-Dalgarno sequence (binding site)
  • Antisense rna complementary to rna near or at this sequence prevents binding of smal ribosomal subunit
  • Binding of translation repressor protein at or near the sequence
• Translation initiation factors (prok):

  Availability of such factors to help position ribosomal subunits controls rate of initiation
• Prokaryotic regulation (post translation)

  • Covalent modification
  • Phosphorylation or dephosphorylation
  • Protein degradation
• Deacetylation: removes acetyl groups, restores positive charge on histones (catalyses by histones deacetylase HDAC) → restores electrostatic forces of attraction, condenses dna
• Importance of telomeres:

  • ensure genes are not lost with each round of DNA replication due to end replication problem
  • signal for apoptosis when they are critically short, prevent accumulation of mutations
  • Protect and stabilise ends of chromosomes
• Proteolytic cleavage and activation in euk:

  • newly synthesised polypeptide cannot immediately serve as a functional protein
  • removal of inhibitory portions via cleavage by proteases can activate proteins that are initially inactive
• chemical modifications of proteins in euk:

  • addition of chemical groups via glucosylation (adding carbohydrate chains/sugar monomers)
  • phosphorylation (adding phosphate groups)
• protein degradation in euk:

  • determines how long a protein can maintain in cell to carry out its function
  • proteins to be degraded tagged with ubiquitin molecules (catalysed by ubiquitin ligase), these proteins degraded by proteasomes