aos1 - nucleic acids

• Dna


  deoxyribonucleic acidmade up of deoxyribose sugar, phosphate group and nitrogenous bases (A, T, C, G) double helix structure + synthesised in 5' to 3' 2 strands joined tgth by complementary base pairing → hydrogen bonding b/w nitrogenous bases
• transcription(IET)

  INITIATION, ELONGATION, TERMINATION

  
  

  INITIATION: RNA polymerase binds to the promoter region and the DNA

  ELONGATION: RNA polymerase catalyses the production of the mRNA strand by joining together complementary RNA nucleotides in the 5’ to 3’ direction.

  • mRNA is complementary to the DNA template strand - adenine pairs with uracil in RNA instead.

  TERMINATION: continues until stop sequence reached
• pre-mRNA processing


  METHYL CAP, POLY-A TAIL, SPLICING

  • sometimes grouped with transcription as both occur in nucleus
  • alternative splicing is not compulsory

  1. methyl (guanine) cap is added to the 5’ end
  2. poly-A tail is added to the 3’ end
  3. a process called splicing occurs, where exons are retained and introns are cut out of the mRNA strand by a molecule known as a splicesome
  4. alternative splicing may occur, and exons may be cut out and shuffled, and introns may be retained
• translation (IET)

  INITIATION, ELONGATION, TERMINATION

  
  

  INITIATION: mRNA molecule binds to the small subunit of the ribosome at the 5’ end

  ELONGATION: tRNA anticodons complementary to mRNA codons deliver specific amino acids in correct order + adjacent amino acids joined by condensation polymerisation by ribosome

  TERMINATION: translation ends when the stop codon is reached
• repression in trp operon


  • trp operon controlled by a regulatory gene upstream of the actual operator for the operon itself → prevents start of transcription
  • produces the trp repressor protein → binds to operator
  • repressor only bind to operator when trp bonded to it → this causes a conformational shape change
  • repressor-operator association fade after while → allows production of trp to happen if levels of trp drop in cell later
• attenuation in trp operon


  • occurs “during” transcription and translation (occur at same time in prokaryotes)→ blocks completion
  • leader sequence with 2 trp residues transcribed → form 2 hairpin loops:
  • attenuator loop: prevent completion of transcription and translation
  • anti-terminator loop: allows completion of transcription and translation
  • which loop forms depends on progress of ribosome thru leader sequence
  • low trp → ribosome has to pause + allow time for anti-terminator loop to form
  • high trp → ribosome don’t pause + causes formation of attenuator loop
• protein secretory pathway

  1. translation occurs at ribosome in rER
  2. moves into lumen of rER for some modifications
  3. shifted to golgi apparatus where folded and packaged further until its released
  4. exported via exocytosis
• polymerase chain reaction (PCR)


  cycling DNA alongside a buffer solution + moving along different temperatures

  DAER

  1. denaturing (94 celsius): separate 2 DNA strands by breaking apart hydrogen bonds
  2. annealing (50 celsius): keep strands separate + primers to anneal to DNA strands via 3’ ends → allow polymerase begin replication
  3. elongation (72 celsius): taq polymerase function optimally + replicate DNA strand → extend primers until end of sample or termination sequence
  4. steps repeated many times to generate a large sample of DNA
• gel electrophoresis


  1. DNA samples placed at 1 end in wells in agarose gel

  • one of the wells includes fragments of known sizes (standard ladder)

  1. gel immersed in buffer solution - ions allow for conducting charge + electric current passed using 2 electrodes (negative one at the end with the wells) → DNA moves to positive electrode
  2. smaller = move faster, larger = move slower → separated
  3. dye applied to visualise results (commonly ethidium bromide)→ DNA fragments and sample appear as fluorescent bands under UV light
• CRISPR-Cas9 in bacteria


  1. virus inserts DNA into bacterium + spacer is cut out + incorporated into CRISPR locus (bacterial chromosome)
  2. transcribed to form crRNA → combined w tracrRNA + forms guide RNA

  • crRNA + tracrRNA = guide RNA

  1. Cas9 enzyme + guide RNA → forms CRISPR-Cas9 complex + floats around cell until encounters complementary viral DNA

  • complementary to guide RNA

  1. viral DNA cut + inactivated
• CRISPR-Cas9 in gene editing


  describe how CRISPR Cas-9 could be used to fix [problem]

  1. identify target sequence for a cut + develop single guide RNA (sgRNA) complementary to it
  2. combine this w Cas9 enzyme, altered w PAM to suit the target
  3. inject into target cell, and then sgRNA will bind to target DNA, and signal the cut
  4. cell repair mechanisms try and repair causing errors, or will repair using gene u want to insert

  • e.g. use for increasing crop yields + increasing photosynthesis efficiencies
• recombinant plasmids


  1. plasmid w antibiotic resistance gene, reporter gene + endonuclease restriction site prepared (plasmid vector)
  2. both plasmid + gene of interest cut w same endonuclease → create (same) sticky ends = allow for specificity in insertion of gene of interest (ensures inserted in correct orientation)
  3. DNA ligase joins gene of interest + plasmid together
  4. bacteria transformed thru heat shock or electroporation
  5. transformed bacteria selected using antibiotics + recombinant plasmids selected for using reporter gene