Gene expression

• Complementary base pairing:

  • adenine to thymine (DNA), adenine to uracil (RNA), 2 hydrogen bonds
  • cytosine to guanine, 3 hydrogen bonds
• semi-conservative dna replication:

  • both strands of the dna molecule unwound and unzipped
  • each strand acts as template for synthesis of new daughter strand
  • both new dna molecules have one original parent strand and one new daughter strand
• Conservative DNA replication

  • both strands act as template for synthesis of new dna molecule
  • one dna molecule containing both parent strands, the other dna molecule containing both daughter strands
• Dispersive DNA replication

  • parental dna broken into segments, each segment acts as template for synthesis of new dna
  • segments then joined tgt to form 2 molecules with sections of both original and newly synthesised dna
• Role of helicase: unwinds and unzips dna molecules by breaking hydrogen bonds
• Role of DNA Topoisomerase: cuts one strand of dna, unwinds and reseals it to prevent supercoiling and dna strand from breaking due to increased pressure
• Role of single-stranded DNA binding proteins: stabilises single stranded dna when exposed
• Role of primase: catalyses formation of RNA primer
• Role of RNA primer:

  • 10 nucleotides long
  • provides a 3’ OH group for dna polymerase to add dna nucleotides
• Role of DNA polymerase III:

  • adds dna nucleotides to 3’ end of rna primer via complementary base pairing
  • catalyses formation of phosphodiester bonds between nucleotides
  • proofreading ability (to replace wrongly added nucleotide)
  • can only synthesise in the 5’ to 3’ direction wrt growing daughter strand
• Leading strand:

  • strand that is synthesised continuously towards to replication fork
  • strand that is running from 5’ to 3’ direction
• Lagging strand:

  • strand that is running from 3’ to 5’ direction
  • synthesised discontinuously away from the replication fork
  • requires rna primer to be added when more dna is unzipped
  • dna is synthesised as Okazaki fragments
• Role of DNA polymerase I:

  • removes rna primers
  • adds complementary dna nucleotides to where rna primers were
  • catalyses formation of phosphodiester bonds between adjacent dna nucleotides (but not between where rna primer was and other nucleotides)
  • Can only synthesise in the 5’ to 3’ direction
• Role of DNA ligase:

  • seals nicks between Okazaki fragments
  • catalyses formation of phosphodiester bonds between dna nucleotides (of adjacent Okazaki fragments)
• End replication problem

  • At 5’ end of lagging strand, DNA polymerase I removes the last rna primer
  • Cannot be replaced since its a 5’ end, no 3’ OH group for dna polymerase to add dna nucleotides
  • Results in a 3’ overhang
• Transcription initiation in prokaryotes:

  Sigma factor (subunit of RNA polymerase) binds to Pribnow box (TATAAT) in promoter
• Transcription initiation in eukaryotes:

  • TATA box recognised by TATA-binding protein of transcription factor II D (TFIID)
  • TFIID recruits other general transcription factors and RNA polymerase to bind to promoter —> transcription initiation complex
• Transcription begins at the transcription start site (25 to 35 base pairs after the TATA box)
• Role of RNA polymerase:

  • Takes free ribonucleotides that bind to DNA template (one strand) via complementary base pairing
  • Catalyses formation of phosphodiester bonds between adjacent nucleotides
  • Moves in 5’ to 3’ direction wrt growing RNA
• Transcription termination:

  • Stops upon reaching terminator sequence
  • RNA polymerase falls off the DNA temple and releases RNA strand
• Post-transcriptional modifications

  • Takes place in the nucleus

  1. Addition of 5’ cap
  2. Splicing
  3. Addition of poly-A-tail at 3’ end
• Function of 5’ cap

  • Signals for export of mRNA out of nucleus
  • Signal for ribosome to initiate translation (5’ end is ribosome binding site)
  • Prevents degradation by nucleases
• Splicing

  • introns spliced out, exons spliced together
  • Done by spliceosome that recognises splice sites at each end of introns
• Function of poly-A-tail

  • Signal for export of mRNA from nucleus
  • Delays degradation by nucleases in the cytoplasm
• Role of aminoacyl-tRNA synthetase

  • Catalyses formation of amino acid + corresponding tRNA → aminoacyl-tRNA complex (bound by ester bond) At 3’ end of tRNA (process is also known as amino acid activation)
• Translation initiation

  • Small ribosomal subunit binds to 5’ end of mRNA, moves down mRNA until reaches start codon AUG
  • Methionine-tRNA complex binds to start codon via complementary codon-anticodon base pairing (tRNA complex contains UAC)
  • Large ribosomal subunit binds, forms translation initiation complex
• Translation elongation

  • 2nd aminoacyl-tRNA complex binds to mRNA at aminoacyl-tRNA binding site (A site)
  • Peptidyl transferase catalyses peptide bond between adjacent amino acids, amino acid at P site transfers to tRNA at A site
  • Ribosome translocates 1 codon down (5’ to 3’)
  • tRNA without amino acid moved from P to E site, ejected
  • A site can take another aminoacyl-tRNA complex
• Translation termination

  • Elongation repeats until ribosome reaches stop codons (UAA, UAG, UGA)
  • Stop codons do not have complementary tRNA
  • Release factor binds to ribosome at A site, hydrolyses ester bond between last amino acid and its tRNA
  • mRNA and polypeptide released
• A site: aminoacyl-tRNA binding site
• P site: peptidyl-tRNA binding site
• Stop codons: UAA, UAG, UGA
• Function of release factor: hydrolyses ester bond between last amino acid and its tRNA
• Role of non-template strand:

  • can act as template for repair of damaged template strand
  • act as template for synthesis of daughter strand during dna replication
• phosphodiester bond is between 3' hydroxyl group of one nucleotide and 5' phosphate group of another nucleotide
• how non-coding dna can have a role in evolution:

  • introns at where crossing over occurs between exons of alleles of a gene can create new alleles
  • mutations in splice sites can cause introns to be expressed, new alleles
  • mutations in regulatory sequences can alter gene expression
  • can confer selective advantages and increase allele frequency